Immunisation against Chlamydia pneumoniae

Abstract

The published genomic sequence of Chlamydia pneumoniae reveals over 1000 putative encoded proteins but does not itself indicate which of these might be useful antigens for immunisation and vaccination or for diagnosis. This difficulty is addressed by the invention, which provides a number of C. pneumoniae protein sequences suitable for vaccine production and development and/or for diagnostic purposes.

Description

TECHNICAL FIELD

This invention is in the field of immunisation against chlamydial infection, in particular against infection by Chlamydia pneumoniae.

BACKGROUND ART

Chlamydiae are obligate intracellular parasites of eukaryotic cells which are responsible for endemic sexually transmitted infections and various other disease syndromes. They occupy an exclusive eubacterial phylogenic branch, having no close relationship to any other known organisms—they are classified in their own order (Chlamydiales) which contains a single family (Chlamydiaceae) which in turn contains a single genus (Chlamydia). A particular characteristic of the Chlamydiae is their unique life cycle, in which the bacterium alternates between two morphologically distinct forms: an extracellular infective form (elementary bodies, EB) and an intracellular non-infective form (reticulate bodies, RB). The life cycle is completed with the re-organization of RB into EB, which subsequently leave the disrupted host cell ready to infect further cells.

Four chlamydial species are currently known—C. trachomatis, C. pneumoniae, C. pecorum and C. psittaci [e.g. Raulston (1995) Mol Microbiol 15:607-616; Everett (2000) Vet Microbiol 75:109-126]. C. pneumoniae is closely related to C. trachomatis, as the whole genome comparison of at least two isolates from each species has shown [Kalman et al. (1999) Nature Genetics 21:385-389; Read et al. (2000) Nucleic Acids Res 28:1397-406; Stephens et al. (1998) Science 282:754-759]. Based on surface reaction with patient immune sera, the current view is that only one serotype of C. pneumoniae exists world-wide.

C. pneumoniae is a common cause of human respiratory disease. It was first isolated from the conjunctiva of a child in Taiwan in 1965, and was established as a major respiratory pathogen in 1983. In the USA, C. pneumoniae causes approximately 10% of community-acquired pneumonia and 5% of pharyngitis, bronchitis, and sinusitis.

More recently, the spectrum of C. pneumoniae infections has been extended to include atherosclerosis, coronary heart disease, carotid artery stenosis, myocardial infarction, cerebrovascular disease, aortic aneurysm, claudication, and stroke. The association of C. pneumoniae with atherosclerosis is corroborated by the presence of the organism in atherosclerotic lesions throughout the arterial tree and the near absence of the organism in healthy arterial tissue. C. pneumoniae has also been isolated from coronary and carotid atheromatous plaques. The bacterium has also been associated with other acute and chronic respiratory diseases (e.g. otitis media, chronic obstructive pulmonary disease, pulmonary exacerbation of cystic fibrosis) as a result of sero-epidemiologic observations, case reports, isolation or direct detection of the organism in specimens, and successful response to anti-chlamydial antibiotics. To determine whether chronic infection plays a role in initiation or progression of disease, intervention studies in humans have been initiated, and animal models of C. pneumoniae infection have been developed.

Considerable knowledge of the epidemiology of C. pneumoniae infection has been derived from serologic studies using the C. pneumoniae-specific microimmunofluorescence test. Infection is ubiquitous, and it is estimated that virtually everyone is infected at some point in life, with common re-infection. Antibodies against C. pneumoniae are rare in children under the age of 5, except in developing and tropical countries. Antibody prevalence increases rapidly at ages 5 to 14, reaching 50% at the age of 20, and continuing to increase slowly to ˜80% by age 70.

A current hypothesis is that C. pneumoniae can persist in an asymptomatic low-grade infection in very large sections of the human population. When this condition occurs, it believed that the presence of C. pneumoniae, and/or the effects of the host reaction to the bacterium, can cause or help progress of cardiovascular illness.

It is not yet clear whether C. pneumoniae is actually a causative agent of cardiovascular disease, or whether it is just artefactually associated with it. It has been shown, however, that C. pneumoniae infection can induce LDL oxidation by human monocytes [Kalayoglu et al. (1999) J. Infect. Dis. 180:780-90; Kalayoglu et al. (1999) Am. Heart J. 138:S488-490]. As LDL oxidation products are highly atherogenic, this observation provides a possible mechanism whereby C. pneumoniae may cause atheromatous degeneration. If a causative effect is confirmed, vaccination (prophylactic and therapeutic) will be universally recommended.

Genomic sequence information has been published for C. pneumoniae [Kalman et al. (1999) supra; Read et al. (2000) supra; Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527; WO99/27105; WO00/27994] and is available from GenBank. Sequencing efforts have not, however, focused on vaccination, and the availability of genomic sequence does not in itself indicate which of the >1000 genes might encode useful antigens for immunisation and vaccination. WO99/27105, for instance, implies that every one of the 1296 ORFs identified in the C. pneumoniae strain CM1 genome is a useful vaccine antigen.

It is thus an object of the present invention to identify antigens useful for vaccine production and development from amongst the many proteins present in C. pneumoniae. It is a further object to identify antigens useful for diagnosis (e.g. immunodiagnosis) of C. pneumoniae.

DISCLOSURE OF THE INVENTION

The invention provides proteins comprising the C. pneumoniae amino acid sequences disclosed in the examples.

It also provides proteins comprising sequences which share at least x% sequence identity with the C. pneumoniae amino acid sequences disclosed in the examples. Depending on the particular sequence, x is preferably 50% or more (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more). These include mutants and allelic variants. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.

The invention further provides proteins comprising fragments of the C. pneumoniae amino acid sequences disclosed in the examples. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 75, 100 or more). Preferably the fragments comprise one or more epitope(s) from the sequence. Other preferred fragments omit a signal peptide.

The proteins of the invention can, of course, be prepared by various means (e.g. native expression, recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (e.g. native, fusions etc.). They are preferably prepared in substantially pure form (ie. substantially free from other C. pneumoniae or host cell proteins). Heterologous expression in E. coli is a preferred preparative route.

According to a further aspect, the invention provides nucleic acid comprising the C. pneumoniae nucleotide sequences disclosed in the examples. In addition, the invention provides nucleic acid comprising sequences which share at least x% sequence identity with the C. pneumoniae nucleotide sequences disclosed in the examples. Depending on the particular sequence, x is preferably 50% or more (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more).

Furthermore, the invention provides nucleic acid which can hybridise to the C. pneumoniae nucleic acid disclosed in the examples, preferably under “high stringency” conditions (e.g. 65° C. in a 0.1×SSC, 0.5% SDS solution).

Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the C. pneumoniae sequences and, depending on the particular sequence, n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 75, 100, 200, 300 or more).

According to a further aspect, the invention provides nucleic acid encoding the proteins and protein fragments of the invention.

It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing purposes).

Nucleic acid according to the invention can, of course, be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (e.g. single stranded, double stranded, vectors, probes etc.).

In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

According to a further aspect, the invention provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors) and host cells transformed therewith.

According to a further aspect, the invention provides immunogenic compositions comprising protein and/or nucleic acid according to the invention. These compositions are suitable for immunisation and vaccination purposes. Vaccines of the invention may be prophylactic or therapeutic, and will typically comprise an antigen which can induce antibodies capable of inhibiting (a) chlamydial adhesion, (b) chlamydial entry, and/or (c) successful replication within the host cell. The vaccines preferably induce any cell-mediated T-cell responses which are necessary for chlamydial clearance from the host.

The invention also provides nucleic acid or protein according to the invention for use as medicaments (e.g. as vaccines). It also provides the use of nucleic acid or protein according to the invention in the manufacture of a medicament (e.g. a vaccine or an immunogenic composition) for treating or preventing infection due to C. pneumoniae.

The invention also provides a method of treating (e.g. immunising) a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid or protein according to the invention.

According to further aspects, the invention provides various processes.

A process for producing proteins of the invention is provided, comprising the step of culturing a host cell according to the invention under conditions which induce protein expression.

A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.

A process for detecting C. pneumoniae in a sample is provided, wherein the sample is contacted with an antibody which binds to a protein of the invention.

A summary of standard techniques and procedures which may be employed in order to perform the invention (e.g. to utilise the disclosed sequences for immunisation) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.

General

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature e.g. Sambrook Molecular Cloning, A Laboratory Manual, Second Edition (1989) and Third Edition (2001); DNA Cloning, Volumes I and ii (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification. Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification.

Definitions

A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.

The term “comprising” means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional to X, such as X+Y.

The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a Chlamydial sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.

An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.

A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (e.g. see U.S. Pat. No. 5,753,235).

Expression Systems

The Chlamydial nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning. A Laboratory Manual, 2nd ed.].

Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.

The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236.1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982) PNAS USA 79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning. A Laboratory Manual].

Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposomes, direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. Hep G2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter “Summers and Smith”).

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (e.g. plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′ to 3′) transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.

Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between ˜1% and ˜5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. “Current Protocols in Microbiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers & Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, e.g. Summers and Smith supra.

The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, e.g. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, e.g. proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.

iii. Plant Systems

There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987)

Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.

The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.

iv. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistakes.” In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34]. The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning. A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat. No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].

Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g. plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl₂ or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See e.g. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect. Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].

v. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80: 1].

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds. K. N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See e.g. EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (e.g. WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the genes for invertase (EP-A-0012873; JPO 62,096,086) and A-factor (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin exit, such as an interferon leader, that also provide for secretion in yeast (EP-A-0060057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (e.g. see WO 89/02463.)

Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See e.g. Brake et al., supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See e.g. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 & 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75; 1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach & Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

Pharmaceutical Compositions

Pharmaceutical compositions can comprise polypeptides and/or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.

The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (e.g. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Vaccines

Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).

Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59™ are preferred.

As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

The immunogenic compositions (e.g. the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The immunogenic compositions are conventionally administered parenterally, e.g. by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (e.g. WO98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may be employed [e.g. Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; see later herein].

Gene Delivery Vehicles

Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.

The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retroviruses e.g. MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat. No. 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (e.g. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J. Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md. or isolated from known sources using commonly available techniques.

Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740, U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No. 4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983)Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors are those disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat. No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors are described in U.S. Pat. No. 5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S. Pat. No. 5,252,479.

The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 & WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, those alpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md. or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser. No. 08/679,640).

DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See WO95/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat. No. 5,166,057 and in Enami (1990) Proc Natl Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEJ Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; ONyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.

Particle mediated gene transfer may be employed, for example see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in U.S. Ser. No. 60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033

Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

Delivery Methods

Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for recombinant protein expression. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (e.g. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g. WO93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Polynucleotide and Polypeptide Pharmaceutical Compositions

In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.

A. Polypeptides

One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.

B. Hormones, Vitamins, etc.

Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, etc.

Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide)

D. Lipids, and Liposomes

The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.

Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See e.g. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem. Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.

E. Lipoproteins

In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C, & E, over time these lipoproteins lose A and acquire C and E apoproteins. VLDL comprises A, B, C, & E apoproteins, LDL comprises apoprotein B; HDL comprises apoproteins A, C, & E.

The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.

Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.

Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey (1979) J. Clin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Mass., USA. Further description of lipoproteins can be found in Zuckermann et al. PCT/US97/14465.

F. Polycationic Agents

Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, and purtrescine.

The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene. Lipofectin™, and lipofectAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.

Nucleic Acid Hybridisation

“Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al. [supra] vol. 2, chapt. 9, pp. 9.47 to 9.57.

“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 g for a plasmid or phage digest to 10⁻⁹ to 10⁻⁸ g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 10⁸ cpm/μg. For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10⁸ cpm/μg, resulting in an exposure time of 24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation: Tm=81+16.6(log₁₀Ci)+0.4[%(G+C)]−0.6(%formamide)−600/n−1.5(%mismatch). where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal. Biochem. 138: 267-284).

In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.

Nucleic Acid Probe Assays

Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.

The nucleic acid probes will hybridize to the Chlamydial nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native Chlamydial sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.

The probe sequence need not be identical to the Chlamydial sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional Chlamydial sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a Chlamydial sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Chlamydial sequence in order to hybridize therewith and thereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like. For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably ≧30 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.

Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. [J. Am. Chem. Soc. (1981) 103:3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated e.g. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [e.g. see Agrawal & Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [e.g. see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384-386].

Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acids. The assay is described in: Mullis et al. [Meth. Enzymol. (1987) 155: 335-350]; U.S. Pat. Nos. 4,683,195 & 4,683,202. Two ‘primers’ hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired Chlamydial sequence.

A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labelled probe will hybridize to the Chlamydial sequence (or its complement).

Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra]. mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled with a radioactive moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C, 2A-2C, 3A-3C, 4A-4C, 5A-5C, 6A-6C, 7A-7C, 8A-8C, 9A-9C, 10A-10B, 11A-11C, 12A-12C, 13A-13B, 14A-14B, 15A-15C, 16A-16C, 17A-17C, 18A-18C, 19A-19B, 20A-20B, 21A-21C, 22A-22C, 23A-23C, 24A-24C, 25A-25C, 26A-26B, 27A-27C, 28A-28C, 29A-29C, 30A-30C, 31A-31B, 32A-32C, 33A-33B, 34A-34C, 35A-35C, 36A-36B, 37A-37D, 38A-38B, 39A-39D, 40A-40B, 41A-41C, 42A-42C, 43A-43C, 44A-44C, 45A-45C, 46A-46B, 47A-47C, 48A-48C, 49A-49C, 50A-50C, 51A-51C, 52A-52C, 53A-53B, 54A-54C, 55A-55C, 56A-56D, 57A-57C, 58A-58C, 59A-59C, 60A-60C, 61A-61C, 62A-62C, 63A-63C, 64A-64D, 65A-65C, 66A-66B, 67A-67B, 68A-68B, 69A-69B, 70A-70B, 71A-71B, 72A-72B, 73A-73B, 74A-74C, 75A-75B, 76A-76B, 77A-77B, 78A-78B, 79A-79B, 80A-80B, 81A-81B, 82A-82B, 83A-83B, 848A-84B, 85A-85B, 86A-86B, 87A-87B, 88A-88B, 89A-89B, 90A-90B, 91A-91B, 92A-92B, 93A-93C, 99A-99C, 95A-95C, 96A-96D, 97A-97C, 98A-98C, 99A-99C, 100A-100C, 101A-101C, 102A-102B, 103A-103C, 104A-104C, 105A-105B, 106A-106B, 107, 108A-108B, 109A-109B, 110A-110B, 111A-111B, 112A-112B, 113A-113B, 114A-114B, 115A-115B, 116A-116B, 117A-117B, 118A-118B, 119A-119B, 120A-120B, 121A-121B, 122A-122B, 123A-123B, 124A-124B, 125A-125B, 126A-126B, 127A-127B, 128A-128B, 129A-129B, 130A-130B, 131A-131B, 132A-132B, 133A-133B, 134A-134B, 135A-135B, 136A-136B, 137A-137B, 138A-138B, 139A-139B, 140A-140B, 141A-141B, 142A-142B, 143A-143B, 144A-144B, 145A-145B, 146A-146B, 147A-147B, 148A-148B, 149A-149B, 150A-150B, 151A-151B, 152A-152B, 153, 154A-154B, 155, 156, 157, 158, 159A-159B, 160, 161A-161B, 162, 163, 164A-164B, 165, 166, 167A-167B, 168, 169, 170, 171A-171B, 172, 173, 174A-174B, 175, 176, 177, 178, 179A-179B, 180A-180B, 181, 182, 183, 184, 185, 186A-186B, 187A-187B, 188A-188B, 189A-189B show data pertaining to examples 1-189, respectively.

FIG. 190 shows a representative 2D gel of proteins in elementary bodies.

FIG. 191 shows an alignment of sequences in five (six) proteins of the invention.

EXAMPLES

The examples indicate C. pneumoniae proteins, together with evidence to support the view that the proteins are useful antigens for vaccine production and development or for diagnostic purposes. This evidence takes the form of:

• • Computer prediction based on sequence information from CWL029 strain (e.g. using the PSORT algorithm available from www.psort.nibb.ac.jp).
  • Data on recombinant expression and purification of the proteins cloned from IOL207 strain.
  • Western blots to demonstrate immunoreactivity in serum (typically a blot of an EB extract of C. pneumoniae strain FB/96 stained with mouse antiserum against the recombinant protein).
  • FACS analysis of C. pneumoniae bacteria or purified EBs to confirm accessibility of the antigen to the immune system (see also table III).
  • An indication if the protein was identified by MALDI-TOF from a 2D gel electrophoresis map of proteins from purified elementary bodies from strain FB/96. This confirms that the protein is expressed in vivo (see also table V).

Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the examples. For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot. A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie. the protein is an immunogen. This method can also be used to identify immunodominant proteins.

The recombinant protein can also be conveniently used to prepare antibodies e.g. in a mouse. These can be used for direct confirmation that a protein is located on the cell-surface. Labelled antibody (e.g. fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.

In particular, the following methods (A) to (O) were used to express, purify and biochemically characterise the proteins of the invention:

Cloning of Cpn ORFs for Expression in E. coli

ORFs of Chlamydia pneumoniae (Cpn) were cloned in such a way as to potentially obtain three different kind of proteins:

• • a) proteins having an hexa-histidine tag at the C-terminus (cpn-His)
  • b) proteins having a GST fusion partner at the N-terminus (Gst-cpn)
  • c) proteins having both hexa-histidine tag at the C-terminus and GST at the N-terminus (GST/His fusion; NH₂-GST-cpn-(His)₆-COOH)

The type a) proteins were obtained upon cloning in the pET21b+ (Novagen). The type b) and c) proteins were obtained upon cloning in modified pGEX-KG vectors [Guan & Dixon (1991) Anal. Biochem. 192:262]. For instance pGEX-KG was modified to obtain pGEX-NN, then by modifying pGEX-NN to obtain pGEX-NNH. The Gst-cpn and Gst-cpn-His proteins were obtained in pGEX-NN and pGEX-NNH respectively.

The modified versions of pGEX-KG vector were made with the aim of allowing the cloning of single amplification products in all three vectors after only one double restriction enzyme digestion and to minimise the presence of extraneous amino acids in the final recombinant proteins.

(A) Construction of pGEX-NN and pGEX-NNH Expression Vectors

Two couples of complementary oligodeoxyribonucleotides were synthesised using the DNA synthesiser ABI394 (Perkin Elmer) and the reagents from Cruachem (Glasgow, Scotland). Equimolar amounts of the oligo pairs (50 ng each oligo) were annealed in T4 DNA ligase buffer (New England Biolabs) for 10 min in a final volume of 50 μl and then were left to cool slowly at room temperature. With the described procedure the following DNA linkers were obtained:

gexNN linker:
NdeI  NheI XmaI  EcoRI   NcoI       SalI     XhoI       SacI             NotI
GATCCCATATGGCTAGCCCGGGGAATTCGTCCATGGAGTGAGTCGACTGACTCGAGTGATCGAGCTCCTGAGCGGCCGCATGAA
GGTATACCGATCGGGCCCCTTAAGCAGGTACCTCACTCAGCTGACTGAGCTCACTAGCTCGAGGACTCGCCGGCGTACTTTCGA
gexNNH linker:
HindIII NotI  XhoI   --Hexa-Histidine--
TCGACAAGCTTGCGGCCGCACTCGAGCATCACCATCACCATCACTGAT
GTTCGAACGCCGGCGTGAGCACGTAGAGGTAGTGGTAGTGACTATCGA

The plasmid pGEX-KG was digested with BamHI and HindIII and 100 ng were ligated overnight at 16° C. to the linker gexNN with a molar ratio of 3:1 linker/plasmid using 200 units of T4 DNA ligase (New england Biolabs). After transformation of the ligation product in E. coli DH5, a clone containing the pGEX-NN plasmid, having the correct linker, was selected by means of restriction enzyme analysis and DNA sequencing.

The new plasmid pGEX-NN was digested with SalI and HindIII and ligated to the linker gexNNH. After transformation of the ligation product in E. coli DH5, a clone containing the pGEX-NNH plasmid, having the correct linker, was selected by means of restriction enzyme analysis and DNA sequencing.

(B) Chromosomal DNA Preparation

The chromosomal DNA of elementary bodies (EB) of C. pneumoniae strain 10L-207 was prepared by adding 1.5 ml of lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 0.6% SDS, 100 μg/ml Proteinase K, pH 8) to 450 μl EB suspension (400.000/μl) and incubating overnight at 37° C. After sequential extraction with phenol, phenol-chloroform, and chloroform, the DNA was precipitated with 0.3 M sodium acetate, pH 5.2 and 2 volumes of absolute ethanol. The DNA pellet was washed with 70% ethanol. After solubilization with distilled water and treatment with 20 μg/ml RNAse A for 1 hour at RT, the DNA was extracted again with phenol-chloroform, alcohol precipitated and suspended with 300 μl 1 mM Tris-HCl pH 8.5. The DNA concentration was evaluated by measuring OD₂₆₀ of the sample.

(C) Oligonucleotide Design

Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF using the sequence of C. pneumoniae strain CWL029. Any predicted signal peptide were omitted, by deducing the 5′ end amplification primer sequence immediately downstream from the predicted leader sequence. For most ORFs, the 5′ tail of the primers (table I) included only one restriction enzyme recognition site (NdeI, or NheI, or SpeI depending on the gene's own restriction pattern); the 3′ primer tails (table I) included a XhoI or a NotI or a HindIII restriction site.

5′ tails	3′ tails
NdeI	5′ GTGCGTCATATG 3′	XhoI	5′ GCGTCTCGAG 3′
NheI	5′ GTGCGTGCTAGC 3′	NotI	5′
			ACTCGCTAGCGGCCGC 3′
SpeI	5′ GTGCGTACTAGT 3′	HindIII	5′ GCGTAAGCTT 3′

Table I. Oligonucleotide Tails of the Primers Used to Amplify Cpn Genes.

As well as containing the restriction enzyme recognition sequences, the primers included nucleotides which hybridized to the sequence to be amplified. The number of hybridizing nucleotides depended on the melting temperature of the primers which was determined as described [(Breslauer et al. (1986) PNAS USA 83:3746-50]. The average melting temperature of the selected oligos was 50-55° C. for the hybridizing region alone and 65-75° C. for the whole oligos. Table II shows the forward and reverse primers used for each amplification.

(D) Amplification

The standard PCR protocol was as follow: 50 ng genomic DNA were used as template in the presence of 0.2 μM each primer, 200 μM each dNTP, 1.5 mM MgCl₂, 1×PCR buffer minus Mg (Gibco-BRL), and 2 units of Taq DNA polymerase (Platinum Taq, Gibco-BRL) in a final volume of 100 μl. Each sample underwent a double-step amplification: the first 5 cycles were performed using as the hybridizing temperature the one of the oligos excluding the restriction enzyme tail, followed by 25 cycles performed according to the hybridization temperature of the whole length primers. The standard cycles were as follow:

denaturation:  94° C., 2 min
denaturation:  94° C., 30 seconds
               {close oversize brace} 5 cycles
hybridization: 51° C., 50 seconds
elongation:    72° C., 1 min or 2 min and 40 sec
denaturation:  94° C., 30 seconds
               {close oversize brace} 25 cycles
hybridization: 70° C., 50 seconds
elongation:    72° C., 1 min or 2 min and 40 sec
               72° C., 7 min
               4° C.

The elongation time was 1 min for ORFs shorter than 2000 bp, and 2 min and 40 seconds for ORFs longer than 2000 bp. The amplifications were performed using a Gene Amp PCR system 9600 (Perkin Elmer).

To check the amplification results, 4 μl of each PCR product was loaded onto 1-1.5 agarose gel and the size of amplified fragments compared with DNA molecular weight standards (DNA markers III or IX, Roche). The PCR products were loaded on agarose gel and after electrophoresis the right size bands were excised from the gel. The DNA was purified from the agarose using the Gel Extraction Kit (Qiagen) following the instruction of the manufacturer. The final elution volume of the DNA was 50 μl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8). One μl of each purified DNA was loaded onto agarose gel to evaluate the yield.

(E) Digestion of PCR Fragments

One-two μg of purified PCR product were double digested overnight at 37° C. with the appropriate restriction enzymes (60 units of each enzyme) using the appropriate restriction buffer in 100 μl final volume. The restriction enzymes and the digestion buffers were from New England Biolabs. After purification of the digested DNA (PCR purification Kit, Qiagen) and elution with 30 μl TE, 1 μl was subjected to agarose gel electrophoresis to evaluate the yield in comparison to titrated molecular weight standards (DNA markers III or IX, Roche).

(F) Digestion of the Cloning Vectors (pET21b+, pGEX-NN, and pGEX-NNH)

10 μg of plasmid was double digested with 100 units of each restriction enzyme in 400 μl reaction volume in the presence of appropriate buffer by overnight incubation at 37° C. After electrophoresis on a 1% agarose gel, the band corresponding to the digested vector was purified from the gel using the Qiagen Qiaex II Gel Extraction Kit and the DNA was eluted with 50 μl TE. The DNA concentration was evaluated by measuring OD₂₆₀ of the sample.

(G) Cloning

75 ng of the appropriately digested and purified vectors and the digested and purified fragments corresponding to each ORF, were ligated in final volumes of 10-20 μl with a molar ratio of 1:1 fragment/vector, using 400 units T4 DNA ligase (New England Biolabs) in the presence of the buffer supplied by the manufacturer. The reactions were incubated overnight at 16° C.

Transformation in E. coli DH5 competent cells was performed as follow: the ligation reaction was mixed with 200 μl of competent DH5 cells and incubated on ice for 30 min and then at 42° C. for 90 seconds. After cooling on ice, 0.8 ml LB was added and the cells were incubated for 45 min at 37° C. under shaking. 100 and 900 μl of cell suspensions were plated on separate plates of agar LB 100 μg/ml Ampicillin and the plates were incubated overnight at 37° C. The screening of the transformants was done by growing randomly chosen clones in 6 ml LB 100 μg/ml Ampicillin, by extracting the DNA using the Qiagen Qiaprep Spin Miniprep Kit following the manufacturer instructions, and by digesting 2 μl of plasmid minipreparation with the restriction enzymes specific for the restriction cloning sites. After agarose gel electrophoresis of the digested plasmid mini-preparations, positive clones were chosen on the basis of the correct size of the restriction fragments, as evaluated by comparison with appropriate molecular weight markers (DNA markers III or IX, Roche).

(H) Expression

1 μl of each right plasmid mini-preparation was transformed in 200 μl of competent E. coli strain suitable for expression of the recombinant protein. All pET21b+recombinant plasmids were transformed in BL21 DE3 (Novagen) E. coli cells, whilst all pGEX-NN and all pGEX-NNH recombinant plasmids were transformed in BL21 cells (Novagen). After plating transformation mixtures on LB/Amp agar plates and incubation overnight at 37° C., single colonies were inoculated in 3 ml LB 100 μg/ml Ampicillin and grown at 37° C. overnight. 70 μl of the overnight culture was inoculated in 2 ml LB/Amp and grown at 37° C. until OD₆₀₀ of the pET clones reached the 0.4-0.8 value or until OD₆₀₀ of the pGEX clones reached the 0.8-1 value. Protein expression was then induced by adding IPTG (Isopropil β-D thio-galacto-piranoside) to the mini-cultures. pET clones were induced using 1 mM IPTG, whilst pGEX clones were induced using 0.2 mM IPTG. After 3 hours incubation at 37° C. the final OD₆₀₀ was checked and the cultures were cooled on ice. After centrifugation of 0.5 ml culture, the cell pellet was suspended in 50 μl of protein Loading Sample Buffer (60 mM TRIS-HCl pH 6.8, 5% w/v SDS, 10% v/v glycerin, 0.1% w/v Bromophenol Blue, 100 mM DTT) and incubated at 100° C. for 5 min. A volume of boiled sample corresponding to 0.1 OD₆₀₀ culture was analysed by SDS-PAGE and Coomassie Blue staining to verify the presence of induced protein band.

Purification of the Recombinant Proteins

Single colonies were inoculated in 25 ml LB 100 μg/ml Ampicillin and grown at 37° C. overnight. The overnight culture was inoculated in 500 ml LB/Amp and grown under shaking at 25° C. until OD₆₀₀ 0.4-0.8 value for the pET clones, or until OD₆₀₀ 0.8-1 value for the pGEX clones. Protein expression was then induced by adding IPTG to the cultures. pET clones were induced using 1 mM IPTG, whilst pGEX clones were induced using 0.2 mM IPTG. After 4 hours incubation at 25° C. the final OD₆₀₀ was checked and the cultures were cooled on ice. After centrifugation at 6000 rpm (JA10 rotor, Beckman), the cell pellet was processed for purification or frozen at −20° C.

(I) Procedure for the Purification of Soluble His-Tagged Proteins from E. coli

• 1. Transfer the pellets from −20° C. to ice bath and reconstitute with 10 ml 50 mM NaHPO₄ buffer, 300 mM NaCl, pH 8.0, pass in 40-50 ml centrifugation tubes and break the cells as per the following outline:
• 2. Break the pellets in the French Press performing three passages with in-line washing.
• 3. Centrifuge at about 30-40000×g per 15-20 min. If possible use rotor JA 25.50 (21000 rpm, 15 min.) or JA-20 (18000 rpm, 15 min.)
• 4. Equilibrate the Poly-Prep columns with 1 ml Fast Flow Chelating Sepharose resin with 50 mM phosphate buffer, 300 mM NaCl, pH 8.0.
• 5. Store the centrifugation pellet at −20° C., and load the supernatant in the columns.
• 6. Collect the flow through.
• 7. Wash the columns with 10 ml (2 ml+2 ml+4 ml) 50 mM phosphate buffer, 300 mM NaCl, pH 8.0.
• 8. Wash again with 10 ml 20 mM imidazole buffer, 50 mM phosphate, 300 mM NaCl, pH 8.0.
• 9. Elute the proteins bound to the columns with 4.5 ml (1.5 ml+1.5 ml+1.5 ml) 250 mM imidazole buffer, 50 mM phosphate, 300 mM NaCl, pH 8.0 and collect the 3 corresponding fractions of 1.5 ml each. Add to each tube 15 μl DTT 200 mM (final concentration 2 mM)
• 10. Measure the protein concentration of the first two fractions with the Bradford method, collect a 10 μg aliquot of proteins from each sample and analyse by SDS-PAGE. (N.B.: should the sample be too diluted, load 21 μl+7 μl loading buffer).
• 11. Store the collected fractions at +4° C. while waiting for the results of the SDS-PAGE analysis.
• 12. For immunisation prepare 4-5 aliquots of 100 μg each in 0.5 ml in 40% glycerol. The dilution buffer is the above elution buffer, plus 2 mM DTT. Store the aliquots at −20° C. until immunisation. (J) Purification of His-Tagged Proteins from Inclusion Bodies

Purifications were carried out essentially according the following protocol:

• 1. Bacteria are collected from 500 ml cultures by centrifugation. If required store bacterial pellets at −20° C. For extraction, resuspend each bacterial pellet in 10 ml 50 mM TRIS-HCl buffer, pH 8.5 on an ice bath.
• 2. Disrupt the resuspended bacteria with a French Press, performing two passages.
• 3. Centrifuge at 35000×g for 15 min and collect the pellets. Use a Beckman rotor JA 25.50 (21000 rpm, 15 min.) or JA-20 (18000 rpm, 15 min.).
• 4. Dissolve the centrifugation pellets with 50 mM TRIS-HCl, 1 mM TCEP {Tris(2-carboxyethyl)-phosphine hydrochloride, Pierce}, 6M guanidium chloride, pH 8.5. Stir for ˜10 min. with a magnetic bar.
• 5. Centrifuge as described above, and collect the supernatant.
• 6. Prepare an adequate number of Poly-Prep (Bio-Rad) columns containing 1 ml of Fast Flow Chelating Sepharose (Pharmacia) saturated with Nichel according to manufacturer recommendations. Wash the columns twice with 5 ml of H₂0 and equilibrate with 50 mM TRIS-HCl, 1 mM TCEP, 6M guanidinium chloride, pH 8.5.
• 7. Load the supernatants from step 5 onto the columns, and wash with 5 ml of 50 mM TRIS-Hcl buffer, 1 mM TCEP, 6M urea, pH 8.5
• 8. Wash the columns with 10 ml of 20 mM imidazole, 50 mM TRIS-HCl, 6M urea, 1 mM TCEP, pH 8.5. Collect and set aside the first 5 ml for possible further controls.
• 9. Elute the proteins bound to the columns with 4.5 ml of a buffer containing 250 mM imidazole, 50 mM TRIS-HCl, 6M urea, 1 mM TCEP, pH 8.5. Add the elution buffer in three 1.5 ml aliquots, and collect the corresponding 3 fractions. Add to each fraction 15 μl DTT (final concentration 2 mM).
• 10. Measure eluted protein concentration with the Bradford method, and analyze aliquots of ca 10 μg of protein by SDS-PAGE.
• 11. Store proteins at −20° C. in 40% (v/v) glycerol, 50 mM TRIS-HCl, 2M urea, 0.5 M arginine, 2 mM DTT, 0.3 mM TCEP, 83.3 mM imidazole, pH 8.5 (K) Procedure for the Purification of GST-Fusion Proteins from E. coli
• 1. Transfer the bacterial pellets from −20° C. to an ice bath and resuspend with 7.5 ml PBS, pH 7.4 to which a mixture of protease inhibitors (COMPLETE™—Boehringer Mannheim, 1 tablet every 25 ml of buffer) has been added. Transfer to 40-50 ml centrifugation tubes and sonicate according to the following procedure:
  • a) Position the probe at about 0.5 cm from the bottom of the tube
  • b) Block the tube with the clamp
  • c) Dip the tube in an ice bath
  • d) Set the sonicator as follows: Timer→Hold, Duty Cycle→55, Out. Control→6.
  • e) perform 5 cycles of 10 impulses at a time lapse of 1 minute (i.e. one cycle=10 impulses+˜45″ hold; b. 10 impulses+˜45″ hold; c. 10 impulses+˜45″ hold; d. 10 impulses+˜45″ hold; e. 10 impulses+˜45″ hold)
• 2. Centrifuge at about 30-40000×g for 15-20 min. E.g.: use rotor Beckman JA 25.50 at 21000 rpm, for 15 min.
• 3. Store the centrifugation pellets at −20° C., and load the supernatants on the chromatography columns, as follows
• 4. Equilibrate the Poly-Prep (Bio-Rad) columns with 0.5 ml (≅1 ml suspension) of Glutathione-Sepharose 4B resin, wash with 2 ml (1+1) H₂O, and then with 10 ml (2+4+4) PBS, pH 7.4.
• 5. Load the supernatants on the columns and discard the flow through.
• 6. Wash the columns with 10 ml (2+4+4) PBS, pH 7.4.
• 7. Elute the proteins bound to the columns with 4.5 ml of 50 mM TRIS buffer, 10 mM reduced glutathione, pH 8.0, adding 1.5 ml+1.5 ml+1.5 ml and collecting the respective 3 fractions of ˜1.5 ml each.
• 8. Measure the protein concentration of the first two fractions with the Bradford method, analyse a 10 μg aliquot of proteins from each sample by SDS-PAGE. (N.B.: if the sample is too diluted load 21 μl (+7 μl loading buffer).
• 9. Store the collected fractions at +4° C. while waiting for the results of the SDS-PAGE analysis.
• 10. For each protein destined to the immunisation prepare 4-5 aliquots of 100 μg each in 0.5 ml of 40% glycerol. The dilution buffer is 50 mM TRIS.HCl, 2 mM DTT, pH 8.0. Store the aliquots at −20° C. until immunisation. Serology (L) Protocol of Immunization
• 1. Groups of four CD1 female mice aged between 6 and 7 weeks were immunized with 20 μg of recombinant protein resuspended in 100 μl.
• 2. Four mice for each group received 3 doses with a 14 days interval schedule.
• 3. Immunization was performed through intra-peritoneal injection of the protein with an equal volume of Complete Freund's Adjuvant (CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for the following two doses.
• 4. Sera were collected before each immunization. Mice were sacrified 14 days after the third immunization and the collected sera were pooled and stored at −20° C. (M) Western Blot Analysis of Cpn Elementary Body Proteins with Mouse Sera

Aliquots of elementary bodies containing approximately 4 μg of proteins, mixed with SDS loading buffer (1×: 60 mM TRIS-HCl pH 6.8, 5% w/v SDS, 10% v/v glycerin, 0.1% Bromophenol Blue, 100 mM DTT) and boiled 5 minutes at 95° C., were loaded on a 12% SDS-PAGE gel. The gel was run using a SDS-PAGE running buffer containing 250 mM TRIS, 2.5 mM Glycine and 0.1% SDS. The gel was electroblotted onto nitrocellulose membrane at 200 mA for 30 minutes. The membrane was blocked for 30 minutes with PBS, 3% skimmed milk powder and incubated O/N at 4° C. with the appropriate dilution (1/100) of the sera. After washing twice with PBS+0.1% Tween (Sigma) the membrane was incubated for 2 hours with peroxidase-conjugated secondary anti-mouse antibody (Sigma) diluted 1:3000. The nitrocellulose was washed twice for 10 minutes with PBS+0.1% Tween-20 and once with PBS and thereafter developed by Opti-4CN Substrate Kit (Biorad).

Lanes shown in Western blots are: (P)=pre-immune control serum; (I)=immune serum.

(N) FACS Analysis of Chlamydia pneumoniae Elementary Bodies with Mouse Sera

• 1. 2×10⁵ Elementary Bodies (EB)/well were washed with 200 μl of PBS-01% BSA in a 96 wells U bottom plate and centrifuged for 10 min. at 1200 rpm, at 4° C.
• 2. The supernatant was discarded and the E.B. resuspended in 10 μl of PBS-0.1% BSA.
• 3. 10 μl mouse sera diluted in PBS-01% BSA were added to the E.B. suspension to a final dilution of 1:400, and incubated on ice for 30 min.
• 4. EB were washed by adding 180 μl PBS-0.1% BSA and centrifuged for 10 min. at 1200 rpm, 4° C.
• 5. The supernatant was discarded and the E.B. resuspended in 10 l of PBS-0.1% BSA.
• 6. 10 μl of a goat anti-mouse IgG, F(ab′)₂ fragment specific-R-Phycoerythrin-conjugated (Jackson Immunoresearch Laboratories Inc., cat. No 115-116-072) was added to the EB suspension to a final dilution of 1:100, and incubated on ice for 30 min. in the dark.
• 7. EB were washed by adding 180 μl PBS-0.1% BSA and centrifuged for 10 min. at 1200 rpm, 4° C.
• 8. The supernatant was discarded and the E.B. resuspended in 150 μl of PBS-0.1% BSA.
• 9. E.B. suspension was passed through a cytometric chamber of a FACS Calibur (Becton Dikinson, Mountain View, Calif. USA) and 10.000 events were acquired.
• 10. Data were analysed using Cell Quest Software (Becton Dikinson, Mountain View, Calif. USA) by drawing a morphological dot plot (using forward and side scatter parameters) on E.B. signals. An histogram plot was then created on FL2 intensity of fluorescence log scale recalling the morphological region of EB.

NB: the results of FACS depend not only on the extent of accessibility of the native antigens but also on the quality of the antibodies elicited by the recombinant antigens, which may have structures with a variable degree of correct folding as compared with the native protein structures. Therefore, even if a FACS assay appears negative this does not necessarily mean that the protein is not abundant or accessible on the surface. PorB antigen, for instance, gave negative results in FACS but is a surface-exposed neutralising antigen [Kubo & Stephens (2000) Mol. Microbiol. 38:772-780].

(O) Mass Spectrometry Analysis of Two-Dimensional Electrophoretic Protein Maps

Gradient purified EBs from strain FB/96 were solubilized at a final concentration of 5.5 mg/ml with immobiline rehydratation buffer (7M urea, 2M thiourea, 2% (w/v) CHAPS, 2% (w/v) ASB 14 [Chevallet et al. (1998) Electrophor. 19:1901-9], 2% (v/v) C.A 3-10NL (Amersham Pharmacia Biotech), 2 mM tributyl phosphine, 65 mM DTT). Samples (250 μg protein) were adsorbed overnight on Immobiline DryStrips (7 cm, pH 3-10 non linear). Electrophocusing was performed in a IPGphor Isoelectric Focusing Unit (Amersham Pharmacia Biotech). Before PAGE separation, the focused strips were incubated in 4M urea, 2M thiourea, 30% (v/v) glycerol, 2% (w/v) SDS, 5 mM tributyl phosphine 2.5% (w/v) acrylamide, 50 mM Tris-HCl pH 8.8, as described [Herbert et al. (1998) Electrophor. 19:845-51]. SDS-PAGE was performed on linear 9-16% acrylamide gradients. Gels were stained with colloidal Coomassie (Novex, San Diego) [Doherty et al. (1998) Electrophor. 19:355-63]. Stained gels were scanned with a Personal Densitometer SI (Molecular Dynamics) at 8 bits and 50 μm per pixel. Map images were annotated with the software Image Master 2D Elite, version 3.10 (Amersham Pharmacia Biotech). Protein spots were excised from the gel, using an Ettan Spot picker (Amersham Pharmacia Biotech), and dried in a vacuum centrifuge. In-gel digestion of samples for mass spectrometry and extraction of peptides were performed as described by Wilm et al. [Nature (1996) 379:466-9]. Samples were desalted with a ZIP TIP (Millipore), eluted with a saturated solution of alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% TFA and directly loaded onto a SCOUT 381 multiprobe plate (Bruker). Spectra were acquired on a Bruker Biflex II MALDI-TOF. Spectra were calibrated using a combination of known standard peptides, located in spots adjacent to the samples. Resulting values for monoisotopic peaks were used for database searches using the computer program Mascot (www.matrixscience.com). All searches were performed using an error of 200-500 ppm as constraint. A representative gel is shown in FIG. 190.

Example 1

The following C. pneumoniae protein (PID 4376552) was expressed <SEQ ID 1; cp6552>:

1   MKKKLSLLVG LIFVLSSCHK EDAQNKIRIV ASPTPHAELL
    ESLQEEAKDL
51  GIKLKILPVD DYRIPNRLLL DKQVDANYFQ HQAFLDDECE
    RYDCKGELVV
101 IAKVHLEPQA IYSKKHSSLE RLKSQKKLTI AIPVDRTNAQ
    RALHLLEECG
151 LIVCKGPANL NMTAKDVCGK ENRSINILEV SAPLLVGSLP
    DVDAAVIPGN
201 FAIAANLSPK KDSLCLEDLS VSKYTNLVVI RSEDVGSPKM
    IKLQKLFQSP
251 SVQHFFDTKY HGNILTMTQD NG*

A predicted signal peptide is highlighted.

The cp6552 nucleotide sequence <SEQ ID 2> is:

1   ATGAAAAAAA AATTATCATT ACTTGTAGGT TTAATTTTTG
    TTTTGAGTTC
51  TTGCCATAAG GAAGATGCTC AGAATAAAAT ACGTATTGTA
    GCCAGTCCGA
101 CACCTCATGC GGAATTATTG GAGAGTTTAC AGGAAGAGGC
    TAAAGATCTT
151 GGAATCAAGC TGAAAATACT TCCAGTAGAT GATTATCGTA
    TTCCTAATCG
201 TTTGCTTTTG GATAAACAAG TAGATGCAAA TTACTTTCAA
    CATCAAGCTT
251 TTCTTGATGA CGAATGCGAG CGTTATGATT GTAAGGGTGA
    ATTAGTTGTT
301 ATCGCTAAAG TTCATTTGGA ACCTCAAGCA ATTTATTCTA
    AGAAACATTC
351 TTCTTTAGAG CGCTTAAAAA GCCAGAAGAA ACTGACTATA
    GCGATTCCTG
401 TGGATCGTAC GAATGCTCAG CGTGCTCTAC ACTTGTTAGA
    AGAGTGCGGA
451 CTCATTGTTT GCAAAGGGCC TGCTAATTTA AATATGACAG
    CTAAAGATGT
501 CTGTGGGAAA GAAAATAGAA GTATCAACAT ATTAGAGGTG
    TCAGCTCCTC
551 TTCTTGTCGG ATCTCTTCCT GACGTTGATG CTGCTGTCAT
    TCCTGGAAAT
601 TTTGCTATAG CAGCAAACCT TTCTCCAAAG AAAGATAGTC
    TTTGTTTAGA
651 GGATCTTTCG GTATCTAAGT ATACAAACCT TGTTGTCATT
    CGTTCTGAAG
701 ACGTAGGTTC TCCTAAAATG ATAAAATTAC AGAAGCTGTT
    TCAATCTCCT
751 TCTGTACAAC ATTTTTTTGA TACAAAATAT CATGGGAATA
    TTTTGACAAT
801 GACTCAAGAC AATGGTTAG

The PSORT algorithm predicts an inner membrane location (0.127).

The protein was expressed in E. coli and purified as a his-tag product, as shown in FIG. 1A, and also as a GST-fusion. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 1B) and for FACS analysis (FIG. 1C).

The cp6552 protein was also identified in the 2D-PAGE experiment (Cpn0278).

These experiments show that cp6552 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 2

The following C. pneumoniae protein (PID 4376736) was expressed <SEQ ID 3; cp6736>:

1   MKTSIRKFLI STTLAPCFAS TAFTVEVIMP SENFDGSSGK
    IFRYTTLSDP
51  RGTLCIFSGD LYIANLDNAI SRTSSSCFSN RAGALQILGK
    GGVFSFLNIR
101 SSADGAAISS VITQNPELCP LSFSGFSQMI FDNCESLTSD
    TSASNVIPHA
151 SAIYATTPML FTNNDSILFQ YNRSAGFGAA IRGTSITIEN
    TKKSLLFNGN
201 GSISNGGALT GSAAINLINN SAPVIFSTNA TGIYGGAIYL
    TGGSMLTSGN
251 LSGVLFVNNS SRSGGAIYAN GNVTFSNNSD LTFQNNTASP
    QNSLPAPTPP
301 PTPPAVTPLL GYGGAIFCTP PATPPPTGVS LTISGENSVT
    FLENIASEQG
351 GALYGKKISI DSNKSTIFLG NTAGKGGAIA IPESGELSLS
    ANQGDILFNK
401 NLSITSGTPT RNSIHFGKDA KFATLGATQG YTLYFYDPIT
    SDDLSAASAA
451 ATVVVNPKAS ADGAYSGTIV FSGETLTATE AATPANATST
    LNQKLELEGG
501 TLALRNGATL NVHNFTQDEK SVVIMDAGTT LATTNGANNT
    DGAITLNKLV
551 INLDSLDGTK AAVVNVQSTN GALTISGTLG LVKNSQDCCD
    NHGMFNKDLQ
601 QVPILELKAT SNTVTTTDFS LGTNGYQQSP YGYQGTWEFT
    IDTTTHTVTG
651 NWKKTGYLPH PERLAPLIPN SLWANVIDLR AVSQASAADG
    EDVPGKQLSI
701 TGITNFFHAN HTGDARSYRH MGGGYLINTY TRITPDAALS
    LGFGQLFTKS
751 KDYLVGHGHS NVYFATVYSN ITKSLFGSSR FFSGGTSRVT
    YSRSNEKVKT
801 SYTKLPKGRC SWSNNCWLGE LEGNLPITLS SRILNLKQII
    PFVKAEVAYA
851 THGGIQENTP EGRIFGHGHL LNVAVPVGVR FGKNSHNRPD
    FYTIIVAYAP
901 DVYRHNPDCD TTLPINGATW TSIGNNLTRS TLLVQASSHT
    SVNDVLEIFG
951 HCGCDIRRTS RQYTIDIGSK LRF*

A predicted signal peptide is highlighted.

The cp6736 nucleotide sequence <SEQ ID 4> is:

1    ATGAAAACGT CTATTCGTAA GTTCTTAATT TCTACCACAC
     TGGCGCCATG
51   TTTTGCTTCA ACAGCGTTTA CTGTAGAAGT TATCATGCCT
     TCCGAGAACT
101  TTGATGGATC GAGTGGGAAG ATTTTTCCTT ACACAACACT
     TTCTGATCCT
151  AGAGGGACAC TCTGTATTTT TTCAGGGGAT CTCTACATTG
     CGAATCTTGA
201  TAATGCCATA TCCAGAACCT CTTCCACTTG CTTTAGCAAT
     AGGGCGGGAG
251  CACTACAAAT CTTAGGAAAA GGTGGGGTTT TCTCCTTCTT
     AAATATCCGT
301  TCTTCAGCTG ACGGAGCCGC GATTAGTAGT GTAATCACCC
     AAAATCCTGA
351  ACTATGTCCC TTGAGTTTTT CAGGATTTAG TCAGATGATC
     TTCGATAACT
401  GTGAATCTTT GACTTCAGAT ACCTCAGCGA GTAATGTCAT
     ACCTCACGCA
451  TCGGCGATTT ACGCTACAAC GCCCATGCTC TTTACAAACA
     ATGACTCCAT
501  ACTATTCCAA TACAACCGTT CTGCAGGATT TGGAGCTGCC
     ATTCGAGGCA
551  CAAGCATCAC AATAGAAAAT ACGAAAAAGA GCCTTCTCTT
     TAATGGTAAT
601  GGATCCATCT CTAATGGAGG GGCCCTCACG GGATCTGCAG
     CGATCAACCT
651  CATCAACAAT AGCGCTCCTG TGATTTTCTC AACGAATGCT
     ACAGGGATCT
701  ATGGTGGGGC TATTTACCTT ACCGGAGGAT CTATGCTCAC
     CTCTGGGAAC
751  CTCTCAGGAG TCTTGTTCGT TAATAATAGC TCGCGCTCAG
     GAGGCGCTAT
801  CTATGCTAAC GGAAATGTCA CATTTTCTAA TAACAGCGAC
     CTGACTTTCC
851  AAAACAATAC AGCATCTCCA CAAAACTCCT TACCTGCACC
     TACACCTCCA
901  CCTACACCAC CAGCAGTCAC TCCTTTGTTA GGATATGGAG
     GCGCCATCTT
951  CTGTACTCCT CCAGCTACCC CCCCACCAAC AGGTGTTAGC
     CTGACTATAT
1001 CTGGAGAAAA CAGCGTTACA TTCCTAGAAA ACATTGCCTC
     CGAACAAGGA
1051 GGAGCCCTCT ATGGCAAAAA GATCTCTATA GATTCTAATA
     AATCTACAAT
1101 ATTTCTTGGA AGTACAGCTG GAAAAGGAGG CGCTATTGCT
     ATTCCCGAAT
1151 CTGGGGAGCT CTCTCTATCC GCAAATCAAG GTGATATCCT
     CTTTAACAAG
1201 AACCTCAGCA TCACTAGTGG GACACCTACT CGCAATAGTA
     TTCACTTCGG
1251 AAAAGATGCC AAGTTTGCCA CTCTAGGAGC TACGCAAGGC
     TATACCCTAT
1301 ACTTCTATGA TCCGATTACA TCTGATGATT TATCTGCTGC
     ATCCGCAGCC
1351 GCTACTGTGG TCGTCAATCC CAAAGCCAGT GCAGATGGTG
     CGTATTCAGG
1401 GACTATTGTC TTTTCAGGAG AAACCCTCAC TGCTACCGAA
     GCAGCAACCC
1451 CTGCAAATGC TACATCTACA TTAAACCAAA AGCTAGAACT
     TGAAGGCGGT
1501 ACTCTCGCTT TAAGAAACGG TGCTACCTTA AATGTTCATA
     ACTTCACGCA
1551 AGATGAAAAG TCCGTCGTCA TCATGGATGC AGGGACCACA
     TTAGCAACTA
1601 CAAATGGAGC TAATAATACT GACGGTGCTA TCACCTTAAA
     CAAGCTTGTA
1651 ATCAATCTGG ATTCTTTGGA TGGCACTAAA GCGGCTGTCG
     TTAATGTGCA
1701 GAGTACCAAT GGAGCTCTCA CTATATCCGG AACTTTAGGA
     CTTGTGAAAA
1751 ACTCTCAAGA TTGCTGTGAC AACCACGGGA TGTTTAATAA
     AGATTTACAG
1801 CAAGTTCCGA TTTTAGAACT CAAAGCGACT TCAAATACTG
     TAACCACTAC
1851 GGACTTCAGT CTCGGCACAA ACGGCTATCA GCAATCTCCC
     TATGGGTATC
1901 AAGGAACTTG GGAGTTTACC ATAGACACGA CAACCCATAC
     GGTCACAGGA
1951 AATTGGAAAA AAACCGGTTA TCTTCCTCAT CCGGAGCGTC
     TTGCTCCCCT
2001 CATTCCTAAT AGCCTACGGG CAAACGTCAT AGATTTACGA
     GCTGTAAGTC
2051 AAGCGTCAGC AGCTGATGGC GAAGATGTCC CTGGGAAGCA
     ACTGAGCATC
2101 ACAGGAATTA CAAATTTCTT CCATGCGAAT CATACCGGTG
     ATGCACGCAG
2151 CTACCGCCAT ATGGGTGGAG GCTACCTCAT CAATACCTAC
     ACACGCATCA
2201 CTCCAGATGC TGCGTTAAGT CTAGGTTTTG GACAGCTGTT
     TACAAAATCT
2251 AAGGATTACC TCGTAGGTCA CGGTCATTCT AACGTTTATT
     TCGCTACAGT
2301 ATACTCTAAC ATCACCAAGT CTCTGTTTGG ATCATCGAGA
     TTCTTCTCAG
2351 GAGGCACTTC TCGAGTTACC TATAGCCGTA GCAATGAGAA
     AGTAAAGACT
2401 TCATATACAA AATTGCCTAA AGGGCGCTGC TCTTGGAGTA
     ACAATTGCTG
2451 GTTAGGAGAA CTCGAAGGGA ACCTTCCCAT CACTCTCTCT
     TCTCGCATCT
2501 TAAACCTCAA GCAGATCATT CCCTTTGTAA AAGCTGAAGT
     TGCTTACGCG
2551 ACTCATGGGG GCATCCAAGA AAATACCCCC GAGGGGAGGA
     TTTTTGGACA
2601 CGGTCATCTA CTCAACGTTG CAGTTCCCGT AGGCGTCCGC
     TTTGGTAAAA
2651 ATTCTCATAA TCGACCAGAT TTTTACACTA TAATCGTAGC
     CTATGCTCCT
2701 GATGTCTATC GTCACAATCC TGATTGCGAT ACGACATTAC
     CTATTAATGG
2751 AGCTACGTGG ACCTCTATAG GGAATAATCT AACCAGAAGT
     ACTTTGCTAG
2801 TACAAGCATC CAGCCATACT TCAGTAAATG ATGTTCTAGA
     GATCTTCGGG
2851 CACTGTGGAT GTGATATTCG CAGAACCTCC CGTCAATATA
     CTCTAGATAT
2901 AGGAAGCAAA TTACGATTTT AA

The PSORT algorithm predicts an outer membrane location (0.917).

The protein was expressed in E. coli and purified as a his-tag product, as shown in FIG. 2A, and also as a GST-fusion. Both proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 2B) and for FACS analysis (FIG. 2C).

The cp6736 protein was also identified in the 2D-PAGE experiment (Cpn0453) and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6736 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 3

The following C. pneumoniae protein (PID 4376751) was expressed <SEQ ID 5; cp6751>:

1   MRFFCFGMLL PFTFVLANEG LQLPLETYIT LSPEYQAAPQ
    VGFTHNQNQD
51  LAIVGNHNDF ILDYKYYRSN GGALTCKNLL ISENIGNVFF
    EKNVCPNSGG
101 AIYAAQNCTI SKNQNYAFTT NLVSDNPTAT AGSLLGGALF
    AINCSITNNL
151 GQGTFVDNLA LNKGGALYTE TNLSIKDNKG PIIIKQNRAL
    NSDSLGGGIY
201 SGNSLNIEGN SGAIQITSNS SGSGGGIFST QTLTISSNKK
    LIEISENSAF
251 ANNYGSNFNP GGGGLTTTFC TILNNREGVL FNNNQSQSNG
    GAIHAKSIII
301 KENGPVYFLN NTATRGGALL NLSAGSGNGS FILSADNGDI
    IFNNNTASKH
351 ALNPPYRNAI HSTPNMNLQI GARPGYRVLF YDPIEHELPS
    SFPILFNFET
401 GHTGTVLFSG EHVHQNFTDE NMFFSYLRNT SELRQGVLAV
    EDGAGLACYK
451 FFQRGGTLLL GQGAVITTAG TIPTPSSTPT TVGSTITLNH
    IAIDLPSILS
501 FQAQAPKIWI YPTKTGSTYT EDSNPTITIS GTLTLRNSNN
    EDPYDSLDLS
551 HSLEKVPLLY IVDVAAQKIN SSQLDLSTLN SGEHYGYQGI
    WSTYWVETTT
601 ITNPTSLLGA NTKHKLLYAN WSPLGYRPHP ERRGEFITNA
    LWQSAYTALA
651 GLHSLSSWDE EKGHAASLQG IGLLVHQKDK NGFKGFRSHM
    TGYSATTEAT
701 SSQSPNFSLG FAQFFSKAKE HESQNSTSSH HYFSGMCIEN
    TLFKEWIRLS
751 VSLAYMFTSE HTHTMYQGLL EGNSQGSFHN HTLAGALSCV
    FLPQPHGESL
801 QIYPFITALA IRGNLAAFQE SGDHAREFSL HRPLTDVSLP
    VGIRASWKNH
851 HRVPLVWLTE ISYRSTLYRQ DPELHSKLLI SQGTWTTQAT
    PVTYNALGIK
901 VKNTMQVFPK VTLSLDYSAD ISSSTLSHYL NVASRMRF*

A predicted signal peptide is highlighted.

The cp6751 nucleotide sequence <SEQ ID 6> is:

1    ATGCGCTTTT TTTGCTTCGG AATGTTGCTT CCTTTTACTT
     TTGTATTGGC
51   TAATGAAGGT CTCCAACTTC CTTTGGAGAC CTATATTACA
     TTAAGTCCTG
101  AATATCAAGC AGCCCCTCAA GTAGGGTTTA CTCATAACCA
     AAATCAAGAT
151  CTCGCAATTG TCGGGAATCA CAATGATTTC ATCTTGGACT
     ATAAGTACTA
201  TCGGTCGAAT GGAGGTGCTC TTACCTGTAA GAATCTTCTG
     ATCTCTGAAA
251  ATATAGGGAA TGTCTTCTTT GAGAAGAATG TCTGTCCCAA
     TTCTGGCGGG
301  GCAATTTATG CTGCTCAAAA TTGCACGATC TCCAAGAATC
     AGAACTATGC
351  ATTTACTACA AACTTGGTCT CTGACAATCC TACAGCCACT
     GCGGGATCAC
401  TATTGGGTGG AGCTCTCTTT GCCATAAATT GCTCTATTAC
     TAATAACCTA
451  GGACAGGGAA CTTTCGTTGA CAATCTCGCT TTAAATAAGG
     GGGGTGCCCT
501  CTATACTGAG ACGAACTTAT CTATTAAAGA CAATAAAGGC
     CCGATCATAA
551  TCAAGCAGAA TCGGGCACTA AATTCGGACA GTTTAGGAGG
     AGGGATTTAT
601  AGTGGGAACT CTCTAAATAT AGAGGGAAAT TCTGGAGCTA
     TACAGATCAC
651  AAGCAACTCT TCAGGATCTG GGGGAGGCAT ATTTTCTACC
     CAAACACTCA
701  CGATCTCCTC GAATAAAAAA CTCATAGAAA TCAGTGAAAA
     TTCCGCGTTC
751  GCAAATAACT ATGGATCGAA CTTCAATCCA GGAGGAGGAG
     GTCTTACTAC
801  CACCTTTTGC ACGATATTGA ACAACCGAGA AGGGGTACTC
     TTTAACAATA
851  ACCAAAGCCA GAGCAACGGT GGAGCCATTC ATGCGAAATC
     TATCATTATC
901  AAAGAAAATG GTCCTGTATA CTTTTTAAAT AACACTGCAA
     CTCGGGGAGG
951  GGCTCTCCTC AACTTACCAG CAGGTTCTGG AAACGGAAGC
     TTCATCTTAT
1001 CTGCAGATAA TGGAGATATT ATCTTTAACA ATAATACGGC
     CTCCAAGCAT
1051 GCCCTCAATC CTCCATACAG AAACGCCATT CACTCGACTC
     CTAATATGAA
1101 TCTGCAAATA GGAGCCCGTC CCGGCTATCG AGTGCTGTTC
     TATGATCCCA
1151 TAGAACATGA GCTCCCTTCC TCCTTCCCCA TACTCTTTAA
     TTTCGAAACC
1201 GGTCATACAG GTACAGTTTT ATTTTCAGGG GAACATGTAC
     ACCAGAACTT
1251 TACCGATGAA ATGAATTTCT TTTCCTATTT AAGGAACACT
     TCGGAACTAC
1301 GTCAAGGAGT CCTTGCTGTT GAAGATGGTG CGGGGCTGGC
     CTGCTATAAG
1351 TTCTTCCAAC GAGGAGGCAC TCTACTTCTA GGTCAAGGTG
     CGGTGATCAC
1401 GACAGCAGGA ACGATTCCCA CACCATCCTC AACACCAACG
     ACAGTAGGAA
1451 GTACTATAAC TTTAAATCAC ATTGCCATTG ACCTTCCTTC
     TATTCTTTCT
1501 TTTCAAGCTC AGGCTCCAAA AATTTGGATT TACCCCACAA
     AAACAGGATC
1551 TACCTATACT GAAGATTCCA ACCCGACAAT CACAATCTCA
     GGAACTCTCA
1601 CCTTACGCAA CAGCAACAAC GAAGATCCCT ACGATAGTCT
     GGATCTCTCG
1651 CACTCTCTTG AGAAAGTTCC CCTTCTTTAT ATTGTCGATG
     TCGCTGCACA
1701 AAAAATTAAC TCTTCGCAAC TGGATCTATC CACATTAAAT
     TCTGGCGAAC
1751 ACTATGGGTA TCAAGGCATC TGGTCGACCT ATTGGGTAGA
     AACTACAACA
1801 ATCACGAACC CTACATCTCT ACTAGGCGCG AATACAAAAC
     ACAAGCTGCT
1851 CTATGCAAAC TGGTCTCCTC TAGGCTACCG TCCTCATCCC
     GAACGTCGAG
1901 GAGAATTCAT TACGAATGCC TTGTGGCAAT CGGCATATAC
     GGCTCTTGCA
1951 GGACTCCACT CCCTCTCCTC CTGGGATGAA GAGAAGGGTC
     ATGCAGCTTC
2001 CCTACAAGGC ATTGGTCTTC TGGTTCATCA AAAAGACAAA
     AACGGTTTTA
2051 AGGGATTTCG TAGTCATATG ACAGGTTATA GTGCTACCAC
     CGAAGCAACC
2101 TCTTCTCAAA GTCCGAATTT CTCTTTAGGA TTTGCTCAGT
     TCTTCTCCAA
2151 AGCTAAAGAA CATGAATCTC AAAATAGCAC GTCCTCTCAC
     CACTATTTCT
2201 CTGGAATGTG CATAGAAAAT ACTCTCTTCA AAGAGTGGAT
     ACGTCTATCT
2251 GTGTCTCTTG CTTATATGTT TACCTCGGAA CTACCCCATA
     CAATGTATCA
2301 GGGTCTCCTG GAAGGGAACT CTCAGGGATC TTTCCACAAC
     CATACCTTAG
2351 CAGGGGCTCT CTCCTGTGTT TTCTTACCTC AACCTCACGG
     CGAGTCCCTG
2401 CAGATCTATC CCTTTATTAC TGCCTTAGCC ATCCGAGGAA
     ATCTTGCTGC
2451 GTTTCAAGAA TCTGGAGACC ATGCTCGGGA ATTTTCCCTA
     CACCGCCCCC
2501 TAACGGACGT CTCCCTCCCT GTAGGAATCC GCGCTTCTTG
     GAAGAACCAC
2551 CACCGAGTTC CCCTAGTCTG GCTCACAGAA ATTTCCTATC
     GCTCTACTCT
2601 CTATAGGCAA GATCCTGAAC TCCACTCGAA ATTACTGATT
     AGCCAAGGTA
2651 CGTGGACGAC GCAGGCCACT CCTGTGACCT ACAATGCTTT
     AGGGATCAAA
2701 GTGAAAAATA CCATGCAGGT GTTTCCTAAA GTCACTCTCT
     CCTTAGATTA
2751 CTCTGCGGAT ATTTCTTCCT CCACGCTGAG TCACTACTTA
     AACGTGGCGA
2801 GTAGAATGAG ATTTTAA

The PSORT algorithm predicts an outer membrane location (0.923).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 3A, and also in his-tagged form. The GST-fusion recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 3B) and for FACS analysis (FIG. 3C).

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6751 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 4

The following C. pneumoniae protein (PID 4376752) was expressed <SEQ ID 7; cp6752>:

1   MFGMTPAVYS LQTDSLEKFA LERDEEFRTS FPLLDSLSTL
    TGFSPITTFV
51  GNRHNSSQDI VLSNYKSIDN ILLLWTSAGG AVSCNNFLLS
    NVEDHAFFSK
101 NLAIGTGGAI ACQGACTITK NRGPLIFFSN RGLNNASTGG
    ETRGGAIACN
151 GDFTISQNQG TFYFVNNSVN NWGGALSTNG HCRIQSNRAP
    LLFFNNTAPS
201 GGGALRSENT TISDNTRPIY FKNNCGNNGG AIQTSVTVAI
    KNNSGSVIFN
251 NNTALSGSIN SGNGSGGAIY TTNLSIDDNP GTILFNNKYC
    IRDGGAICTQ
301 FLTIKNSGHV YFTNNQGNWG GALMLLQDST CLLFAEQGNI
    AFQNNEVFLT
351 TFGRYNAIHC TPNSNLQLGA NKGYTTAFFD PIEHQHPTTN
    PLIFNPNANH
401 QGTILFSSAY IPEASDYENN FISSSKNTSE LRNGVLSIED
    RAGWQFYKFT
451 QKGGILKIGH AASIATTANS ETPSTSVGSQ VIINNLAINL
    PSILAKGKAP
501 TLWIRPLQSS APFTEDNNPT ITLSGPLTLL NEENRDPYDS
    IDLSEPLQNI
551 HLLSLSDVTA RHINTDNFHP ESLNATEHYG YQGIWSPYWV
    ETITTTNNAS
601 IETANTLYRA IYANWTPLGY KVNPEYQGDL ATTPLWQSFH
    TMFSLLRSYN
651 RTGDSDIERP FLEIQGIADG LFVHQNSIPG APGFRIQSTG
    YSLQASSETS
701 HLQKISLGFA QFFTRTKEIG SSNNVSAHNT VSSLYVELPW
    FQEAFATSTV
751 LAYGYGDHHL HSLHPSHQEQ AEGTCYSHTL AAAIGCSFPW
    QQKSYLHLSP
801 FVQAIAIRSH QTAFEEIGDN PRKFVSQKPF YNLTLPLGIQ
    GKWQSKFHVP
851 TEWTLELSYQ PVLYQQNPQI GVTLLASGGS WDILGHNYVR
    NALGYKVHNQ
901 TALFRSLDLF IDYQGSVSSS TSTHHLQAGS TLKF*

The cp6752 nucleotide sequence <SEQ ID 8> is:

1    ATGTTCGGGA TGACTCCTGC AGTGTATAGT TTACAAACGG
     ACTCCCTTGA
51   AAAGTTTGCT TTAGAGAGGG ATGAAGAGTT TCGTACGAGC
     TTTCCTCTCT
101  TAGACTCTCT CTCCACTCTT ACAGGATTTT CTCCAATAAC
     TACGTTTGTT
151  GGAAATAGAC ATAATTCCTC TCAAGACATT GTACTTTCTA
     ACTACAAGTC
201  TATTGATAAC ATCCTTCTTC TTTGGACATC GGCTGGGGGA
     GCTGTGTCCT
251  GTAATAATTT CTTATTATCA AATGTTGAAG ACCATGCCTT
     CTTCAGTAAA
301  AATCTCGCGA TTGGGACTGG AGGCGCGATT GCTTGCCAGG
     GAGCCTGCAC
351  AATCACGAAG AATAGAGGAC CCCTTATTTT TTTCAGCAAT
     CGAGGTCTTA
401  ACAATGCGAG TACAGGAGGA GAAACTCGTG GGGGTGCGAT
     TGCCTGTAAT
451  GGAGACTTCA CGATTTCTCA AAATCAAGGG ACTTTCTACT
     TTGTCAACAA
501  TTCCGTCAAC AACTGGGGAG GAGCCCTCTC CACCAATGGA
     CACTGCCGCA
551  TCCAAAGCAA CAGGGCACCT CTACTCTTTT TTAACAATAC
     AGCCCCTAGT
601  GGAGGGGGTG CGCTTCGTAG TGAAAATACA ACGATCTCTG
     ATAACACGCG
651  TCCTATTTAT TTTAAGAACA ACTGTGGGAA CAATGGCGGG
     GCCATTCAAA
701  CAAGCGTTAC TGTTGCGATA AAAAATAACT CCGGGTCGGT
     GATTTTCAAT
751  AACAACACAG CGTTATCTGG TTCGATAAAT TCAGGAAATG
     GTTCAGGAGG
801  GGCGATTTAT ACAACAAACC TATCCATAGA CGATAACCCT
     GGAACTATTC
851  TTTTCAATAA TAACTACTGC ATTCGCGATG GCGGAGCTAT
     CTGTACACAA
901  TTTTTGACAA TCAAAAATAG TGGCCACGTA TATTTCACCA
     ACAATCAAGG
951  AAACTGGGGA GGTGCTCTTA TGCTCCTACA GGACAGCACC
     TGCCTACTCT
1001 TCGCGGAACA AGGAAATATC GCATTTCAAA ATAATGAGGT
     TTTCCTCACC
1051 ACATTTGGTA GATACAACGC CATACATTGT ACACCAAATA
     GCAACTTACA
1101 ACTTGGAGCT AATAAGGGGT ATACGACTGC TTTTTTTGAT
     CCTATAGAAC
1151 ACCAACATCC AACTACAAAT CCTCTAATCT TTAATCCCAA
     TGCGAACCAT
1201 CAGGGAAGCA TCTTATTTTC TTCAGCCTAT ATCCCAGAAG
     CTTCTGACTA
1251 CGAAAATAAT TTCATTAGCA GCTCGAAAAA TACCTCTGAA
     CTTCGCAATG
1301 GTGTCCTCTC TATCGAGGAT CGTGCGGGAT GGCAATTCTA
     TAAGTTCACT
1351 CAAAAAGGAG GTATCCCTAA ATTAGGGCAT GCGGCGAGTA
     TTGCAACAAC
1401 TGCCAACTCT GAGACTCCAT CAACTAGTGT AGGCTCCCAG
     GTCATCATTA
1451 ATAACCTTGC GATTAACCTC CCCTCGATCT TAGCAAAAGG
     AAAAGCTCCT
1501 ACCTTGTGGA TCCGTCCTCT ACAATCTAGT GCTCCTTTCA
     CAGAGGACAA
1551 TAACCCTACA ATTACTTTAT CAGGTCCTCT GACACTCTTA
     AATGAGGAAA
1601 ACCGCGATCC CTACGACAGT ATAGATCTCT CTGAGCCTTT
     ACAAAACATT
1651 CATCTTCTTT CTTTATCGGA TGTAACAGCA CGTCATATCA
     ATACCGATAA
1701 CTTTCATCCT GAAAGCTTAA ATGCGACTGA GCATTACGGT
     TATCAAGGCA
1751 TCTGGTCTCC TTATTGGGTA GAGACGATAA CAACAACAAA
     TAACGCTTCT
1801 ATAGAGACGG CAAACACCCT CTACAGAGCT CTGTATGCCA
     ATTGGACTCC
1851 CTTAGGATAT AAGGTCAATC CTGAATACCA AGGAGATCTT
     GCTACGACTC
1901 CCCTATGGCA ATCCTTTCAT ACTATGTTCT CTCTATTAAG
     AAGTTATAAT
1951 CGAACTGGTG ATTCTGATAT CGAGAGGCCT TTCTTAGAAA
     TTCAAGGGAT
2001 TGCCGACGGC CTCTTTGTTC ATCAAAATAG CATCCCCGGG
     GCTCCAGGAT
2051 TCCGTATCCA ATCTACAGGG TATTCCTTAC AAGCATCCTC
     CGAAACTTCT
2101 TTACATCAGA AAATCTCCTT AGGTTTTGCA CAGTTCTTCA
     CCCGCACTAA
2151 AGAAATCGGA TCAAGCAACA ACGTCTCGGC TCACAATACA
     GTCTCTTCAC
2201 TTTATGTTGA GCTTCCGTGG TTCCAAGAGG CCTTTGCAAC
     ATCCACAGTG
2251 TTAGCGTATG GCTATGGGGA CCATCACCTC CACAGCCTAC
     ATCCCTCACA
2301 TCAAGAACAG GCAGAAGGGA CGTGTTATAG CCATACATTA
     GCAGCAGCTA
2351 TCGGCTGTTC TTTCCCTTGG CAACAGAAAT CCTATCTTCA
     CCTCAGCCCG
2401 TTCGTTCAGG CAATTGCAAT ACGTTCTCAC CAAACAGCGT
     TCGAAGAGAT
2451 TGGTGACAAT CCCCGAAAGT TTGTCTCTCA AAAGCCTTTC
     TATAATCTGA
2501 CCTTACCTCT AGGAATCCAA GGAAAATGGC AGTCAAAATT
     CCACGTACCT
2551 ACAGAATGGA CTCTAGAACT TTCTTACCAA CCGGTACTCT
     ATCAACAAAA
2601 TCCCCAAATC GGTCTCACGC TACTTGCGAG CGGAGGTTCC
     TGGGATATCC
2651 TAGGCCATAA CTATGTTCGC AATGCTTTAG GGTACAAAGT
     CCACAATCAA
2701 ACTGCGCTCT TCCGTTCTCT CGATCTATTC TTGGATTACC
     AAGGATCGGT
2751 CTCCTCCTCG ACATCTACGC ACCATCTCCA AGCAGGAAGT
     ACCTTAAAAT
2801 TCTAA

The PSORT algorithm predicts a cytoplasmic location (0.138).

The protein was expressed in E. coli and purified as a his-tag product, as shown in FIG. 4A, and also as a GST-fusion. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (4B) and the his-tagged protein was used for FACS analysis (4C).

The cp6752 protein was also identified in the 2D-PAGE experiment (Cpn0467).

These experiments show that cp6752 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 5

The following C. pneumoniae protein (PID 4376850) was expressed <SEQ ID 9; cp6850>:

1  MKKAVLIAAM FCGVVSLSSC CRIVDCCFED PCAPSSCNPC
   EVIRKKERSC
51 GGNACGSYVP SCSNPCGSTE CNSQSPQVKG CTSPDGRCKQ *

A predicted signal peptide is highlighted.

The cp6850 nucleotide sequence <SEQ ID 10> is:

1   ATGAAGAAAG CTGTTTTAAT TGCTGCAATG TTTTGTGGAG
    TAGTTAGCTT
51  AAGTAGCTGC TGCCGCATTG TAGATTGTTG TTTTGAGGAT
    CCTTGCGCAC
101 CCTCTTCTTG CAATCCTTGT GAAGTAATAA GAAAAAAAGA
    AAGATCTTGC
151 GGCGGTAATG CTTGTGGGTC CTACGTTCCT TCTTGTTCTA
    ATCCATGTGG
201 TTCAACAGAG TGTAACTCTC AAAGCCCACA AGTTAAAGGT
    TGTACATCAC
251 CTGATGGCAG ATGCAAACAG TAA

The PSORT algorithm predicts an inner membrane location (0.329).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 5A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 5B) and for FACS analysis (FIG. 5B). A his-tagged protein was also expressed.

These experiments show that cp6850 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 6

The following C. pneumoniae protein (PID 4376900) was expressed <SEQ ID 11; cp6900>:

1   MKIKFSWKVN FLICLLAVGL IFFGCSRVKR EVLVGRDATW
    FPKQFGIYTS
51  DTNAFLNDLV SEINYKENLN INIVNQDWVH LFENLDDKKT
    QGAFTSVLPT
101 LEMLEHYQFS DPILLTGPVL VVAQDSPYQS IEDLKGRLIG
    VYKFDSSVLV
151 AQNIPDAVIS LYQHVPIALE ALTSNCYDAL LAPVIEVTAL
    IETAYKGRLK
201 IISKPLNADG LRLAILKGTN GDLLEGFNAG LVKTRRSGKY
    DAIKQRYRLP

The cp6900 nucleotide sequence <SEQ ID 12> is:

1   GTGAAGATAA AATTTTCTTG GAAGGTAAAT TTTTTAATAT
    GTTTACTGGC
51  TGTGGGACTG ATCTTTTTCG GGTGCTCTCG AGTAAAAAGA
    GAAGTTCTCG
101 TAGGTCGTGA TGCCACCTGG TTTCCAAAAC AATTCGGCAT
    TTATACATCC
151 GATACCAACG CATTTTTAAA CGATCTTGTT TCTGAGATTA
    ACTATAAAGA
201 GAATCTAAAT ATTAATATTG TAAATCAAGA TTGGGTGCAT
    CTCTTTGAGA
251 ATTTAGATGA TAAAAAGACC CAAGGAGCAT TTACATCTGT
    ATTGCCTACT
301 CTTGAGATGC TCGAACACTA TCAATTTTCT GATCCCATTT
    TACTCACAGG
351 TCCTGTCCTT GTCGTCGCTC AAGACTCTCC TTACCAATCT
    ATAGAGGATC
401 TTAAAGGTCG TCTTATTGGA GTGTATAAGT TTGACTCTTC
    AGTTCTTGTA
451 GCTCAAAATA TCCCTGACGC TGTGATTAGC CTCTACCAAC
    ATGTTCCAAT
501 AGCATTGGAA GCCTTAACAT CGAATTGTTA CGACGCTCTT
    CTAGCTCCTG
551 TAATTGAAGT GACCGCGCTA ATAGAAACAG CATATAAAGG
    AAGACTGAAA
601 ATTATTTCAA AACCCTTAAA CGCAGATGGT TTGCGGCTTG
    CAATACTGAA
651 AGGGACAAAC GGAGATTTGC TTGAAGGGTT TAACGCAGGA
    CTTGTGAAAA
701 CACGACGCTC AGGAAAATAC GATGCTATAA AACAGCGGTA
    TCGTCTTCCC
751 TAA

The PSORT algorithm predicts an inner membrane location (0.452).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 6A. The recombinant protein was used to immunise mice, whose sera were used for FACS analysis (FIG. 6B). A his-tagged protein was also expressed.

The cp6900 protein was also identified in the 2D-PAGE experiment (Cpn0604).

These experiments show that cp6900 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 7

The following C. pneumoniae protein (PID 4377033) was expressed <SEQ ID 13; cp7033>:

1   MVNPIGPGPI DETERTPPAD LSAQGLEASA ANKSAEAQRI
    AGAEAKPKES
51  KTDSVERWSI LRSAVNALMS LADKLGIASS NSSSSTSRSA
    DVDSTTATAP
101 TPPPPTFDDY KTQAQTAYDT IFTSTSLADI QAALVSLQDA
    VTNIKDTAAT
151 DEETAIAAEW ETKNADAVKV GAQITELAKY ASDNQAILDS
    LGKLTSFDLL
201 QAALLQSVAN NNKAAELLKE MQDNPVVPGK TPAIAQSLVD
    QTDATATQIE
251 KDGNAIRDAY FAGQNASGAV ENAKSNNSIS NIDSAKAAIA
    TAKTQIAEAQ
301 KKFPDSPILQ EAEQMVIQAE KDLKNIKPAD GSDVPNPGTT
    VGGSKQQGSS
351 IGSIRVSMLL DDAENETASI LMSGFRQMIH MFNTENPDSQ
    AAQQELAAQA
401 RAAKAAGDDS AAAALADAQK ALEAALGKAG QQQGILNALG
    QIASAAVVSA
451 GVPPAAASSI GSSVKQLYKT SKSTGSDYKT QISAGYDAYK
    SINDAYGRAR
501 NDATRDVINN VSTPALTRSV PRARTEARGP EKTDQALARV
    ISGNSRTLGD
551 VYSQVSALQS VMQIIQSNPQ ANNEEIRQKL TSAVTKPPQF
    GYPYVQLSND
601 STQKFIAKLE SLFAEGSRTA AEIKALSFET NSLFIQQVLV
    NIGSLYSGYL
651 Q*

The cp7033 nucleotide sequence <SEQ ID 14> is:

1    ATGGTTAATC CTATTGGTCC AGGTCCTATA GACGAAACAG
     AACGCACACC
51   TCCCGCAGAT CTTTCTGCTC AAGGATTGGA GGCGAGTGCA
     GCAAATAAGA
101  GTGCGGAAGC TCAAAGAATA GCAGGTGCGG AAGCTAAGCC
     TAAAGAATCT
151  AAGACCGATT CTGTAGAGCG ATGGAGCATC TTGCGTTCTG
     CAGTGAATGC
201  TCTCATGAGT CTGGCAGATA AGCTGGGTAT TGCTTCTAGT
     AACAGCTCGT
251  CTTCTACTAG CAGATCTGCA GACGTGGACT CAACGACAGC
     GACCGCACCT
301  ACGCCTCCTC CACCCACGTT TGATGATTAT AAGACTCAAG
     CGCAAACAGC
351  TTACGATACT ATCTTTACCT CAACATCACT AGCTGACATA
     CAGGCTGCTT
401  TGGTGAGCCT CCAGGATGCT GTCACTAATA TAAAGGATAC
     AGCGGCTACT
451  GATGAGGAAA CCGCAATCGC TGCGGAGTGG GAAACTAAGA
     ATGCCGATGC
501  AGTTAAAGTT GGCGCGCAAA TTACAGAATT AGCGAAATAT
     GCTTCGGATA
551  ACCAAGCGAT TCTTGACTCT TTAGGTAAAC TGACTTCCTT
     CGACCTCTTA
601  CAGGCTGCTC TTCTCCAATC TGTAGCAAAC AATAACAAAG
     CAGCTGAGCT
651  TCTTAAAGAG ATGCAAGATA ACCCAGTAGT CCCAGGGAAA
     ACGCCTGCAA
701  TTGCTCAATC TTTAGTTGAT CAGACAGATG CTACAGCGAC
     ACAGATAGAG
751  AAAGATGGAA ATGCGATTAG GGATGCATAT TTTGCAGGAC
     AGAACGCTAG
801  TGGAGCTGTA GAAAATGCTA AATCTAATAA CAGTATAAGC
     AACATAGATT
851  CAGCTAAAGC AGCAATCGCT ACTGCTAAGA CACAAATAGC
     TGAAGCTCAG
901  AAAAAGTTCC CCGACTCTCC AATTCTTCAA GAAGCGGAAC
     AAATGGTAAT
951  ACAGGCTGAG AAAGATCTTA AAAATATCAA ACCTGCAGAT
     GGTTCTGATG
1001 TTCCAAATCC AGGAACTACA GTTGGAGGCT CCAAGCAACA
     AGGAAGTAGT
1051 ATTGGTAGTA TTCGTGTTTC CATGCTGTTA GATGATGCTG
     AAAATGAGAC
1101 CGCTTCCATT TTGATGTCTG GGTTTCGTCA GATGATTCAC
     ATGTTCAATA
1151 CGGAAAATCC TGATTCTCAA GCTGCCCAAC AGGAGCTCGC
     AGCACAAGCT
1201 AGAGCAGCGA AAGCCGCTGG AGATGACAGT GCTGCTGCAG
     CGCTGGCAGA
1251 TGCTCAGAAA GCTTTAGAAG CGGCTCTAGG TAAAGCTGGG
     CAACAACAGG
1301 GCATACTCAA TGCTTTAGGA CAGATCGCTT CTGCTGCTGT
     TGTGAGCGCA
1351 GGAGTTCCTC CCGCTGCAGC AAGTTCTATA GGGTCATCTG
     TAAAACAGCT
1401 TTACAAGACC TCAAAATCTA CAGGTTCTGA TTATAAAACA
     CAGATATCAG
1451 CAGGTTATGA TGCTTACAAA TCCATCAATG ATGCCTATGG
     TAGGGCACGA
1501 AATGATGCGA CTCGTGATGT GATAAACAAT GTAAGTACCC
     CCGCTCTCAC
1551 ACGATCCGTT CCTAGAGCAC GAACAGAAGC TCGAGGACCA
     GAAAAAACAG
1601 ATCAAGCCCT CGCTAGGGTG ATTTCTGGCA ATAGCAGAAC
     TCTTGGAGAT
1651 GTCTATAGTC AAGTTTCGGC ACTACAATCT GTAATGCAGA
     TCATCCAGTC
1701 GAATCCTCAA GCGAATAATG AGGAGATCAG ACAAAAGCTT
     ACATCGGCAG
1751 TGACAAAGCC TCCACAGTTT GGCTATCCTT ATGTGCAACT
     TTCTAATGAC
1801 TCTACACAGA AGTTCATAGC TAAATTAGAA AGTTTGTTTG
     CTGAAGGATC
1851 TAGGACAGCA GCTGAAATAA AAGCACTTTC CTTTGAAACG
     AACTCCTTGT
1901 TTATTCAGCA GGTGCTGGTC AATATCGGCT CTCTATATTC
     TGGTTATCTC
1951 CAATAA

The PSORT algorithm predicts a cytoplasmic location (0.272).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 7A. A his-tagged protein was also expressed. The recombinant proteins were used to immunise mice, whose sera were used for FACS (FIG. 7B) and Western blot (7C) analyses.

The cp7033 protein was also identified in the 2D-PAGE experiment (Cpn0728) and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp7033 a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 8

The following C. pneumoniae protein (PID 6172321) was expressed <SEQ ID 15; cp0017>:

1   MGIKGTGIIV WVDDATAKTK NATLTWTKTG YKPNPERQGP
    LVPNSLWGSF
51  VDVRSIQSLM DRSTSSLSSS TNLWVSGIAD FLHEDQKGNQ
    RSYRHSSAGY
101 ALGGGFFTAS ENFFNFAFCQ LFGYDKDHLV AKNHTHVYAG
    AMSYRHLGES
151 KTLAKILSGN SDSLPFVFNA RFAYGHTDNN MTTKYTGYSP
    VKGSWGNDAF
201 GIECGGAIPV VASGRRSWVD THTPFLNLEM IYAHQNDFKE
    NGTEGRSFQS
251 EDLFNLAVPV GIKFEKFSDK STYDLSIAYV PDVIRNDPGC
    TTTLMVSGDS
301 WSTCGTSLSR QALLVRAGNH HAFASNFEVF SQFEVELRGS
    SRSYAIDLGG
351 RFGF*

The cp0017 nucleotide sequence <SEQ ID 16> is:

1    ATGGGTATCA AGGGAACTGG AATAATTGTT TGGGTCGACG
     ATGCAACTGC
51   AAAAACAAAA AATGCTACCT TAACTTGGAC TAAAACAGGA
     TACAAGCCGA
101  ATCCAGAACG TCAGGGACCT TTGGTTCCTA ATAGCCTGTG
     GGGTTCTTTT
151  GTCGATGTCC GCTCCATTCA GAGCCTCATG GACCGGAGCA
     CAAGTTCGTT
201  ATCTTCGTCA ACAAATTTGT GGGTATCAGG AATCGCGGAC
     TTTTTGCATG
251  AAGATCAGAA AGGAAACCAA CGTAGTTATC GTCATTCTAG
     CGCGGGTTAT
301  GCATTAGGAG GAGGATTCTT CACGGCTTCT GAAAATTTCT
     TTAATTTTGC
351  TTTTTGTCAG CTTTTTGGCT ACGACAAGGA CCATCTTGTG
     GCTAAGAACC
401  ATACCCATGT ATATGCAGGG GCAATGAGTT ACCGACACCT
     CGGAGAGTCT
451  AAGACCCTCG CTAAGATTTT GTCAGGAAAT TCTGACTCCC
     TACCTTTTGT
501  CTTCAATGCT CGGTTTGCTT ATGGCCATAC CGACAATAAC
     ATGACCACAA
551  AGTACACTGG CTATTCTCCT GTTAAGGGAA GCTGGGGAAA
     TGATGCCTTC
601  GGTATAGAAT GTGGAGGAGC TATCCCGGTA GTTGCTTCAG
     GACGTCGGTC
651  TTGGGTGGAT ACCCACACGC CATTTCTAAA CCTAGAGATG
     ATCTATGCAC
701  ATCAGAATGA CTTTAAGGAA AACGGCACAG AAGGCCGTTC
     TTTCCAAAGT
751  GAAGACCTCT TCAATCTAGC GGTTCCTGTA GGGATAAAAT
     TTGAGAAATT
801  CTCCGATAAG TCTACGTATG ATCTCTCCAT AGCTTACGTT
     CCCGATGTGA
851  TTCGTAATGA TCCAGGCTGC ACGACAACTC TTATGGTTTC
     TGGGGATTCT
901  TGGTCGACAT GTGGTACAAG CTTGTCTAGA CAAGCTCTTC
     TTGTACGTGC
951  TGGAAATCAT CATGCCTTTG CTTCAAACTT TGAAGTTTTC
     AGTCAGTTTG
1001 AAGTCGAGTT GCGAGGTTCT TCTCGTAGCT ATGCTATCGA
     TCTTGGAGGA
1051 AGATTCGGAT TTTAA

This sequence is frame-shifted with respect to cp0016.

The PSORT algorithm predicts a cytoplasmic location (0.075).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 8A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 8B) and for FACS analysis (FIG. 8C). A his-tagged protein was also expressed.

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp0017 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 9

The following C. pneumoniae protein (PID 6172315) was expressed <SEQ ID 17; cp0014>:

1   MKSSFPKFVF STFAIFPLSM IATETVLDSS ASFDGNKNGN
    FSVRESQEDA
51  GTTYLFKGNV TLENIPGTGT AITKSCFNNT KGDLTFTGNG
    NSLLFQTVDA
101 GTVAGAAVNS SVVDKSTTFI GFSSLSFIAS PGSSITTGKG
    AVSCSTGSLS
151 LTKMSVCSSA KTFQRIMAVL SPQKLFH*

The cp0014 nucleotide sequence <SEQ ID 18> is:

1   ATGAAGTCTT CTTTCCCCAA GTTTGTATTT TCTACATTTG
    CTATTTTCCC
51  TTTGTCTATG ATTGCTACCG AGACAGTTTT GGATTCAAGT
    GCGAGTTTCG
101 ATGGGAATAA AAATGGTAAT TTTTCAGTTC GTGAGAGTCA
    GGAAGATGCT
151 GGAACTACCT ACCTATTTAA GGGAAATGTC ACTCTAGAAA
    ATATTCCTGG
201 AACAGGCACA GCAATCACAA AAAGCTGTTT TAACAACACT
    AAGGGCGATT
251 TGACTTTCAC AGGTAACGGG AACTCTCTAT TGTTCCAAAC
    GGTGGATGCA
301 GGGACTGTAG CAGGGGCTGC TGTTAACAGC AGCGTGGTAG
    ATAAATCTAC
351 CACGTTTATA GGGTTTTCTT CGCTATCTTT TATTGCGTCT
    CCTGGAAGTT
401 CGATAACTAC CGGCAAAGGA GCCGTTAGCT GCTCTACGGG
    TAGCTTGAGT
451 TTGACAAAAA TCTCAGTTTG CTCTTCAGCA AAAACTTTTC
    AACGGATAAT
501 GGCGGTGCTA TCACCGCAAA AACTCTTTCA TTAA

This protein is frame-shifted with respect to cp0015.

The PSORT algorithm predicts an inner membrane location (0.047).

The protein was expressed in E. coli and purified as a his-tag product, as shown in FIG. 9A. A GST-fusion was also expressed. The recombinant proteins were used to immunise mice, whose sera were used in an immunoassay (FIG. 9B) and for FACS analysis (FIG. 9C).

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments suggest that cp0014 is a useful immunogen. These properties are not evident from the sequence alone.

Example 10

The following C. pneumoniae protein (PID 6172317) was expressed <SEQ ID 19; cp0015>:

1   MSALFSENTS SKKGGAIQTS DALTITGNQG EVSFSDNTSS
    DSGAAIFTEA
51  SVTISNNAKV SFIDNKVTGA SSSTTGDMSG GAICAYKTST
    DTKVTLTGNQ
101 MLLFSNNTST TAGGAIYVKK LELASGGLTL FSRNSVNGGT
    APKGGAIAIE
151 DSGELSLSAD SGDIVFLGNT VTSTTPGTNR SSIDLGTSAK
    MTALRSAAGR
201 AIYFYDPITT GSSTTVTDVL KVNETPADSA LQYTGNIIFT
    GEKLSETEAA
251 DSKNLTSKLL QPVTLSGGTL SLKHGVTLQT QAFTQQADSR
    LEMDVGTTLE
301 PADTSTINNL VINISSIDGA KKAKIETKAT SKNLTLSGTI
    TLLDPTGTFY
351 ENHSLRNPQS YDILELKASG TVTSTAVTPD PIMGEKFEYG
    YQGTWGPIVW
401 GTGASTTATF NWTKTGYIPN PERIGSLVPN SLWNAFIDIS
    SLHYLMETAN
451 EGLQGDRAFW CAGLSNFFHK DSTKTRRGFR HLSGGYVIGG
    NLHTCSDKIL
501 SAAFCQLFGR DRDYFVAKNQ GTVYGGTLYY QHNETYISLP
    CKLRPCSLSY
551 VPTEIPVLFS GNLSYTHTDN DLKTKYTTYP TVKGSWGNDS
    FALEFGGRAP
601 ICLDESALFE QYMPFMKLQF VYAHQEGFKE QGTEAREFGS
    SRLVNLALPI
651 GIRFDKESDC QDATYNLTLG YTVDLVRSNP DCTTTLRISG
    DSWKTFGTNL
701 ARQALVLRAG VHFCFNSNFE AFSQFSFELR GSSRNYNVDL
    GAKYQF*

This sequence is frame-shifted with respect to cp0014.

The cp0015 nucleotide sequence <SEQ ID 20> is:

1    ATGTCAGCTC TGTTTTCTGA AAATACCTCC TCAAAGAAAG
     GCGGAGCCAT
51   TCAGACTTCC GATGCCCTTA CCATTACTGG AAACCAAGGG
     GAAGTCTCTT
101  TTTCTGACAA TACTTCTTCG GATTCTGGAG CTGCAATTTT
     TACAGAAGCC
151  TCGGTGACTA TTTCTAATAA TGCTAAAGTT TCCTTTATTG
     ACAATAAGGT
201  CACAGGAGCG AGCTCCTCAA CAACGGGGGA TATGTCAGGA
     GGTGCTATCT
251  GTGCTTATAA AACTAGTACA GATACTAAGG TCACCCTCAC
     TGGAAATCAG
301  ATGTTACTCT TCAGCAACAA TACATCGACA ACAGCGGGAG
     GAGCTATCTA
351  TGTGAAAAAG CTCGAACTGG CTTCCGGAGG ACTTACCCTA
     TTCAGTAGAA
401  ATAGTGTCAA TGGAGGTACA GCTCCTAAAG GTGGAGCCAT
     AGCTATCGAA
451  GATAGTGGGG AATTGAGTTT ATCCGCCGAT AGTGGTGACA
     TTGTCTTTTT
501  AGGGAATACA GTCACTTCTA CTACTCCTGG GACGAATAGA
     AGTAGTATCG
551  ACTTAGGAAC GAGTGCAAAG ATGACAGCTT TGCGTTCTGC
     TGCTGGTAGA
601  GCCATCTACT TCTATGATCC CATAACTACA GGATCATCCA
     CAACAGTTAC
651  AGATGTCTTA AAAGTTAATG AGACTCCGGC AGATTCTGCA
     CTACAATATA
701  CAGGGAACAT CATCTTCACA GGAGAAAAGT TATCAGAGAC
     AGAGGCCGCA
751  GATTCTAAAA ATCTTACTTC GAAGCTACTA CAGCCTGTAA
     CTCTTTCAGG
801  AGGTACTCTA TCTTTAAAAC ATGGAGTGAC TCTGCAGACT
     CAGGCATTCA
851  CTCAACAGGC AGATTCTCGT CTCGAAATGG ACGTAGGAAC
     TACTCTAGAA
901  CCTGCTGATA CTAGCACCAT AAACAATTTG GTCATTAACA
     TCAGTTCTAT
951  AGACGGTGCA AAGAAGGCAA AAATAGAAAC CAAAGCTACG
     TCAAAAAATC
1001 TGACTTTATC TGGAACCATC ACTTTATTGG ACCCGACGGG
     CACGTTTTAT
1051 GAAAATCATA GTTTAAGAAA TCCTCAGTCC TACGACATCT
     TAGAGCTCAA
1101 AGCTTCTGGA ACTGTAACAA GCACCGCAGT GACTCCAGAT
     CCTATAATGG
1151 GTGAGAAATT CCATTACGGC TATCAGGGAA CTTGGGGCCC
     AATTGTTTGG
1201 GGGACAGGGG CTTCTACGAC TGCAACCTTC AACTGGACTA
     AAACTGGCTA
1251 TATTCCTAAT CCCGAGCGTA TCGGCTCTTT AGTCCCTAAT
     AGCTTATGGA
1301 ATGCATTTAT AGATATTAGC TCTCTCCATT ATCTTATGGA
     GACTGCAAAC
1351 GAAGGGTTGC AGGGAGACCG TGCTTTTTGG TGTGCTGGAT
     TATCTAACTT
1401 CTTCCATAAG GATAGTACAA AAACACGACG CGGGTTTCGC
     CATTTGAGTG
1451 GCGGTTATGT CATAGGAGGA AACCTACATA CTTGTTCAGA
     TAAGATTCTT
1501 AGTGCTGCAT TTTGTCAGCT CTTTGGAAGA GATAGAGACT
     ACTTTGTAGC
1551 TAAGAATCAA GGTACAGTCT ACGGAGGAAC TCTCTATTAC
     CAGCACAACG
1601 AAACCTATAT CTCTCTTCCT TGCAAACTAC GGCCTTGTTC
     GTTGTCTTAT
1651 GTTCCTACAG AGATTCCTGT TCTCTTTTCA GGAAACCTTA
     GCTACACCCA
1701 TACGGATAAC GATCTGAAAA CCAAGTATAC AACATATCCT
     ACTGTTAAAG
1751 GAAGCTGGGG GAATGATAGT TTCGCTTTAG AATTCGGTGG
     AAGAGCTCCG
1801 ATTTGCTTAG ATGAAAGTGC TCTATTTGAG CAGTACATGC
     CCTTCATGAA
1851 ATTGCAGTTT GTCTATGCAC ATCAGGAAGG TTTTAAAGAA
     CAGGGAACAG
1901 AAGCTCGTGA ATCTGGAAGT AGCCGTCTTG TGAATCTTGC
     CTTACCTATC
1951 GGGATCCGAT TTGATAAGGA ATCAGACTGC CAAGATGCAA
     CGTACAATCT
2001 AACTCTTGGT TATACTGTGG ATCTTGTTCG TAGTAACCCC
     GACTGTACGA
2051 CAACACTGCG AATTAGCGGT GATTCTTGGA AAACCTTCGG
     TACGAATTTG
2101 GCAAGACAAG CTTTAGTCCT TCGTGCAGGG AACCATTTTT
     GCTTTAACTC
2151 AAATTTTGAA GCCTTTAGCC AATTTTCTTT TGAATTGCGT
     GGGTCATCTC
2201 GCAATTACAA TCTAGACTTA GGAGCAAAAT ACCAATTCTA A

The PSORT algorithm predicts a cytoplasmic location (0.274).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 10A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 10B) and for FACS analysis. A his-tagged protein was also expressed.

These experiments show that cp0015 is a useful immunogen. These properties are not evident from the sequence alone.

Example 11

The following C. pneumoniae protein (PID 6172325) was expressed <SEQ ID 21; cp0019>:

1   LQDSQDYSFV KLSPGAGGTI ITQDASQKPL EVAPSRFHYG
    YQGHWNVQVI
51  PGTGTQPSQA NLEWVRTGYL PNPERQGSLV PNSLWGSFVD
    QRAIQEIMVN
101 SSQILCQERG VWGAGIANFL HRDKINEHGY RHSGVGYLVG
    VGTHAFSDAT
151 INAAFCQLFS RDKDYVVSKN HGTSYSGVVF LEDTLEFRSP
    QGFYTDSSSE
201 ACCNQVVTID MQLSYSHRNN DMKTKYTTYP EAQGSWANDV
    FGLEFGATTY
251 YYPNSTFLFD YYSPFLRLQC TYAHQEDFKE TGGEVRHFTS
    GDLFNLAVPI
301 GVKFERFSDC KRGSYELTLA YVPDVIRKDP KSTATLASGA
    TWSTHGNNLS
351 RQGLQLRLGN HCLINPGIEV FSHGAIELRG SSRNYNINLG
    GKYRF*

This sequence is frame-shifted with respect to cp0018.

The cp0019 nucleotide sequence <SEQ ID 22> is:

1    TTGCAAGACT CTCAAGACTA TAGCTTTGTA AAGTTATCTC
     CAGGAGCGGG
51   AGGGACTATA ATTACTCAAG ATGCTTCTCA GAAGCCTCTT
     GAAGTAGCTC
101  CTTCTAGACC ACATTATGGC TATCAAGGAC ATTGGAATGT
     GCAAGTCATC
151  CCAGGAACGG GAACTCAACC GAGCCAGGCA AATTTAGAAT
     GGGTGCGGAC
201  AGGATACCTT CCGAATCCCG AACGGCAAGG ATCTTTAGTT
     CCCAATAGCC
251  TGTGGGGTTC TTTTGTTGAT CAGCGTGCTA TCCAAGAAAT
     CATGGTAAAT
301  AGTAGCCAAA TCTTATGTCA GGAACGGGGA GTCTGGGGAG
     CTGGAATTGC
351  TAATTTCCTA CATAGAGATA AAATTAATGA GCACGGCTAT
     CGCCATAGCG
401  GTGTCGGTTA TCTTGTGGGA GTTGGCACTC ATGCTTTTTC
     TGATGCTACG
451  ATAAATGCGG CTTTTTGCCA GCTCTTCAGT AGAGATAAAG
     ACTACGTAGT
501  ATCCAAAAAT CATGGAACTA GCTACTCAGG GGTCGTATTT
     CTTGAGGATA
551  CCCTAGAGTT TAGAAGTCCA CAGGGATTCT ATACTGATAG
     CTCCTCAGAA
601  GCTTGCTGTA ACCAAGTCGT CACTATAGAT ATGCAGTTGT
     CTTACAGCCA
651  TAGAAATAAT GATATGAAAA CCAAATACAC GACATATCCA
     GAAGCTCAGG
701  GATCTTGGGC AAATGATGTT TTTGGTCTTG AGTTTGGAGC
     GACTACATAC
751  TACTACCCTA ACAGTACTTT TTTATTTGAT TACTACTCTC
     CGTTTCTCAG
801  GCTGCAGTGC ACCTATGCTC ACCAGGAAGA CTTCAAAGAG
     ACAGGAGGTG
851  AGGTTCGTCA CTTTACTAGC GGAGATCTTT TCAATTTAGC
     AGTTCCTATT
901  GGCGTGAAGT TTGAGAGATT TTCAGACTGT AAAAGGGGAT
     CTTATGAACT
951  TACCCTTGCT TATGTTCCTG ATGTGATTCG CAAAGATCCC
     AAGAGCACGG
1001 CAACATTGGC TAGTGGAGCT ACGTGGAGCA CCCACGGAAA
     CAATCTCTCC
1051 AGACAAGGAT TACAACTGCG TTTAGGGAAC CACTGTCTCA
     TAAATCCTGG
1101 AATTGAGGTG TTCAGTCACG GAGCTATTGA ATTGCGGGGA
     TCCTCTCGTA
1151 ATTATAACAT CAATCTCGGG GGTAAATACC GATTTTAA

The PSORT algorithm predicts a cytoplasmic location (0.189).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 11A. This protein was used to immunise mice, whose sera were used in a Western blot (FIG. 11B) and an immunoblot assay (FIG. 11C). A his-tagged protein was also expressed.

These experiments show that cp0019 is a useful immunogen. These properties are not evident from the sequence alone.

Example 12

The following C. pneumoniae protein (PID 4376466) was expressed <SEQ ID 23; cp6466>:

1   MRKISVGICI TILLSLSVVL QGCKESSHSS TSRGELAINI
    RDEPRSLDPR
51  QVRLLSEISL VKHIYEGLVQ ENNLSGNIEP ALAEDYSLSS
    DGLTYTFKLK
101 SAFWSNGDPL TAEDFIESWK QVATQEVSGI YAFALNPIKN
    VRKIQEGHLS
151 IDHFGVHSPN ESTLVVTLES PTSHFLKLLA LPVFFPVEKS
    QRTLQSKSLP
201 IASGAFYPKN IKQKQWIKLS KNPHYYNQSQ VETKTITIHF
    IPDANTAAKL
251 FNQGKLNWQG PPWGERIPQE TLSNLQSKGH LHSFDVAGTS
    WLTFNINKFP
301 LNNMKLREAL ASALDKEALV STIFLGRAKT ADHLLPTNIH
    SYPEHQKQEM
351 AQRQAYAKKL FKAELEELQI TAKDLEHLNL IFPVSSSASS
    LLVQLIREQW
401 KESLGFAIPI VGKEFALLQA DLSSGNFSLA TGGWFADFAD
    PMAFLTIFAY
451 PSGVPPYAIN HKDFIEILQN IEQEQDHQKR SELVSQASLY
    LETFHIIEPI
501 YHDAFQFAMN KKLSNLGVSP TGVVDFRYAK EN*

A predicted signal peptide is highlighted.

The cp6466 nucleotide sequence <SEQ ID 24> is:

1    ATGCGCAAGA TATCAGTGGG AATCTGTATC ACCATTCTCC
     TTAGCCTCTC
51   CGTAGTCCTC CAAGGCTGCA AGGAGTCCAG TCACTCCTCT
     ACATCTCGGG
101  GAGAACTCGC TATTAATATA AGAGATGAAC CCCGTTCTTT
     AGATCCAAGA
151  CAAGTGCGAC TTCTTTCAGA AATCAGCCTT GTCAAACATA
     TCTATGAGGG
201  ATTAGTTCAA GAAAATAATC TTTCAGGAAA TATAGAGCCT
     GCTCTTGCAG
251  AAGACTACTC TCCTTCCTCG GACGGACTCA CTTATACTTT
     TAAACTGAAA
301  TCAGCTTTTT GGAGTAATGG CGACCCCTTA ACAGCTGAAG
     ACTTTATAGA
351  ATCTTGGAAA CAAGTAGCTA CTCAAGAAGT CTCAGGAATC
     TATGCTTTTG
401  CCTTGAATCC AATTAAAAAT GTACGAAAGA TCCAAGAGGG
     ACACCTCTCC
451  ATAGACCATT TTGGAGTGCA CTCTCCTAAT GAATCTACAC
     TTGTTGTTAC
501  CCTGGAATCC CCAACCTCGC ATTTCTTAAA ACTTTTAGCT
     CTTCCAGTCT
551  TTTTCCCCGT TCATAAATCT CAAAGAACCC TGCAATCCAA
     ATCTCTACCT
601  ATAGCAAGCG GAGCTTTCTA TCCTAAAAAT ATCAAACAAA
     AACAATGGAT
651  AAAACTCTCA AAAAACCCTC ACTACTATAA TCAAAGTCAG
     GTGGAAACTA
701  AAACGATTAC GATTCACTTC ATTCCCGATG CAAACACAGC
     AGCAAAACTA
751  TTTAATCAGG GAAAACTCAA TTGGCAAGGA CCTCCTTGGG
     GAGAACGCAT
801  TCCTCAAGAA ACCCTATCCA ATTTACAGTC TAAGGGGCAC
     TTACACTCTT
851  TTGATGTCGC AGGAACCTCA TGGCTCACCT TCAATATCAA
     TAAATTCCCC
901  CTCAACAATA TGAAGCTTAG AGAAGCCTTA GCATCAGCCT
     TAGATAAGGA
951  AGCTCTTGTC TCAACTATAT TCTTAGGCCG TGCAAAAACT
     GCCGATCATC
1001 TCCTACCTAC AAATATTCAT AGCTATCCCG AACATCAAAA
     ACAAGAGATG
1051 GCACAACGCC AAGCTTACGC TAAAAAACTC TTTAAAGAAG
     CTTTAGAAGA
1101 ACTCCAAATC ACTGCTAAAG ATCTCGAACA TCTTAATCTT
     ATCTTTCCCG
1151 TTTCCTCGTC AGCAAGTTCT TTACTAGTCC AACTTATACG
     AGAACAGTGG
1201 AAAGAAAGTT TAGGGTTCGC TATCCCTATT GTCGGAAAGG
     AATTTGCTCT
1251 TCTCCAAGCA GACCTATCTT CAGGGAACTT CTCTTTAGCT
     ACAGGAGGAT
1301 GGTTCGCAGA CTTTGCTGAT CCTATGGCAT TTCTAACGAT
     CTTTGCTTAT
1351 CCATCAGGAG TTCCTCCTTA TGCAATCAAC CATAAGGACT
     TCCTAGAAAT
1401 TCTACAAAAC ATAGAACAAG AGCAAGATCA CCAAAAACGC
     TCGGAATTAG
1451 TGTCGCAAGC TTCTCTTTAC CTAGAGACCT TTCATATTAT
     TGAGCCGATC
1501 TACCACGACG CATTTCAATT TGCTATGAAT AAAAAACTTT
     CTAATCTAGG
1551 AGTCTCACCA ACAGGAGTTG TGGACTTCCG TTATGCTAAG
     GAAAATTAG

The PSORT algorithm predicts that the protein is an outer membrane lipoprotein (0.790).

The protein was expressed in E. coli and purified both as a GST-fusion product and a His-tag fusion product. Purification of the protein as a GST-fusion product is shown in FIG. 12A. The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 12B and 12C). FACS analysis was also performed.

These experiments show that cp6466 is a useful immunogen. These properties are not evident from the sequence alone.

Example 13

The following C. pneumoniae protein (PID 4376468) was expressed <SEQ ID 25; cp6468>:

1   MFSRWITLFL LFISLTGCSS YSSKHKQSLI IPIHDDPVAF
    SPEQAKRAMD
51  LSIAQLLFDG ITRETHRESN DLELAIASRY TVSEDFCSYT
    FFIKDSALWS
101 DGTPITSEDI RNAWEYAQEN SPHIQIFQGL NFSTPSSNAI
    TIHLDSPNPD
151 FPKLLAFPAF AIFKPENPKL FSGPYTLVEY FPGENIHLKK
    NPNYYDYHCV
201 SINSIKLLII PDIYTAIHLL NRGKVDWVGQ PWHQGIPWEL
    HKQSQYHYYT
251 YPVEGAFWLC INTKSPHLND LQNRHRLATC IDKRSIIEEA
    LQGTQQPAET
301 LSRGAPQPNQ YKKQKPLTPQ EKLVLTYPSD ILRCQRIAEI
    LKEQWKAAGI
351 DLILEGLEYH LFVNKRKVQD YAIATQTGVA YYPGANLISE
    EDKLLQNFEI
401 IPIYYLSYDY LTQDFIEGVI YNASGAVDLK YTYFP*

A predicted signal peptide is highlighted.

The cp6468 nucleotide sequence <SEQ ID 26> is:

1    ATGTTTTCAC GATGGATCAC CCTCTTTTTA TTATTCATTA
     GCCTTACTGG
51   ATGCTCCTCC TACTCTTCAA AACATAAACA ATCTTTAATT
     ATTCCCATAC
101  ATGACGACCC TCTAGCTTTT TCTCCTGAAC AAGCAAAACG
     GGCCATGGAC
151  CTTTCTATTG CCCAACTTCT TTTTGATGGT CTGACTAGAG
     AAACTCATCG
201  CGAATCCAAT GATTTGGAAT TAGCGATTGC CAGTCGCTAT
     ACAGTCTCTG
251  AAGACTTTTG CTCTTATACG TTCTTTATCA AAGACAGCGC
     TTTATGGAGC
301  GACGGAACAC CAATCACCTC CGAAGATATC CGTAACGCTT
     GGGAGTATGC
351  ACAGGAGAAC TCTCCCCACA TACAGATCTT CCAAGGACTT
     AACTTCTCAA
401  CTCCTTCATC AAATGCAATT ACGATTCATC TCGACTCGCC
     CAACCCCGAT
451  TTTCCTAAGC TTCTTGCCTT TCCTGCATTT GCTATCTTTA
     AACCAGAAAA
501  CCCGAAGCTC TTTAGCGGTC CGTATACTCT TGTAGAGTAT
     TTCCCAGGGC
551  ATAACATTCA TTTAAAGAAA AACCCTAACT ATTACGACTA
     CCACTGCGTC
601  TCCATCAACT CCATCAAACT GCTCATTATT CCTGATATAT
     ATACAGCCAT
651  CCACCTCCTA AACAGAGGCA AGGTGGACTG GGTAGGACAA
     CCCTGGCATC
701  AAGGGATTCC TTGGGAGCTC CATAAACAAT CGCAATATCA
     CTACTACACC
751  TATCCTGTAG AAGGTGCCTT CTGGCTTTGT CTAAATACAA
     AATCCCCACA
801  CTTAAATGAT CTTCAAAACA GACATAGACT CGCTACTTGT
     ATTGATAAAC
851  GTTCTATCAT TGAAGAAGCT CTTCAAGGAA CCCAACAACC
     AGCGGAAACA
901  CTGTCCCGAG GAGCTCCACA ACCAAATCAA TATAAAAAAC
     AAAAGCCTCT
951  AACTCCACAA GAAAAACTCG TGCTTACCTA TCCCTCAGAT
     ATTCTAAGAT
1001 GCCAACGCAT AGCAGAAATC TTAAAGGAAC AATGGAAAGC
     TGCTGGAATA
1051 GATTTAATCC TTGAAGGACT CGAATACCAT CTGTTTGTTA
     ACAAACGAAA
1101 AGTCCAAGAC TACGCCATAG CAACACAGAC TGGAGTTGCT
     TATTACCCAG
1151 GAGCAAATCT AATTTCTGAA GAAGACAAGC TCCTGCAAAA
     CTTTGAGATT
1201 ATCCCGATCT ACTATCTGAG CTATGACTAT CTCACTCAAG
     ATTTTATAGA
1251 GGGAGTAATC TATAATGCTT CTGGAGCTGT AGATCTCAAA
     TATACCTATT
1301 TCCCCTAG

The PSORT algorithm predicts that this protein is an outer membrane lipoprotein (0.790).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 13A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 13B) and for FACS analysis. A his-tagged protein was also expressed.

These experiments show that cp6468 is a useful immunogen. These properties are not evident from the sequence alone.

Example 14

The following C. pneumoniae protein (PID 4376469) was expressed <SEQ ID 27; cp6469>:

1   MKMHRLKPTL KSLIPNLLFL LLTLSSCSKQ KQEPLGKELV
    IAMSHDLADL
51  DPRNAYLSRD ASLAKALYEG LTRETDQGIA LALAESYTLS
    KDHKVYTFKL
101 RPSVWSDGTP LTAYDFEKSI KQLYFEEFSP SIHTLLGVIK
    NSSAIHNAQK
151 SLETLGIQAK DDLTLVITLE QPFPYFLTLI ARPVFSPVHH
    TLRESYKKGT
201 PPSTYISNGP FVLKKHEHQN YLILEKNPHY YDHESVKLDR
    VTLKIIPDAS
251 TATKLFKSKS IDWIGSPWSA PISNEDQKVL SQEKILTYSV
    SSTTLLIYNL
301 QKPLIQNKAL RKAIAHAIDR KSILRLVPSG QEAVTLVPPN
    LSQLNLQKEI
351 STEFRQTKAR AYFQEAKETL SEKELAELSI LYPIDSSNSS
    IIAQEIQRQL
401 KDTLGLKIKI QGMEYHCFLK KRRQGDFFIA TGGWIAEYVS
    PVAFLSILGN
451 PRDLTQWRNS DYEKTLEKLY LPHAYKENLK RAEMIIEEET
    PIIPLYHGKY
501 IYAIHPKIQN TFGSLLGHTD LKNIDILS*

A predicted signal peptide is highlighted.

The cp6469 nucleotide sequence <SEQ ID 28> is:

1    ATGAAGATGC ATAGGCTTAA ACCTACCTTA AAAAGTCTGA
     TCCCTAATCT
51   TCTTTTCTTA TTGCTCACTC TTTCAAGCTG CTCAAAGCAA
     AAACAAGAAC
101  CCTTAGGAAA ACATCTCGTT ATTGCGATGA GCCATGATCT
     CGCCGACCTA
151  GATCCTCGCA ATGCCTATTT AAGCAGAGAT GCTTCCCTAG
     CAAAAGCCCT
201  CTATGAAGGA CTGACAAGAG AAACTGATCA AGGAATCGCA
     CTGGCTCTTG
251  CAGAAAGTTA TACCCTGTCA AAAGATCATA AGGTCTATAC
     CTTTAAACTC
301  AGACCTTCTG TGTGGAGCGA TGGCACTCCA CTCACTGCTT
     ATGACTTTGA
351  AAAATCTATA AAACAACTGT ACTTCGAAGA ATTTTCACCT
     TCCATACATA
401  CTTTACTCGG CGTGATTAAA AATTCTTCGG CAATCCACAA
     TGCTCAAAAA
451  TCTCTGGAAA CTCTTGGGAT ACAGGCAAAA GATGATCTTA
     CTTTGGTGAT
501  TACCCTAGAG CAACCTTTCC CATACTTTCT CACACTTATC
     GCTCGCCCCG
551  TATTCTCCCC TGTTCATCAC ACCCTTAGGG AATCCTATAA
     GAAAGGAACA
601  CCCCCATCCA CATACATCTC CAATGGGCCC TTTGTCTTAA
     AAAAACATGA
651  ACACCAAAAC TACTTAATTT TAGAAAAAAA TCCTCACTAC
     TATGATCATG
701  AATCAGTAAA GTTAGACCGA GTCACCTTAA AAATTATCCC
     AGACGCCTCC
751  ACAGCCACGA AACTTTTCAA AAGTAAATCT ATAGATTGGA
     TTGGCTCACC
801  TTGGAGCGCT CCGATATCTA ACGAAGACCA AAAAGTTCTC
     TCCCAAGAAA
851  AGATTCTTAC CTATTCTGTT TCAAGCACCA CCCTTCTTAT
     CTATAACCTG
901  CAAAAACCTC TAATACAAAA TAAAGCCCTC AGGAAAGCCA
     TTGCTCATGC
951  TATTGATAGA AAATCTATCT TAAGACTCGT GCCTTCAGGA
     CAAGAAGCTG
1001 TAACTCTAGT TCCCCCAAAT CTTTCACAAC TCAATCTTCA
     AAAAGAGATC
1051 TCAACAGAAG AACGACAAAC AAAAGCCAGA GCATATTTTC
     AAGAAGCTAA
1101 AGAAACACTT TCTGAAAAAG AACTCGCAGA ACTCAGCATC
     CTCTATCCTA
1151 TAGATTCCTC GAATTCCTCC ATCATAGCTC AAGAAATCCA
     AAGACAACTT
1201 AAAGATACCT TAGGATTGAA AATCAAAATC CAAGGCATGG
     AGTACCACTG
1251 CTTTTTAAAG AAACGTCGTC AAGGAGATTT CTTCATAGCG
     ACAGGAGGAT
1301 GGATTGCGGA ATACGTAAGC CCCGTAGCCT TCCTATCTAT
     TCTAGGCAAC
1351 CCCAGAGACC TCACACAATG GAGAAACAGT GATTACGAAA
     AGACTTTAGA
1401 GAAACTCTAT CTCCCTCATG CCTACAAAGA GAATTTAAAA
     CGCGCAGAAA
1451 TGATAATAGA AGAAGAAACC CCGATTATCC CCCTGTATCA
     CGGCAAATAT
1501 ATTTACGCTA TACATCCTAA AATCCAGAAT ACATTCGGAT
     CTCTTCTAGG
1551 CCACACAGAT CTCAAAAATA TCGATATCTT AAGTTAG

The PSORT algorithm predicts a periplasmic location (0.934).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 14A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 14B) and for FACS analysis. A his-tagged protein was also expressed.

These experiments show that cp6469 is a useful immunogen. These properties are not evident from the sequence alone.

Example 15

The following C. pneumoniae protein (PID 4376602) was expressed <SEQ ID 29; cp6602>:

1   MAASGGTGGL GGTQGVNLAA VEAAAAKADA AEVVASQEGS
    EMNMIQQSQD
51  LTNPAAATRT KKKEEKFQTL ESRKKGEAGK AEKKSESTEE
    KPDTDLADKY
101 ASGNSEISGQ ELRGLRDAIG DDASPEDILA LVQEKIKDPA
    LQSTALDYLV
151 QTTPPSQGKL KEALIQARNT HTEQFGRTAI GAKNILFASQ
    FYADQLNVSP
201 SGLRSLYLEV TGDTHTCDQL LSMLQDRYTY QDMAIVSSFL
    MKGMATELKR
251 QGPYVPSAQL QVLMTETRNL QAVLTSYDYF ESRVPILLDS
    LKAEGIQTPS
301 DLNFVKVAES YHKIINDKFP TASKVEREVR NLIGDDVDSV
    TGVLNLFFSA
351 LRQTSSRLFS SADKRQQLGA MIANALDAVN INNEDYPKAS
    DFPKPYPWS*

The cp6602 nucleotide sequence <SEQ ID 30> is:

1    ATGGCAGCAT CAGGAGGCAC AGGTGGTTTA GGAGGCACTC
     AGGGTGTCAA
51   CCTTGCAGCT GTAGAAGCTG CAGCTGCAAA AGCAGATGCA
     GCAGAAGTTG
101  TAGCCAGCCA AGAAGGTTCT GAGATGAACA TGATTCAACA
     ATCTCAGGAC
151  CTGACAAATC CCGCAGCAGC AACACGCACG AAAAAAAAGG
     AAGAGAAGTT
201  TCAAACTCTA GAATCTCGGA AAAAAGGAGA AGCTGGAAAG
     GCTGAGAAAA
251  AATCTGAATC TACAGAAGAG AAGCCTGACA CAGATCTTGC
     TGATAAGTAT
301  GCTTCTGGGA ATTCTGAAAT CTCTGGTCAA GAACTTCGCG
     GCCTGCGTGA
351  TGCAATAGGA GACGATGCTT CTCCAGAAGA CATTCTTGCT
     CTTGTACAAG
401  AGAAAATTAA AGACCCAGCT CTGCAATCCA CAGCTTTGGA
     CTACCTGGTT
451  CAAACGACTC CACCCTCCCA AGGTAAATTA AAAGAAGCGC
     TTATCCAAGC
501  AAGGAATACT CATACGGAGC AATTCGGACG AACTGCTATT
     GGTGCGAAAA
551  ACATCTTATT TGCCTCTCAA GAATATGCAG ACCAACTGAA
     TGTTTCTCCT
601  TCAGGGCTTC GCTCTTTGTA CTTAGAAGTG ACTGGAGACA
     CACATACCTG
651  TGATCAGCTA CTCTCTATGC TTCAAGACCG CTATACCTAC
     CAAGATATGG
701  CTATTGTCAG CTCCTTTCTA ATGAAAGGAA TGGCAACAGA
     ATTAAAAAGG
751  CAGGGTCCCT ACGTACCCAG TGCGCAACTA CAAGTTCTCA
     TGACAGAAAC
801  TCGTAACCTG CAAGCAGTTC TTACCTCGTA CGATTACTTT
     GAAAGTCGCG
851  TTCCTATTTT ACTCGATAGC TTAAAAGCTG AGGGAATCCA
     AACTCCTTCT
901  GATCTAAACT TTGTGAAGGT AGCTGAGTCC TACCATAAAA
     TCATTAACGA
951  TAAGTTCCCA ACAGCATCTA AAGTAGAACG AGAAGTCCGC
     AATCTCATAG
1001 GAGACGATGT TGATTCTGTG ACCGGTGTCT TGAACTTATT
     CTTTTCTGCT
1051 TTACGTCAAA CGTCGTCACG CCTTTTCTCT TCAGCAGACA
     AACGTCAGCA
1101 ATTAGGAGCT ATGATTGCTA ATGCTTTAGA TGCTGTAAAT
     ATAAACAATG
1151 AAGATTATCC CAAACGATCA GACTTCCCTA AACCCTATCC
     TTGGTCATGA

The PSORT algorithm predicts a cytoplasmic location (0.080).

The protein was expressed in E. coli and purified as both a His-tag and a GST-fusion product, as shown in FIG. 15A. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 15B) and for FACS analysis (FIG. 15C).

The cp6602 protein was also identified in the 2D-PAGE experiment (Cpn0324).

These experiments show that cp6602 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 16

The following C. pneumoniae protein (PID 4376727) was expressed <SEQ ID 31; cp6727>:

1    MKYSLPWLLT SSALVFSLHP LMAANTDLSS SDNYENGSSG
     SAAFTAKETS
51   DASGTTYTLT SDVSITNVSA ITPADKSCFT NTGGALSFVG
     ADHSLVLQTI
101  ALTHDGAAIN NTNTALSFSG FSSLLIDSAP ATGTSGGKGA
     ICVTNTEGGT
151  ATFTDNADVT LQKNTSEKDG AAVSAYSIDL AKITTAALLD
     QNTSTKNGGA
201  LCSTANTTVQ GNSGTVTFSS NTATDKGGGI YSKEKDSTLD
     ANTGVVTFKS
251  NTAKTGGAWS SDDNLALTGN TQVLFQENKT TGSAAQANNP
     EGCGGAICCY
301  LATATDKTGL AISQNQEMSF TSNTTTANGG AIYATKCTLD
     GNTTLTFDQN
351  TATAGCGGAI YTETEDFSLK GSTGTVTFST NTAKTGGALY
     SKGNSSLTGN
401  TNLLFSGNKA TGPSNSSANQ EGCGGAILAF IDSGSVSDKT
     GLSIANNQEV
451  SLTSNAATVS GGAIYATKCT LTGNGSLTFD GNTAGTSGGA
     IYTETEDFTL
501  TGSTGTVTFS TNTAKTGGAL YSKGNNSLSG NTNLLFSGNK
     ATGPSNSSAN
551  QEGCGGAILS FLESASVSTK KGLWIEDNEN VSLSGNTATV
     SGGAIYATKC
601  ALHGNTTLTF DGNTAETAGG AIYTETEDFT LTGSTGTVTF
     STNTAKTAGA
651  LHTKGNTSFT KNKALVFSGN SATATATTTT DQEGCGGAIL
     CNISESDIAT
701  KSLTLTENES LSFINNTAKR SGGGIYAPKC VISGSESINF
     DGNTAETSGG
751  AIYSKNLSIT ANGPVSFTNN SGGKGGAIYI ADSGELSLEA
     IDGDITFSGN
801  RATEGTSTPN SIHLGAGAKI TKLAAAPGHT IYFYDPITME
     APASGGTIEE
851  LVINPVVKAI VPPPQPKNGP IASVPVVPVA PANPNTGTIV
     FSSGKLPSQD
901  ASIPANTTTI LNQKINLAGG NVVLKEGATL QVYSFTQQPD
     STVFMDAGTT
951  LETTTTNNTD GSIDLKNLSV NLDALDGKRM ITIAVNSTSG
     GLKISGDLKF
1001 HNNEGSFYDN PGLKANLNLP FLDLSSTSGT VNLDDFNPIP
     SSMAAPDYGY
1051 QGSWTLVPKV GAGGKVTLVA EWQALGYTPK PELRATLVPN
     SLWNAYVNIH
1101 SIQQEIATAM SDAPSHPGIW IGGIGNAFHQ DKQKENAGFR
     LISRGYIVGG
1151 SMTTPQEYTF AVAFSQLFGK SKDYVVSDIK SQVYAGSLCA
     QSSYVIPLHS
1201 SLRRHVLSKV LPELPGETPL VLHGQVSYGR NHHNMTTKLA
     NNTQGKSDWD
1251 SHSFAVEVGG SLPVDLNYRY LTSYSPYVKL QVVSVNQKGF
     QEVAADPRIF
1301 DASHLVNVSI PMGLTFKHES AKPPSALLLT LGYAVDAYRD
     HPHCLTSLTN
1351 GTSWSTFATN LSRQAFFAEA SGHLKLLHGL DCFASGSCEL
     RSSSRSYNAN
1401 CGTRYSF*

A predicted signal peptide is highlighted.

The cp6727 nucleotide sequence <SEQ ID 32> is:

1    ATGAAATATT CTTTACCTTG GCTACTTACC TCTTCGGCTT
     TAGTTTTCTC
51   CCTACATCCA CTAATGGCTG CTAACACGGA TCTCTCATCA
     TCCGATAACT
101  ATGAAAATGG TAGTAGTGGT AGCGCAGCAT TCACTGCCAA
     GGAAACTTCG
151  GATGCTTCAG GAACTACCTA CACTCTCACT AGCGATGTTT
     CTATTACGAA
201  TGTATCTGCA ATTACTCCTG CAGATAAAAG CTGTTTTACA
     ACCACAGGAG
251  GAGCATTGAG TTTTGTTGGA GCTGATCACT CATTGGTTCT
     GCAAACCATA
301  GCGCTTACGC ATGATGGTGC TGCAATTAAC AATACCAACA
     CAGCTCTTTC
351  TTTCTCAGGA TTCTCGTCAC TCTTAATCGA CTCAGCTCCA
     GCAACAGGAA
401  CTTCGGGCGG CAAGGGTGCT ATTTGTGTGA CAAATACAGA
     GGGAGGTACT
451  GCGACTTTTA CTGACAATGC CAGTGTCACC CTCCAAAAAA
     ATACTTCAGA
501  AAAAGATGGA GCTGCAGTTT CTGCCTACAG CATCGATCTT
     GCTAAGACTA
551  CGACAGCAGC TCTCTTAGAT CAAAATACTA GCACAAAAAA
     TGGCGGGGCC
601  CTCTGTAGTA CAGCAAACAC TACAGTCCAA GGAAACTCAG
     GAACGGTGAC
651  CTTCTCCTCA AATACTGCTA CAGATAAAGG TGGGGGGATC
     TACTCAAAAG
701  AAAAGGATAG CACGCTAGAT GCCAATACAG GAGTCGTTAC
     CTTCAAATCT
751  AATACTGCAA AGACGGGGGG TGCTTGGAGC TCTGATGACA
     ATCTTGCTCT
801  TACCGGCAAC ACTCAAGTAC TTTTTCAGGA AAATAAAACA
     ACCGGCTCAG
851  CAGCACAGGC AAATAACCCG GAAGGTTGTG GTGGGGCAAT
     CTGTTGTTAT
901  CTTGCTACAG CAACAGACAA AACTGGATTA GCCATTTCTC
     AGAATCAAGA
951  AATGAGCTTC ACTAGTAATA CAACAACTGC GAATGGTGGA
     GCGATCTACG
1001 CTACTAAATG TACTCTGGAT GGAAACACAA CTCTTACCTT
     CGATCAGAAT
1051 ACTGCGACAG CAGGATGTGG CGGAGCTATC TATACAGAAA
     CTGAAGATTT
1101 TTCTCTTAAG GGAAGTACGG GAACCGTGAC CTTCAGCACA
     AATACAGCAA
1151 AGACAGGCGG CGCCTTATAT TCTAAAGGAA ACAGCTCGCT
     GACTGGAAAT
1201 ACCAACCTGC TCTTTTCAGG GAACAAAGCT ACGGGCCCGA
     GTAATTCTTC
1251 AGCAAATCAA GAGGGTTGCG GTGGGGCAAT CCTAGCCTTT
     ATTGATTCAG
1301 GATCCGTAAG CGATAAAACA GGACTATCGA TTGCAAACAA
     CCAAGAAGTC
1351 AGCCTCACTA GTAATGCTGC AACAGTAAGT GGTGGTGCGA
     TCTATGCTAC
1401 CAAATGTACT CTAACTGGAA ACGGCTCCCT GACCTTTGAC
     GGCAATACTG
1451 CTGGAACTTC AGGAGGGGCG ATCTATACAG AAACTGAAGA
     TTTTACTCTT
1501 ACAGGAAGTA CAGGAACCGT GACCTTCAGC ACAAATACAG
     CAAAGACAGG
1551 CGGCGCCTTA TATTCTAAAG GCAACAACTC TCTGTCTGGT
     AATACCAACC
1601 TGCTCTTTTC AGGGAACAAA GCTACGGGCC CGAGTAATTC
     TTCAGCAAAT
1651 CAAGAGGGTT GCGGTGGGGC AATCCTATCG TTTCTTGAGT
     CAGCATCTGT
1701 AAGTACTAAA AAAGGACTCT GGATTGAAGA TAACGAAAAC
     GTGAGTCTCT
1751 CTGGTAATAC TCGAACAGTA AGTGGCGGTG CGATCTATGC
     GACCAAGTGT
1801 GCTCTGCATG GAAACACGAC TCTTACCTTT GATGGCAATA
     CTGCCGAAAC
1851 TGCAGGAGGA GCGATCTATA CAGAAACCGA AGATTTTACT
     CTTACGGGAA
1901 GTACGGGAAC CGTGACCTTC AGCACAAATA CAGCAAAGAC
     AGCAGGGGCT
1951 CTACATACTA AAGGAAATAC TTCCTTTACC AAAAATAAGG
     CTCTTGTATT
2001 TTCTGGAAAT TCAGCAACAG CAACAGCAAC AACAACTACA
     GATCAAGAAG
2051 GTTGTGGTGG AGCGATCCTC TGTAATATCT CAGAGTCTGA
     CATAGCTACA
2101 AAAAGCTTAA CTCTTACTGA AAATGAGAGT TTAAGTTTCA
     TTAACAATAC
2151 GGCAAAAAGA AGTGGTGGTG GTATTTATGC TCCTAAGTGT
     GTAATCTCAG
2201 GCAGTGAATC CATAAACTTT GATGGCAATA CTGCTGAAAC
     TTCGGGAGGA
2251 GCGATTTATT CGAAAAACCT TTCGATTACA GCTAACGGTC
     CTGTCTCCTT
2301 TACCAATAAT TCTGGAGGCA AGGGAGGCGC CATTTATATA
     GCCGATAGCG
2351 GAGAACTTTC CTTAGAGGCT ATTGATGGGG ATATTACTTT
     CTCAGGGAAC
2401 CGAGCGACTG AGGGAACTTC AACTCCCAAC TCGATCCATT
     TAGGTGCAGG
2451 GGCTAAGATC ACTAAGCTTG CAGCAGCTCC TGGTCATACG
     ATTTATTTTT
2501 ATGATCCTAT TACGATGGAA GCTCCTGCAT CTGGAGGAAC
     AATAGAGGAG
2551 TTAGTCATCA ATCCTGTTGT CAAAGCTATT GTTCCTCCTC
     CCCAACCAAA
2601 AAATGGTCCT ATAGCTTCAG TGCCTGTAGT CCCTGTAGCA
     CCTGCAAACC
2651 CAAACACGGG AACTATAGTA TTTTCTTCTG GAAAACTCCC
     CAGTCAAGAT
2701 GCCTCGATTC CTGCAAATAC TACCACCATA CTGAACCAGA
     AGATCAACTT
2751 AGCAGGAGGA AATGTCGTTT TAAAAGAAGG AGCCACCCTA
     CAAGTATATT
2801 CCTTCACACA GCAGCCTGAT TCTACAGTAT TCATGGATGC
     AGGAACGACC
2851 TTAGAGACCA CGACAACTAA CAATACAGAT GGCAGCATCG
     ATCTAAAGAA
2901 TCTCTCTGTA AATCTGGATG CTTTAGATGG CAAGCGTATG
     ATAACGATTG
2951 CCGTAAACAG CACAAGTGGG GGATTAAAAA TCTCAGGGGA
     TCTGAAATTC
3001 CATAACAATG AAGGAAGTTT CTATGACAAT CCTGGGTTGA
     AAGCAAACTT
3051 AAATCTTCCT TTCTTAGATC TTTCTTCTAC TTCAGGAACT
     GTAAATTTAG
3101 ACGACTTCAA TCCGATTCCT TCTAGCATGG CTGCTCCGGA
     TTATGGGTAT
3151 CAAGGGAGTT GGACTCTGGT TCCTAAAGTA GGAGCTGGAG
     GGAAGGTGAC
3201 TTTGGTCGCG GAATGGCAAG CGTTAGGATA CACTCCTAAA
     CCAGAGCTTC
3251 GTGCGACTTT AGTTCCTAAT AGCCTTTGGA ATGCTTATGT
     AAACATCCAT
3301 TCTATACAGC AGGAGATCGC CACTGCGATG TCGGACGCTC
     CCTCACATCC
3351 AGGGATTTGG ATTGGAGGTA TTGGCAACGC CTTCCATCAA
     GACAAGCAAA
3401 AGGAAAATGC AGGATTCCGT TTGATTTCCA GAGGTTATAT
     TGTTGGTGGC
3451 AGCATGACCA CCCCTCAAGA ATATACCTTT GCTGTTGCAT
     TCAGCCAACT
3501 CTTTGGCAAA TCTAAGGATT ACGTAGTCTC GGATATTAAA
     TCTCAAGTCT
3551 ATGCAGGATC TCTCTGTGCT CAGAGCTCTT ATGTCATTCC
     CCTGCATAGC
3601 TCATTACGTC GCCACGTCCT CTCTAAGGTC CTTCCAGAGC
     TCCCAGGAGA
3651 AACTCCCCTT GTTCTCCATG GTCAAGTTTC CTATGGAAGA
     AACCACCATA
3701 ATATGACGAC AAAGCTTGCG AACAACACAC AAGGGAAATC
     AGACTGGGAC
3751 AGCCATAGCT TCGCTGTTGA AGTCGGTGGT TCTCTTCCTG
     TAGATCTAAA
3801 CTACAGATAC CTTACCAGCT ACTCTCCCTA TGTGAAACTC
     CAAGTTGTGA
3851 GTGTAAATCA AAAAGGATTC CAAGAGGTTG CTGCTGATCC
     ACGTATCTTT
3901 GACGCTAGCC ATCTGGTCAA CGTGTCTATC CCTATGGGAC
     TCACCTTCAA
3951 ACACGAATCA GCAAAGCCCC CCAGTGCTTT GCTTCTTACT
     TTAGGTTACG
4001 CTGTAGATGC TTACCGGGAT CACCCTCACT GCCTGACCTC
     CTTAACAAAT
4051 GGCACCTCGT GGTCTACGTT TGCTACAAAC TTATCACGAC
     AAGCTTTCTT
4101 TGCTGAGGCT TCTGGACATC TGAAGTTACT TCATGGTCTT
     GACTGCTTCG
4151 CTTCTGGAAG TTGTGAACTG CGCAGCTCCT CAAGAAGCTA
     TAATGCAAAC
4201 TGTGGAACTC GTTATTCTTT CTAA

The PSORT algorithm predicts an outer membrane location (0.915).

The protein was expressed in E. coli and purified as a his-tag product, as shown in FIG. 16A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 16B) and for FACS analysis (FIG. 16C). A GST-fusion protein was also expressed.

The cp6727 protein was also identified in the 2D-PAGE experiment (Cpn0444).

These experiments show that cp6727 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 17

The following C. pneumoniae protein (PID 4376731) was expressed <SEQ ID 33; cp6731>:

1   MKSSLHWFLI SSSLALPLSL NFSAFAAVVE INLGPTNSFS
    GPGTYTPPAQ
51  TTNADGTIYN LTGDVSITNA GSPTALTASC FKETTGNLSF
    QGHGYQFLLQ
101 NIDAGANCTF TNTAANKLLS FSGFSYLSLI QTTNATTGTG
    AIKSTGACSI
151 QSNYSCYFGQ NFSNDNGGAL QGSSISLSLN PNLTFAKNKA
    TQKGGALYST
201 GGITINNTLN SASFSENTAA NNGGAIYTEA SSFISSNKAI
    SFINNSVTAT
251 SATGGAIYCS STSAPKPVLT LSDNGELNFI GNTAITSGGA
    IYTDNLVLSS
301 GGPTLFKNNS AIDTAAPLGG AIAIADSGSL SLSALGGDIT
    FEGNTVVKGA
351 SSSQTTTRNS INIGNTNAKI VQLRASQGNT IYFYDPITTS
    ITAALSDALN
401 LNGPDLAGNP AYQGTIVFSG EKLSEAEAAE ADNLKSTIQQ
    PLTLAGGQLS
451 LKSGVTLVAK SFSQSPGSTL LMDAGITLET ADGITINNLV
    LNVDSLKETK
501 KATLKATQAS QTVTLSGSLS LVDPSGNVYE DVSWNNPQVF
    SCLTLTADDP
551 ANIHITDLAA DPLEKNPIHW GYQGNWALSW QEDTATKSKA
    ATLTWTKTGY
601 NPNPERRGTL VANTLWGSFV DVRSIQQLVA TKVRQSQETR
    GIWCEGISNF
651 FHKDSTKINK GFRHISAGYV VGATTTLASD NLITAAFCQL
    FGKDRDHFIN
701 KNRASAYAAS LHLQHLATLS SPSLLRYLPG SESEQPVLFD
    AQISYIYSKN
751 TMKTYYTQAP KGESSWYNDG CALELASSLP HTALSHEGLF
    HAYFPFIKVE
801 ASYIHQDSFK ERNTTLVRSF DSGDLINVSV PIGITFERFS
    RNERASYEAT
851 VIYVADVYRK NPDCTTALLI NNTSWKTTGT NLSRQAGIGR
    AGIFYAFSPN
901 LEVTSNLSME IRGSSRSYNA DLGGKFQF*

A predicted signal peptide is highlighted.

The cp6731 nucleotide sequence <SEQ ID 34> is:

1    ATGAAATCCT CTCTTCATTG GTTTTTAATC TCGTCATCTT
     TAGCACTTCC
51   CTTGTCACTA AATTTCTCTG CGTTTGCTGC TGTTGTTGAA
     ATCAATCTAG
101  GACCTACCAA TAGCTTCTCT GGACCAGGAA CCTACACTCC
     TCCAGCCCAA
151  ACAACAAATG CAGATGGAAC TATCTATAAT CTAACAGGGG
     ATGTCTCAAT
201  CACCAATGCA GGATCTCCGA CAGCTCTAAC CGCTTCCTGC
     TTTAAAGAAA
251  CTACTGGGAA TCTTTCTTTC CAAGGCCACG GCTACCAATT
     TCTCCTACAA
301  AATATCGATG CGGGAGCGAA CTGTACCTTT ACCAATACAG
     CTGCAAATAA
351  GCTTCTCTCC TTTTCAGGAT TCTCCTATTT GTCACTAATA
     CAAACCACGA
401  ATGCTACCAC AGGAACAGGA GCCATCAAGT CCACAGGAGC
     TTGTTCTATT
451  CAGTCGAACT ATAGTTGCTA CTTTGGCCAA AACTTTTCTA
     ATGACAATGG
501  AGGCGCCCTC CAAGGCAGCT CTATCAGTCT ATCGCTAAAC
     CCCAACCTAA
551  CGTTTGCCAA AAACAAAGCA ACGCAAAAAG GGGGTGCCCT
     CTATTCCACG
601  GGAGGGATTA CAATTAACAA TACGTTAAAC TCAGCATCAT
     TTTCTGAAAA
651  TACCGCGGCG AACAATGGCG GAGCCATTTA CACGGAAGCT
     AGCAGTTTTA
701  TTAGCAGCAA CAAAGCAATT AGCTTTATAA ACAATAGTGT
     GACCGCAACC
751  TCAGCTACAG GGGGAGCCAT TTACTGTAGT AGTACATCAG
     CCCCCAAACC
801  AGTCTTAACT CTATCAGACA ACGGGGAACT GAACTTTATA
     GGAAATACAG
851  CAATTACTAG TGGTGGGGCG ATTTATACTG ACAATCTAGT
     TCTTTCTTCT
901  GGAGGACCTA CGCTTTTTAA AAACAACTCT GCTATAGATA
     CTGCAGCTCC
951  CTTAGGAGGA GCAATTGCGA TTGCTGACTC TGGATCTTTG
     AGTCTTTCGG
1001 CTCTTGGTGG AGACATCACT TTTGAAGGAA ACACAGTAGT
     CAAAGGAGCT
1051 TCTTCGAGTC AGACCACTAC CAGAAATTCT ATTAACATCG
     GAAACACCAA
1101 TGCTAAGATT GTACAGCTGC GAGCCTCTCA AGGCAATACT
     ATCTACTTCT
1151 ATGATCCTAT AACAACTAGC ATCACTGCAG CTCTCTCAGA
     TGCTCTAAAC
1201 TTAAATGGTC CTGACCTTGC AGGGAATCCT GCATATCAAG
     GAACCATCGT
1251 ATTTTCTGGA GAGAAGCTCT CGGAAGCAGA AGCTGCAGAA
     GCTGATAATC
1301 TCAAATCTAC AATTCAGCAA CCTCTAACTC TTGCGGGAGG
     GCAACTCTCT
1351 CTTAAATCAG GAGTCACTCT AGTTGCTAAG TCCTTTTCGC
     AATCTCCGGG
1401 CTCTACCCTC CTCATGGATG CAGGGACCAC ATTAGAAACC
     GCTGATGGGA
1451 TCACTATCAA TAATCTTGTT CTCAATGTAG ATTCCTTAAA
     AGAGACCAAG
1501 AAGGCTACGC TAAAAGCAAC ACAAGCAAGT CAGACAGTCA
     CTTTATCTGG
1551 ATCGCTCTCT CTTGTAGATC CTTCTGGAAA TGTCTACGAA
     GATGTCTCTT
1601 GGAATAACCC TCAAGTCTTT TCTTGTCTCA CTCTTACTGC
     TGACGACCCC
1651 GCGAATATTC ACATCACAGA CTTAGCTGCT GATCCCCTAG
     AAAAAAATCC
1701 TATCCATTGG GGATACCAAG GGAATTGGGC ATTATCTTGG
     CAAGAGGATA
1751 CTGCGACTAA ATCCAAAGCA GCGACTCTTA CCTGGACAAA
     AACAGGATAC
1801 AATCCGAATC CTGAGCGTCG TGGAACCTTA GTTGCTAACA
     CGCTATGGGG
1851 ATCCTTTGTT GATGTGCGCT CCATACAACA GCTTGTAGCC
     ACTAAAGTAC
1901 GCCAATCTCA AGAAACTCGC GGCATCTGGT GTGAAGGGAT
     CTCGAACTTC
1951 TTCCATAAAG ATAGCACGAA GATAAATAAA GGTTTTCGCC
     ACATAAGTGC
2001 AGGTTATGTT GTAGGAGCGA CTACAACATT AGCTTCTGAT
     AATCTTATCA
2051 CTGCAGCCTT CTGCCAATTA TTCGGGAAAG ATAGAGATCA
     CTTTATAAAT
2101 AAAAATAGAG CTTCTGCCTA TGCAGCTTCT CTCCATCTCC
     AGCATCTAGC
2151 GACCTTGTCT TCTCCAAGCT TGTTACGCTA CCTTCCTGGA
     TCTGAAAGTG
2201 AGCAGCCTGT CCTCTTCGAT GCTCAGATCA GCTATATCTA
     TAGTAAAAAT
2251 ACTATGAAAA CCCATTACAC CCAAGCACCA AAGGGAGAGA
     GCTCGTGGTA
2301 TAATGACGGT TGCGCTCTGG AACTTGCGAG CTCCCTACCA
     CACACTGCTT
2351 TAAGCCATGA GGGTCTCTTC CACGCGTATT TTCCTTTCAT
     CAAAGTAGAA
2401 GCTTCGTACA TACACCAAGA TAGCTTCAAA GAACGTAATA
     CTACCTTGGT
2451 ACGATCTTTC GATAGCGGTG ATTTAATTAA CGTCTCTGTG
     CCTATTGGAA
2501 TTACCTTCGA GAGATTCTCG AGAAACGAGC GTGCGTCTTA
     CGAAGCTACT
2551 GTCATCTACG TTGCCGATGT CTATCGTAAG AATCCTGACT
     GCACGACAGC
2601 TCTCCTAATC AACAATACCT CGTGGAAAAC TACAGGAACG
     AATCTCTCAA
2651 GACAAGCTGG TATCGGAAGA GCAGGGATCT TTTATGCCTT
     CTCTCCAAAT
2701 CTTGAGGTCA CAAGTAACCT ATCTATGGAA ATTCGTGGAT
     CTTCACGCAG
2751 CTACAATGCA GATCTTGGAG GTAAGTTCCA GTTCTAA

The PSORT algorithm predicts an outer membrane location (0.926).

The protein was expressed in E. coli and purified as a his-tag product, as shown in FIG. 17A. A GST-fusion protein was also expressed. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 17B; his-tag) and for FACS analysis (FIG. 17C; his-tag and GST-fusion).

The GST-fusion protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis. Less cross-reactivity was seen with the his-fusion.

These experiments show that cp6731 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 18

The following C. pneumoniae protein (PID 4376737) was expressed <SEQ ID 35; cp6737>:

1   MPLSFKSSSF CLLACLCSAS CAFAETRLGG NFVPPITNQG
    EEILLTSDFV
51  CSNFLGASFS SSFINSSSNL SLLGKGLSLT FTSCQAPTNS
    NYALLSAAET
101 LTFKNFSSIN FTGNQSTGLG GLIYGKDIVF QSIKDLIFTT
    NRVAYSPASV
151 TTSATPAITT VTTGASALQP TDSLTVENIS QSIKFFGNLA
    NFGSAISSSP
201 TAVVKFINNT ATMSFSHNFT SSGGGVIYGG SSLLFENNSG
    CIIFTANSCV
251 NSLKGVTPSS GTYALGSGGA ICIPTGTFEL KNNQGKCTFS
    YNGTPNDAGA
301 IYAFTCNIVG NQGALLLDSN TAARNGGAIC AKVLNIQGRG
    PIEFSRNRAE
351 KGGAIFIGPS VGDPAKQTST LTILASEGDI AFQGNMLNTK
    PGIRNAITVE
401 AGGEIVSLSA QGGSRLVFYD PITHSLPTTS PSNKDITINA
    NGASGSVVFT
451 SKGLSSTELL LPANTTTILL GTVKIASGEL KITDNAVVNV
    LGFATQGSGQ
501 LTLGSGGTLG LATPTGAPAA VDFTIGKLAF DPFSFLKRDF
    VSASVNAGTK
551 NVTLTGALVL DEHDVTDLYD MVSLQTPVAI PIAVFKGATV
    TKTGFPDGEI
601 ATPSHYGYQG KWSYTWSRPL LIPAPDGGFP GGPSPSANTL
    YAVWNSDTLV
651 RSTYILDPER YGEIVSNSLW ISFLGNQAFS DILQDVLLID
    HPGLSITAKA
701 LGAYVEHTPR QGHEGFSGRY GSYQAALSMN YTDHTTLGLS
    FGQLYGKTNA
751 NPYDSRCSEQ MYLLSFFGQF PIVTQKSEAL ISWKAAYGYS
    KNHLNTTYLR
801 PDKAPKSQGQ WHNNSYYVLI SAEHPFLNWC LLTRPLAQAW
    DLSGFISAEF
851 LGGWQSKFTE TGDLQRSFSR GKGYNVSLPI GCSSQWFTPF
    KKAPSTLTIK
901 LAYKPDIYRV NPHNIVTVVS NQESTSISGA NLRRHGLFVQ
    IHDVVDLTED
951 TQAFLNYTFD GKNGFTNHRV STGLKSTF*

A predicted signal peptide is highlighted.

The cp6737 nucleotide sequence <SEQ ID 36> is:

1    ATGCCTCTTT CTTTCAAATC TTCATCTTTT TGTCTACTTG
     CCTGTTTATG
51   TAGTGCAAGT TGCGCGTTTG CTGAGACTAG ACTCGGAGGG
     AACTTTGTTC
101  CTCCAATTAC GAATCAGGGT GAAGAGATCT TACTCACTTC
     AGATTTTGTT
151  TGTTCAAACT TCTTGGGGGC GAGTTTTTCA AGTTCCTTTA
     TCAATAGTTC
201  CAGCAATCTC TCCTTATTAG GGAAGGGCCT TTCCTTAACG
     TTTACCTCTT
251  GTCAAGCTCC TACAAATAGT AACTATGCGC TACTTTCTGC
     CGCAGAGACT
301  CTGACCTTCA AGAATTTTTC TTCTATAAAC TTTACAGGGA
     ACCAATCGAC
351  AGGACTTGGC GGCCTCATCT ACGGAAAAGA TATTGTTTTC
     CAATCTATCA
401  AAGATTTGAT CTTCACTACG AACCGTGTTG CCTATTCTCC
     AGCATCTGTA
451  ACTACGTCGG CAACTCCCGC AATCACTACA GTAACTACAG
     GAGCCTCTGC
501  TCTCCAACCT ACAGACTCAC TCACTGTCGA AAACATATCC
     CAATCGATCA
551  AGTTTTTTGG GAACCTTGCC AACTTCGGCT CTGCAATTAG
     CAGTTCTCCC
601  ACGGCAGTCG TTAAATTCAT CAATAACACC GCTACCATGA
     GCTTCTCCCA
651  TAACTTTACT TCGTCAGGAG GCGGCGTGAT TTATGGAGGA
     AGCTCTCTCC
701  TTTTTGAAAA CAATTCTGGA TGCATCATCT TCACCGCCAA
     CTCCTGTGTG
751  AACAGCTTAA AAGGCGTCAC CCCTTCATCA GGAACCTATG
     CTTTAGGAAG
801  TGGCGGAGCC ATCTGCATCC CTACGGGAAC TTTCGAATTA
     AAAAACAATC
851  AGGGGAAGTG CACCTTCTCT TATAATGGTA CACCAAATGA
     TGCGGGTGCG
901  ATCTACGCCG AAACCTGCAA CATCGTAGGG AACCAGGGTG
     CCTTGCTCCT
951  AGATAGCAAC ACTGCAGCGA GAAATGGCGG AGCCATCTGT
     GCTAAAGTGC
1001 TCAATATTCA AGGACGCGGT CCTATTGAAT TCTCTAGAAA
     CCGCGCGGAG
1051 AAGGGTGGAG CTATTTTCAT AGGCCCCTCT GTTGGAGACC
     CTGCGAAGCA
1101 AACATCGACA CTTACGATTT TGGCTTCCGA AGGTGATATT
     GCGTTCCAAG
1151 GAAACATGCT CAATACAAAA CCTGGAATCC GCAATGCCAT
     CACTGTAGAA
1201 GCAGGGGGAG AGATTGTGTC TCTATCTGCA CAAGGAGGCT
     CACGTCTTGT
1251 ATTTTATGAT CCCATTACAC ATAGCCTCCC AACCACAAGT
     CCGTCTAATA
1301 AAGACATTAC AATCAACGCT AATGGCGCTT CAGGATCTGT
     AGTCTTTACA
1351 AGTAAGGGAC TCTCCTCTAC AGAACTCCTG TTGCCTGCCA
     AGACGACAAC
1401 TATACTTCTA GGAACAGTCA AGATCGCTAG TGGAGAACTG
     AAGATTACTG
1451 ACAATGCGGT TGTCAATGTT CTTGGCTTCG CTACTCAGGG
     CTCAGGTCAG
1501 CTTACCCTGG GGTCTGGAGG AACCTTAGGG CTGGCAACAC
     CCACGGGAGC
1551 ACCTGCCGCT GTAGACTTTA CGATTGGAAA GTTAGCATTC
     GATCCTTTTT
1601 CCTTCCTAAA AAGAGATTTT GTTTCAGCAT CAGTAAATGC
     AGGCACAAAA
1651 AACGTCACTT TAACAGGAGC TCTGGTTCTT GATGAACATG
     ACGTTACAGA
1701 TCTTTATGAT ATGGTGTCAT TACAAACTCC AGTAGCAATT
     CCTATCGCTG
1751 TTTTCAAAGG AGCAACCGTT ACTAAGACAG GATTTCCTGA
     TGGGGAGATT
1801 GCGACTCCAA GCCACTACGG CTACCAAGGA AAGTGGTCCT
     ACACATGGTC
1851 CCGTCCCCTG TTAATTCCAG CTCCTGATGG AGGATTTCCT
     GGAGGTCCCT
1901 CTCCTAGCGC AAATACTCTC TATGCTGTAT GGAATTCAGA
     CACTCTCGTG
1951 CGTTCTACCT ATATCTTAGA TCCCGAGCGT TACGGAGAAA
     TTGTCAGCAA
2001 CAGCTTATGG ATTTCCTTCT TAGGAAATCA GGCATTCTCT
     GATATTCTCC
2051 AAGATGTTCT TTTGATAGAT CATCCCGGGT TCTCCATAAC
     CGCGAAAGCT
2101 TTAGGAGCCT ATGTCGAACA CACACCAAGA CAAGGACATG
     AGGGCTTTTC
2151 AGGTCGCTAT GGAGGCTACC AAGCTGCGCT ATCTATGAAC
     TACACGGACC
2201 ACACTACGTT AGGACTTTCT TTCGGGCAGC TTTATGGAAA
     AACTAACGCC
2251 AACCCCTACG ATTCACGTTG CTCAGAACAA ATGTATTTAC
     TCTCGTTCTT
2301 TGGTCAATTC CCTATCGTGA CTCAAAAGAG CGAGGCCTTA
     ATTTCCTGGA
2351 AAGCAGCTTA TGGTTATTCC AAAAATCACC TAAATACCAC
     CTACCTCAGA
2401 CCTGACAAAG CTCCAAAATC TCAAGGGCAA TGGCATAACA
     ATAGTTACTA
2451 TGTTCTTATT TCTGCAGAAC ATCCTTTCCT AAACTGGTGT
     CTTCTTACAA
2501 GACCTCTGGC TCAAGCTTGG GATCTTTCAG GTTTTATTTC
     CGCAGAATTC
2551 CTAGGTGGTT GGCAAAGTAA GTTCACAGAA ACTGGAGATC
     TGCAACGTAG
2601 CTTTAGTAGA GGTAAAGGGT ACAATGTTTC CCTACCGATA
     GGATGTTCTT
2651 CTCAATGGTT CACACCATTT AAGAAGGCTC CTTCTACACT
     GACCATCAAA
2701 CTTGCCTACA AGCCTGATAT CTATCGTGTC AACCCTCACA
     ATATTGTGAC
2751 TGTCGTCTCA AACCAAGAGA GCACTTCGAT CTCAGGAGCA
     AATCTACGCC
2801 GCCACGGTTT GTTTGTACAA ATCCATGATG TAGTAGATCT
     CACCGAGGAC
2851 ACTCAGGCCT TTCTAAACTA TACCTTTGAC GGGAAAAATG
     GATTTACAAA
2901 CCACCGAGTG TCTACAGGAC TAAAATCCAC ATTTTAA

The PSORT algorithm predicts an outer membrane location (0.940).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 18A. The recombinant protein was used to immunise mice, whose sera were used in an immunoblot analysis blot (FIG. 18B) and for FACS analysis (FIG. 18C). A his-tagged protein was also expressed.

The cp6737 protein was also identified in the 2D-PAGE experiment (Cpn0454) and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6737 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 19

The following C. pneumoniae protein (PID 4377090) was expressed <SEQ ID 37; cp7090>:

1   MNIHSLWKLC TLLALLALPA CSLSRNYGWE DSCNTCHHTR
    RKKPSSFGFV
51  PLYTEEDFNP NFTFGEYDSK EEKQYKSSQV AAFRNITFAT
    DSYTIKGEEN
101 LAILTNLVHY MKKNPKATLY IEGHTDERGA ASYNLALGAR
    RANAIKEHLR
151 KQGISADRLS TISYGKEHPL NSGHNELAWQ QNRRTEFKIH AR*

A predicted signal peptide is highlighted.

The cp7090 nucleotide sequence <SEQ ID 38> is:

1   ATGAATATAC ATTCCCTATG GAAACTTTGT ACTTTATTGG
    CTTTACTTGC
51  ATTGCCAGCA TGTAGCCTTT CCCCTAATTA TGGCTGGGAG
    GATTCCTGTA
101 ATACATGCCA TCATACAAGA CGAAAAAAGC CTTCTTCTTT
    TGGCTTTGTT
151 CCTCTCTATA CCGAAGAGGA CTTTAACCCT AATTTTACCT
    TCGGTGAGTA
201 TGATTCCAAA GAAGAAAAAC AATACAAGTC AAGCCAAGTT
    GCAGCATTTC
251 GTAATATCAC CTTTGCTACA GACAGCTATA CAATTAAAGG
    TGAAGAGAAC
301 CTTGCGATTC TCACGAACTT GGTTCACTAC ATGAAGAAAA
    ACCCGAAAGC
351 TACACTGTAC ATTGAAGGGC ATACTGACGA GCGTGGAGCT
    GCATCCTATA
401 ACCTTGCTTT AGGAGCACGA CGAGCCAATG CGATTAAAGA
    GCATCTCCGA
451 AAGCAGGGAA TCTCTGCAGA TCGTCTATCT ACTATTTCCT
    ACGGAAAAGA
501 ACATCCTTTA AATTCGGGAC ACAACGAACT AGCATGGCAA
    CAAAATCGCC
551 GTACAGAGTT TAAGATTCAT GCACGCTAA

The PSORT algorithm predicts an outer membrane location (0.790).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 19A. A his-tagged protein was also expressed. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 19B) and for FACS analysis.

These experiments show that cp7090 is useful immunogen. These properties are not evident from the sequence alone.

Example 20

The following C. pneumoniae protein (PID 4377091) was expressed <SEQ ID 39; cp7091>:

1   MLRQLCFQVF FFCFASLVYA EELEVVVRSE HITLPIEVSC
    QTDTKDPKIQ
51  KYLSSLTEIF CKDIALGDCL QPTAASKESS SPLAISLRLH
    VPQLSVVLLQ
101 SSKTPQTLCS FTISQNLSVD RQKIHHAADT VHYALTGIPG
    ISAGKIVFAL
151 SSLGKDQKLK QGELWTTDYD GKNLAPLTTE CSLSITPKWV
    GVGSNFPYLY
201 VSYKYGVPKI FLGSLENTEG KKVLPLKGNQ LMPTFSPRKK
    LLAFVADTYG
251 NPDLFIQPFS LTSGPMGRPR RLLNENFGTQ GNPSFNPEGS
    QLVFISNKDG
301 RPRLYIMSLD PEPQAPRLLT KKYRNSSCPA WSPDGKKIAF
    CSVIKGVRQI
351 CIYDLSSGED YQLTTSPTNK ESPSWAIDSR HLVFSAGNAE
    ESELYLISLV
401 TKKTNKIAIG VGEKRFPSWG AFPQQPIKRT L*

A predicted signal peptide is highlighted.

The cp7091 nucleotide sequence <SEQ ID 40> is:

1    ATGTTACGGC AACTATGCTT CCAAGTTTTT TTCTTTTGCT
     TCGCATCGCT
51   AGTCTATGCT GAAGAATTAG AAGTTGTTGT CCGTTCCGAA
     CATATCACGC
101  TCCCTATTGA GGTCTCTTGC CAGACCGATA CGAAAGATCC
     AAAAATACAG
151  AAATACCTCA GCTCGCTAAC GGAGATATTT TCGAAGGACA
     TTGCCCTAGG
201  AGATTGTCTA CAACCCACAG CGGCTTCTAA AGAATCGTCA
     TCTCCTTTAG
251  CAATATCTTT ACGGTTGCAT GTACCTCAGC TATCTGTAGT
     GCTTTTACAG
301  TCTTCAAAAA CTCCTCAAAC CTTATGTTCT TTTACTATTT
     CTCAAAATCT
351  TTCTGTAGAT CGTCAAAAAA TCCATCACGC TGCTGATACA
     GTTCATTACG
401  CCCTCACAGG GATTCCTGGA ATCAGTGCTG GGAAAATTGT
     TTTTGCTCTA
451  AGTTCTTTAG GAAAAGATCA AAAGCTCAAG CAAGGAGAAT
     TATGGACTAC
501  AGATTACGAT GGGAAAAACC TCGCCCCTTT AACCACAGAA
     TGTTCGCTCT
551  CTATAACTCC AAAATGGGTG GGTGTGGGAT CAAATTTTCC
     CTATCTCTAT
601  GTTTCGTATA AGTATGGTGT GCCTAAAATT TTTCTTGGTT
     CCCTAGAGAA
651  CACTGAAGGT AAAAAAGTCC TTCCGTTAAA AGGCAACCAA
     CTCATGCCTA
701  CGTTTTCTCC AAGAAAAAAG CTTTTAGCTT TCGTTGCTGA
     TACGTATGGA
751  AATCCTGATT TATTTATTCA ACCGTTCTCA CTAACTTCAG
     GACCTATGGG
801  TCGCCCACGT CGCCTCCTTA ATGAGAATTT CGGGACTCAA
     GGGAATCCCT
851  CCTTCAACCC TGAAGGATCC CAGCTTGTCT TTATATCGAA
     CAAAGACGGC
901  CGTCCGCGTC TTTATATTAT GTCCCTCGAT CCTGAACCCC
     AAGCACCTCG
951  CTTGCTGACA AAAAAATACA GAAATAGCAG TTGCCCTGCA
     TGGTCTCCAG
1001 ATGGTAAAAA AATAGCCTTC TGCTCTGTAA TTAAAGGGGT
     GCGACAAATT
1051 TGTATTTACG ATCTCTCCTC TGGAGAGGAT TACCAACTCA
     CTACGTCTCC
1101 CACAAATAAA GAGAGTCCTT CTTGGGCTAT AGACAGCCGT
     CATCTTGTCT
1151 TTAGTGCGGG GAATGCTGAA GAATCAGAGT TATATTTAAT
     CAGTCTAGTC
1201 ACCAAAAAAA CTAACAAAAT TGCTATAGGA GTAGGAGAAA
     AACGGTTCCC
1251 CTCCTGGGGT GCTTTCCCTC AGCAACCGAT AAAGAGAACA
     CTATGA

The PSORT algorithm predicts an inner membrane location (0.109).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 20A. A his-tagged protein was also expressed. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 20B) and for FACS analysis.

These experiments show that cp7091 is a useful immunogen. These properties are not evident from the sequence alone.

Example 21

The following C. pneumoniae protein (PID 4376260) was expressed <SEQ ID 41; cp6260>:

1   MRFSLCGFPL VFSFTLLSVF DTSLSATTIS LTPEDSFEGD
    SQNAERSYNV
51  QAGDVYSLTG DVSISNVDNS ALNKACFNVT SGSVTFAGNH
    HGLYFNNISS
101 GTTKEGAVLC CQDPQATARF SGFSTLSFIQ SPGDIKEQGC
    LYSKNALMLL
151 NNYVVRFEQN QSKTKGGAIS GANVTIVGNY DSVSFYQNAA
    TFGGAIHSSG
201 PLQIAVNQAE IRFAQNTAKN GSGGALYSDG DIDIDQNAYV
    LFRENEALTT
251 AIGKGGAVCC IPTSGSSTPV PIVTFSDNKQ LVFERNHSIM
    GGGAIYARKL
301 SISSGGPTLF INNISYANSQ NLGGAIAIDT GGEISLSAEK
    GTITFQGNRT
351 SLPFLNGIHL LQNAKFLKLQ ARNGYSIEFY DPITSEADGS
    TQLNINGDPK
401 NKEYTGTILF SGEKSLANDP RDFKSTIPQN VNLSAGYLVI
    KEGAFVTVSK
451 FTQSPGSHLV LDLGTKLIAS KEDIAITGLA IDIDSLSSSS
    TAAVIKANTA
501 NKQISVTDSI ELTSPTGNAY EDLRMRNSQT FPLLSLEPGA
    GGSVTVTAGD
551 FLPVSPHYGF QGNWKLAWTG TGNKVGEFFW DKINYKPRPE
    KEGNLVPNIL
601 WGNAVDVRSL MQVQETHASS LQTDRGLWID GIGNFFHVSA
    SEDNIRYRHN
651 SGGYVLSVNN EITPKHYTSM AFSQLFSRDK DYAVSNNEYR
    MYLGSYLYQY
701 TTSLGNIFRY ASRNFNVNVG ILSRRFLQNP LMIFHFLCAY
    GHATNDMKTD
751 YANFPMVKNS WRNNCWAIEC GGSMPLLVFE NGRLFQGAIP
    FMKLQLVYAY
801 QGDFKETTAD GRRFSNGSLT SISVPLGIRF EKLALSQDVL
    YDFSFSYIPD
851 IFRKDPSCEA ALVISGDSWL VPAAHVSRHA FVGSGTGRYH
    FNDYTELLCR
901 GSIECRPHAR NYNINCGSKF RF*

A predicted signal peptide is highlighted.

The cp6260 nucleotide sequence <SEQ ID 42> is:

1    ATGCGATTTT CGCTCTGCGG ATTTCCTCTA GTTTTTTCTT
     TTACATTGCT
51   CTCAGTCTTC GACACTTCTT TGAGTGCTAC TACGATTTCT
     TTAACCCCAG
101  AAGATAGTTT TCATGGAGAT AGTCAGAATG CAGAACGTTC
     TTATAATGTT
151  CAAGCTGGGG ATGTCTATAG CCTTACTGGT GATGTCTCAA
     TATCTAACGT
201  CGATAACTCT GCATTAAATA AAGCCTGCTT CAATGTGACC
     TCAGGAAGTG
251  TGACGTTCGC AGGAAATCAT CATGGGTTAT ATTTTAATAA
     TATTTCCTCA
301  GGAACTACAA AGGAAGGGGC TGTACTTTGT TGCCAAGATC
     CTCAAGCAAC
351  GGCACGTTTT TCTGGGTTCT CCACGCTCTC TTTTATTCAG
     AGCCCCGGAG
401  ATATTAAAGA ACAGGGATGT CTCTATTCAA AAAAGTCACT
     TATGCTCTTA
451  AACAATTATG TAGTGCGTTT TGAACAAAAC CAAAGTAAGA
     CTAAAGGCGG
501  AGCTATTAGT GGGGCGAATG TTACTATAGT AGGCAACTAC
     GATTCCGTCT
551  CTTTCTATCA GAATGCAGCC ACTTTTGGAG GTGCTATCCA
     TTCTTCAGGT
601  CCCCTACAGA TTGCAGTAAA TCAGGCAGAG ATAAGATTTG
     CACAAAATAC
651  TGCCAAGAAT GGTTCTGGAG GGGCTTTGTA CTCCGATGGT
     GATATTGATA
701  TTGATCAGAA TGCTTATGTT CTATTTCGAG AAAATGAGGC
     ATTGACTACT
751  GCTATAGGTA AGGGAGGGGC TGTCTGTTGT CTTCCCACTT
     CAGGAAGTAG
801  TACTCCAGTT CCTATTGTGA CTTTCTCTGA CAATAAACAG
     TTAGTCTTTG
851  AAAGAAACCA TTCCATAATG GGTGGCGGAG CCATTTATGC
     TAGGAAACTT
901  AGCATCTCTT CAGGAGGTCC TACTCTATTT ATCAATAATA
     TATCATATGC
951  AAATTCGCAA AATTTAGGTG GAGCTATTGC CATTGATACT
     GGAGGGGAGA
1001 TCAGTTTATC AGCAGAGAAA GGAACAATTA CATTCCAAGG
     AAACCGGACG
1051 AGCTTACCGT TTTTGAATGG CATCCATCTT TTACAAAATG
     CTAAATTCCT
1101 GAAATTACAG GCGAGAAATG GATACTCTAT AGAATTTTAT
     GATCCTATTA
1151 CTTCTGAAGC AGATGGGTCT ACCCAATTGA ATATCAACGG
     AGATCCTAAA
1201 AATAAAGAGT ACACAGGGAC CATACTCTTT TCTGGAGAAA
     AGAGTCTAGC
1251 AAACGATCCT AGGGATTTTA AATCTACAAT CCCTCAGAAC
     GTCAACCTGT
1301 CTGCAGGATA CTTAGTTATT AAAGAGGGGG CCGAAGTCAC
     AGTTTCAAAA
1351 TTCACGCAGT CTCCAGGATC GCATTTAGTT TTAGATTTAG
     GAACCAAACT
1401 GATAGCCTCT AAGGAAGACA TTGCCATCAC AGGCCTCGCG
     ATAGATATAG
1451 ATAGCTTAAG CTCATCCTCA ACAGCAGCTG TTATTAAAGC
     AAACACCGCA
1501 AATAAACAGA TATCCGTGAC GGACTCTATA GAACTTATCT
     CGCCTACTGG
1551 CAATGCCTAT GAAGATCTCA GAATGAGAAA TTCACAGACG
     TTCCCTCTGC
1601 TCTCTTTAGA GCCTGGAGCC GGGGGTAGTG TGACTGTAAC
     TGCTGGAGAT
1651 TTCCTACCGG TAAGTCCCCA TTATGGTTTT CAAGGCAATT
     GGAAATTAGC
1701 TTGGACAGGA ACTGGAAACA AAGTTGGAGA ATTCTTCTGG
     GATAAAATAA
1751 ATTATAAGCC TAGACCTGAA AAAGAAGGAA ATTTAGTTCC
     TAATATCTTG
1801 TGGGGGAATG CTGTAGATGT CAGATCCTTA ATGCAGGTTC
     AAGAGACCCA
1851 TGCATCGAGC TTACAGACAG ATCGAGGGCT GTGGATCGAT
     GGAATTGGGA
1901 ATTTCTTCCA TGTATCTGCC TCCGAAGACA ATATAAGGTA
     CCGTCATAAC
1951 AGCGGTGGAT ATGTTCTATC TGTAAATAAT GAGATCACAC
     CTAAGCACTA
2001 TACTTCGATG GCATTTTCCC AACTCTTTAG TAGAGACAAG
     GACTATGCGG
2051 TTTCCAACAA CGAATACAGA ATGTATTTAG GATCGTATCT
     CTATCAATAT
2101 ACAACCTCCC TAGGGAATAT TTTCCGTTAT GCTTCGCGTA
     ACCCTAATGT
2151 AAACGTCGGG ATTCTCTCAA GAAGGTTTCT TCAAAATCCT
     CTTATGATTT
2201 TTCATTTTTT GTGTGCTTAT GGTCATGCCA CCAATGATAT
     GAAAACAGAC
2251 TACGCAAATT TCCCTATGGT GAAAAACAGC TGGAGAAACA
     ATTGTTGGGC
2301 TATAGAGTGC GGAGGGAGCA TGCCTCTATT GGTATTTGAG
     AACGGAAGAC
2351 TTTTCCAAGG TGCCATCCCA TTTATGAAAC TACAATTAGT
     TTATGCTTAT
2401 CAGGGAGATT TCAAAGAGAC GACTGCAGAT GGCCGTAGAT
     TTAGTAATGG
2451 GAGTTTAACA TCGATTTCTG TACCTCTAGG CATACGCTTT
     GAGAAGCTGG
2501 CACTTTCTCA GGATGTACTC TATGACTTTA GTTTCTCCTA
     TATTCCTGAT
2551 ATTTTCCGTA AGGATCCCTC ATGTGAAGCT GCTCTGGTGA
     TTAGCGGAGA
2601 CTCCTGGCTT GTTCCGGCAG CACACGTATC AAGACATGCT
     TTTGTAGGGA
2651 GTGGAACGGG TCGGTATCAC TTTAACGACT ATACTGAGCT
     CTTATGTCGA
2701 GGAAGTATAG AATGCCGCCC CCATGCTAGG AATTATAATA
     TAAACTGTGG
2751 AAGCAAATTT CGTTTTTAG

The PSORT algorithm predicts an outer membrane location (0.921).

The protein was expressed in E. coli and purified both as a his-tag and GST-fusion product. The GST-fusion is shown in FIG. 21A. This recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 21B) and for FACS analysis (FIG. 21C).

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6260 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 22

The following C. pneumoniae protein (PID 4376456) was expressed <SEQ ID 43; cp6456>:

1   MSSPVNNTPS APNIPIPAPT TPGIPTTKPR SSFIEKVIIV
    AKYILFAIAA
51  TSGALGTILG LSGALTPGIG IALLVIFFVS MVLLGLILKD
    SISGGEERRL
101 REEVSRFTSE NQRLTVITTT LETEVKDLKA AKDQLTLEIE
    AFRNENGNLK
151 TTAEDLEEQV SKLSEQLEAL ERINQLIQAN AGDAQEISSE
    LKKLISGWDS
201 KVVEQINTSI QALKVLLGQE WVQEAQTHVK AMQEQIQALQ
    AEILGMHNQS
251 TALQKSVENL LVQDQALTRV VGELLESENK LSQACSALRQ
    EIEKLAQHET
301 SLQQRIDAML AQEQNLAEQV TALEKMKQEA QKAESEFIAC
    VRDRTFGRRE
351 TPPPTTPVVE GDESQEEDEG GTPPVSQPSS PVDRATGDGQ *

The cp6456 nucleotide sequence <SEQ ID 44> is:

1    ATGTCATCTC CTGTAAATAA CACACCCTCA GCACCAAACA
     TTCCAATACC
51   AGCGCCCACG ACTCCAGGTA TTCCTACAAC AAAACCTCGT
     TCTAGTTTCA
101  TTGAAAAGGT TATCATTGTA GCTAAGTACA TACTATTTGC
     AATTGCAGCC
151  ACATCAGGAG CACTCGGAAC AATTCTAGGT CTATCTGGAG
     CGCTAACCCC
201  AGGAATAGGT ATTGCCCTTC TTGTTATCTT CTTTGTTTCT
     ATGGTGCTTT
251  TAGGTTTAAT CCTTAAAGAT TCTATAAGTG GAGGAGAAGA
     ACGCAGGCTC
301  AGAGAAGAGG TCTCTCGATT TACAGGTGAG AATCAACGGT
     TGACAGTCAT
351  AACCACAACA CTTGAGACTG AAGTAAAGGA TTTAAAAGCA
     GCTAAAGATC
401  AACTTACACT TGAAATCGAA GCATTTAGAA ATGAAAACGG
     TAATTTAAAA
451  ACAACTGCTG AGGACTTAGA AGAGCAGGTT TCTAAACTTA
     GCGAACAATT
501  AGAAGCACTA GAGCGAATTA ATCAACTTAT CCAAGCAAAC
     GCTGGAGATG
551  CTCAAGAAAT TTCGTCTGAA CTAAAGAAAT TAATAAGCGG
     TTGGGATTCC
601  AAAGTTGTTG AACAGATAAA TACTTCTATT CAAGCATTGA
     AAGTGTTATT
651  GGGTCAAGAG TGGGTGCAAG AGGCTCAAAC ACACGTTAAA
     GCAATGCAAG
701  AGCAAATTCA AGCATTGCAA GCTGAAATTC TAGGAATGCA
     CAATCAATCT
751  ACAGCATTGC AAAAGTCAGT TGAGAATCTA TTAGTACAAG
     ATCAAGCTCT
801  AACAAGAGTA GTAGGTGAGT TGTTAGAGTC TGAGAACAAG
     CTAAGCCAAG
851  CTTGTTCTGC GCTACGTCAA GAAATAGAAA AGTTGGCCCA
     ACATGAAACA
901  TCTTTGCAAC AACGTATTGA TGCGATGCTA GCCCAAGAGC
     AAAATTTGGC
951  AGAGCAGGTC ACAGCCCTTG AAAAAATGAA ACAAGAAGCT
     CAGAAGGCTG
1001 AGTCCGAGTT CATTGCTTGT GTACGTGATC GAACTTTCGG
     ACGTCGTGAA
1051 ACACCTCCAC CAACAACACC TGTAGTTGAA GGTGATGAAA
     GTCAAGAAGA
1101 AGACGAAGGA GGTACTCCCC CAGTATCACA ACCATCTTCA
     CCCGTAGATA
1151 GAGCAACAGG AGATGGTCAG TAA

The PSORT algorithm predicts inner membrane (0.127).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 22A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 22B) and for FACS analysis (FIG. 22C). A his-tag protein was also expressed.

These experiments show that cp6456 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 23

The following C. pneumoniae protein (PID 4376729) was expressed <SEQ ID 45; cp6729>:

1   MKIPLHKLLI SSTLVTRILL SIATYGADAS LSPTDSFDGA
    GGSTFTPKST
51  ADANGTNYVL SGNVYINDAG KGTALTGCCF TETTGDLTFT
    GKGYSFSFNT
101 VDAGSNAGAA ASTTADKALT FTGFSNLSFI AAPGTTVASG
    KSTLSSAGAL
151 NLTDNGTILF SQNVSNEANN NGGAITTKTL SISGNTSSIT
    FTSNSAKKLG
201 GAIYSSAAAS ISGNTGQLVF MNNKGETGGG ALGFEASSSI
    TQNSSLFFSG
251 NTATDAAGKG GAIYCEKTGE TPTLTISGNK SLTFAENSSV
    TQGGAICAHG
301 LDLSAAGPTL FSNNRCGNTA AGKGGAIAIA DSGSLSLSAN
    QGDITFLGNT
351 LTSTSAPTST RNAIYLGSSA KITNLRAAQG QSIYFYDPIA
    SNTTGASDVL
401 TINQPDSNSP IDYSGTIVFS GEKLSADEAK AADNFTSILK
    QPLALASGTL
451 ALKGNVELDV NGFTQTEGST LLMQPGTKLK ADTEAISLTK
    LVVDLSALEG
501 NKSVSIETAG ANKTITLTSP LVFQDSSGNF YESHTINQAF
    TQPLVVFTAA
551 TAASDIYIDA LLTSPVQTPE PHYGYQGHWE ATWADTSTAK
    SGTMTWVTTG
601 YNPNPERRAS VVPDSLWASF TDIRTLQQIM TSQANSIYQQ
    RGLWASGTAN
651 FFHKDKSGTN QAFRHKSYGY IVGGSAEDFS ENIFSVAFCQ
    LFGKDKDLFI
701 VENTSHNYLA SLYLQHRAFL GGLPMPSFGS ITDMLKDIPL
    ILNAQLSYSY
751 TKNDMDTRYT SYPEAQGSWT NNSGALELGG SLALYLPKEA
    PFFQGYFPFL
801 KFQAVYSRQQ NFKESGAEAR AFDDGDLVNC SIPVGIRLEK
    ISEDEKNNFE
851 ISLAYIGDVY RKNPRSRTSL MVSGASWTSL CKNLARQAFL
    ASAGSHLTLS
901 PHVELSGEAA YELRGSAHIY NVDCGLRYSF *

A predicted signal peptide is highlighted.

The cp6729 nucleotide sequence <SEQ ID 46> is:

1    ATGAAAATAC CCTTGCACAA ACTCCTGATC TCTTCGACTC
     TTGTCACTCC
51   CATTCTATTG AGCATTGCAA CTTACGGAGC AGATGCTTCT
     TTATCCCCTA
101  CAGATAGCTT TGATGGAGCG GGCGGCTCTA CATTTACTCC
     AAAATCTACA
151  GCAGATGCCA ATGGAACGAA CTATGTCTTA TCAGGAAATG
     TCTATATAAA
201  CGATGCTGGG AAAGGCACAG CATTAACAGG CTGCTGCTTT
     ACAGAAACTA
251  CGGGTGATCT GACATTTACT GGAAAGGGAT ACTCATTTTC
     ATTCAACACG
301  GTAGATGCGG GTTCGAATGC AGGAGCTGCG GCAAGCACAA
     CTGCTGATAA
351  AGCCCTAACA TTCACAGGAT TTTCTAACCT TTCCTTCATT
     GCAGCTCCTG
401  GAACTACAGT TGCTTCAGGA AAAAGTACTT TAAGTTCTGC
     AGGAGCCTTA
451  AATCTTACCG ATAATGGAAC GATTCTCTTT AGCCAAAACG
     TCTCCAATGA
501  AGCTAATAAC AATGGCGGAG CGATCACCAC AAAAACTCTT
     TCTATTTCTG
551  GGAATACCTC TTCTATAACC TTCACTAGTA ATAGCGCAAA
     AAAATTAGGT
601  GGAGCGATCT ATAGCTCTGC GGCTGCAAGT ATTTCAGGAA
     ACACCGGCCA
651  GTTAGTCTTT ATGAATAATA AAGGAGAAAC TGGGGGTGGG
     GCTCTGGGCT
701  TTGAAGCCAG CTCCTCGATT ACTCAAAATA GCTCCCTTTT
     CTTCTCTGGA
751  AACACTGCAA CAGATGCTGC AGGCAAGGGC GGGGCCATTT
     ATTGTGAAAA
801  AACAGGAGAG ACTCCTACTC TTACTATCTC TGGAAATAAA
     AGTCTGACCT
851  TCGCCGAGAA CTCTTCAGTA ACTCAAGGCG GAGCAATCTG
     TGCCCATGGT
901  CTAGATCTTT CCGCTGCTGG CCCTACCCTA TTTTCAAATA
     ATAGATGCGG
951  GAACACAGCT GCAGGCAAGG GCGGCGCTAT TGCAATTGCC
     GACTCTGGAT
1001 CTTTAAGTCT CTCTGCAAAT CAAGGAGACA TCACGTTCCT
     TGGCAACACT
1051 CTAACCTCAA CCTCCGCGCC AACATCGACA CGGAATGCTA
     TCTACCTGGG
1101 ATCGTCAGCA AAAATTACGA ACTTAAGGGC AGCCCAAGGC
     CAATCTATCT
1151 ATTTCTATGA TCCGATTGCA TCTAACACCA CAGGAGCTTC
     AGACGTTCTG
1201 ACCATCAACC AACCGGATAG CAACTCGCCT TTAGATTATT
     CAGGAACGAT
1251 TGTATTTTCT GGGGAAAAGC TCTCTGCAGA TGAAGCGAAA
     GCTGCTGATA
1301 ACTTCACATC TATATTAAAG CAACCATTGG CTCTAGCCTC
     TGGAACCTTA
1351 GCACTCAAAG GAAATGTCGA GTTAGATGTC AATGGTTTCA
     CACAGACTGA
1401 AGGCTCTACA CTCCTCATGC AACCAGGAAC AAAGCTCAAA
     GCAGATACTG
1451 AAGCTATCAG TCTTACCAAA CTTGTCGTTG ATCTTTCTGC
     CTTAGAGGGA
1501 AATAAGAGTG TGTCCATTGA AACAGCAGGA GCCAACAAAA
     CTATAACTCT
1551 AACCTCTCCT CTTGTTTTCC AAGATAGTAG CGGCAATTTT
     TATGAAAGCC
1601 ATACGATAAA CCAAGCCTTC ACGCAGCCTT TGGTGGTATT
     CACTGCTGCT
1651 ACTGCTGCTA GCGATATTTA TATCGATGCG CTTCTCACTT
     CTCCAGTACA
1701 AACTCCAGAA CCTCATTACG GGTATCAGGG ACATTGGGAA
     GCCACTTGGG
1751 CAGACACATC AACTGCAAAA TCAGGAACTA TGACTTGGGT
     AACTACGGGC
1801 TACAACCCTA ATCCTGAGCG TAGAGCTTCC GTAGTTCCCG
     ATTCATTATG
1851 GGCATCCTTT ACTGACATTC GCACTCTACA GCAGATCATG
     ACATCTCAAG
1901 CGAATAGTAT CTATCAGCAA CGAGGACTCT GGGCATCAGG
     AACTGCGAAT
1951 TTCTTCCATA AGGATAAATC AGGAACTAAC CAAGCATTCC
     GACATAAAAG
2001 CTACGGCTAT ATTGTTGGAG GAAGTGCTGA AGATTTTTCT
     GAAAATATCT
2051 TCAGTGTAGC TTTCTGCCAG CTCTTCGGTA AAGATAAAGA
     CCTGTTTATA
2101 GTTGAAAATA CCTCTCATAA CTATTTAGCG TCGCTATACC
     TGCAACATCG
2151 AGCATTCCTA GGAGGACTTC CCATGCCCTC ATTTGGAAGT
     ATCACCGACA
2201 TGCTGAAAGA TATTCCTCTC ATTTTGAATG CCCAGCTAAG
     CTACAGCTAC
2251 ACTAAAAATG ATATGGATAC TCGCTATACT TCCTATCCTG
     AAGCTCAAGG
2301 CTCTTGGACC AATAACTCTG GGGCTCTAGA GCTCGGAGGA
     TCTCTGGCTC
2351 TATATCTCCC TAAAGAAGCA CCGTTCTTCC AGGGATATTT
     CCCCTTCTTA
2401 AAGTTCCAGG CAGTCTACAG CCGCCAACAA AACTTTAAAG
     AGAGTGGCGC
2451 TGAAGCCCGT GCTTTTGATG ATGGAGACCT AGTGAACTGC
     TCTATCCCTG
2501 TCGGCATTCG GTTAGAAAAA ATCTCCGAAG ATGAAAAAAA
     TAATTTCGAG
2551 ATTTCTCTAG CCTACATTGG TGATGTGTAT CGTAAAAATC
     CCCGTTCGCG
2601 TACTTCTCTA ATGGTCAGTG GAGCCTCTTG GACTTCGCTA
     TGTAAAAACC
2651 TCGCACGACA AGCCTTCTTA GCAAGTGCTG GAAGCCATCT
     GACTCTCTCC
2701 CCTCATGTAG AACTCTCTGG GGAAGCTGCT TATGAGCTTC
     GTGGCTCAGC
2751 ACACATCTAC AATGTAGATT GTGGGCTAAG ATACTCATTC
     TAG

The PSORT algorithm predicts outer membrane (0.927).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 23A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 23B) and for FACS analysis (FIG. 23C). A his-tag protein was also expressed.

The cp6729 protein was also identified in the 2D-PAGE experiment (Cpn0446) and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6729 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 24

The following C. pneumoniae protein (PID 4376849) was expressed <SEQ ID 47; cp6849>:

1   MSKLIRRVVT VLALTSMASC FASGGIEAAV AESLITKIVA
    SAETKPAPVP
51  MTAKKVRLVR RNKQPVEQKS RGAFCDKEFY PCEEGRCQPV
    EAQQESCYGR
101 LYSVKVNDDC NVEICQSVPE YATVGSPYPI EILAIGKKDC
    VDVVITQQLP
151 CEAEFVSSDP ETTPTSDGKL VWKIDRLGAG DKCKITVWVK
    PLKEGCCFTA
201 ATVCACPELR SYTKCGQPAI CIKQEGPDCA CLRCPVCYKI
    EVVNTGSAIA
251 RNVTVDNPVP DGYSHASGQR VLSFNLGDMR PGDKKVFTVE
    FCPQRRGQIT
301 NVATVTYCGG HKCSANVTTV VNEPCVQVNI SGADWSYVCK
    PVEYSISVSN
351 PGDLVLHDVV IQDTLPSGVT VLEAPGGEIC CNKVVWRIKE
    MCPGETLQFK
401 LVVKAQVPGR FTNQVAVTSE SNCGTCTSCA ETTTHWKGLA
    ATHMCVLDTN
451 DPICVGENTV YRICVTNRGS AEDTNVSLIL KFSKELQPIA
    SSGPTKGTIS
501 GNTVVFDALP KLGSKESVEF SVTLKGIAPG DARGEAILSS
    DTLTSPVSDT
551 ENTHVY*

A predicted signal peptide is highlighted.

The cp6849 nucleotide sequence <SEQ ID 48> is:

1    ATGTCCAAAC TCATCAGACG AGTAGTTACG GTCCTTGCGC
     TAACGAGTAT
51   GGCGAGTTGC TTTGCCAGCG GGGGTATAGA GGCCGCTGTA
     GCAGAGTCTC
101  TGATTACTAA GATCGTCGCT AGTGCGGAAA CAAAGCCAGC
     ACCTGTTCCT
151  ATGACAGCGA AGAAGGTTAG ACTTGTCCGT AGAAATAAAC
     AACCAGTTGA
201  ACAAAAAAGC CGTGGTGCTT TTTGTGATAA AGAATTTTAT
     CCCTGTGAAG
251  AGGGACGATG TCAACCTGTA GAGGCTCAGC AAGAGTCTTG
     CTACGGAAGA
301  TTGTATTCTG TAAAAGTAAA CGATGATTGC AACGTAGAAA
     TTTGCCAGTC
351  CGTTCCAGAA TACGCTACTG TAGGATCTCC TTACCCTATT
     GAAATCCTTG
401  CTATAGGCAA AAAAGATTGT GTTGATGTTG TGATTACACA
     ACAGCTACCT
451  TGCGAAGCTG AATTCGTAAG CAGTGATCCA GAAACAACTC
     CTACAAGTGA
501  TGGGAAATTA GTCTGGAAAA TCGATCGCCT GGGTGCAGGA
     GATAAATGCA
551  AAATTACTGT ATGGGTAAAA CCTCTTAAAG AAGGTTGCTG
     CTTCACAGCT
601  GCTACTGTAT GTGCTTGCCC AGAGCTCCGT TCTTATACTA
     AATGCGGTCA
651  ACCAGCCATT TGTATTAAGC AAGAAGGACC TGACTGTGCT
     TGCCTAAGAT
701  GCCCTGTATG CTACAAAATC GAAGTAGTGA ACACAGGATC
     TGCTATTGCC
751  CGTAACGTAA CTGTAGATAA TCCTGTTCCC GATGGCTATT
     CTCATGCATC
801  TGGTCAAAGA GTTCTCTCTT TTAACTTAGG AGACATGAGA
     CCTGGCGATA
851  AAAAGGTATT TACAGTTGAG TTCTGCCCTC AAAGAAGAGG
     TCAAATCACT
901  AACGTTGCTA CTGTAACTTA CTGCGGTGGA CACAAATGTT
     CTGCAAATGT
951  AACTACAGTT GTTAATGAGC CTTGTGTACA AGTAAATATC
     TCTGGTGCTG
1001 ATTGGTCTTA CGTATGTAAA CCTGTGGAGT ACTCTATCTC
     AGTATCGAAT
1051 CCTGGAGACT TGGTTCTTCA TGATGTCGTG ATCCAAGATA
     CACTCCCTTC
1101 TGGTGTTACA GTACTCGAAG CTCCTGGTGG AGAGATCTGC
     TGTAATAAAG
1151 TTGTTTGGCG TATTAAAGAA ATGTGCCCAG GAGAAACCCT
     CCAGTTTAAA
1201 CTTGTAGTGA AAGCTCAAGT TCCTGGAAGA TTCACAAATC
     AAGTTGCAGT
1251 AACTAGTGAG TCTAACTGCG GAACATGTAC ATCTTGCGCA
     GAAACAACAA
1301 CACATTGGAA AGGTCTTGCA GCTACCCATA TGTGCGTATT
     AGACACAAAT
1351 GATCCTATCT GTGTAGGAGA AAATACTGTC TATCGTATCT
     GTGTAACTAA
1401 CCGTGGTTCT GCTGAAGATA CTAACGTATC TTTAATCTTG
     AAGTTCTCAA
1451 AAGAACTTCA GCCAATAGCT TCTTCAGGTC CAACTAAAGG
     AACGATTTCA
1501 GGTAATACCG TTGTTTTCGA CGCTTTACCT AAACTCGGTT
     CTAAGGAATC
1551 TGTAGAGTTT TCTGTTACCT TGAAAGGTAT TGCTCCCGGA
     GATGCTCGCG
1601 GCGAAGCTAT TCTTTCTTCT GATACACTGA CTTCACCAGT
     ATCAGACACA
1651 GAAAATACCC ACGTGTATTA A

The PSORT algorithm predicts periplasmic space (0.93).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 24A, and also as a his-tag protein. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 24B) and for FACS analysis (FIG. 24C).

The cp6849 protein was also identified in the 2D-PAGE experiment (Cpn0557).

These experiments show that cp6849 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 25

The following C. pneumoniae protein (PID 4376273) was expressed <SEQ ID 49; cp6273>:

1   MGLFHLTLFG LLLCSLPISL VAKFPESVGH KILYISTQST
    QQALATYLEA
51  LDAYGDHDFF VLRKIGEDYL KQSIHSSDPQ TRKSTIIGAG
    LAGSSEALDV
101 LSQAMETADP LQQLLVLSAV SGHLGKTSDD LLFKALASPY
    PVIRLEAAYR
151 LANLKNTKVI DHLHSFIHKL PEEIQCLSAA IFLRLETEES
    DAYIRDLLAA
201 KKSAIRSATA LQIGEYQQKR FLPTLRNLLT SASPQDQEAI
    LYALGKLKDG
251 QSYYNIKKQL QKPDVDVTLA AAQALIALGK EEDALPVIKK
    QALEERPRAL
301 YALRHLPSEI GIPIALPIFL KTKNSEAKLN VALALLELGC
    DTPKLLEYIT
351 ERLVQPHYNE TLALSFSKGR TLQNWKRVNI IVPQDPQERE
    RLLSTTRGLE
401 EQILTFLFRL PKEAYLPCIY KLLASQKTQL ATTAISFLSH
    TSHQEALDLL
451 FQAAKLPGEP IIRAYADLAI YNLTKDPEKK RSLHDYAKKL
    IQETLLFVDT
501 ENQRPHPSMP YLRYQVTPES RYKLMLDILE TLATSKSSED
    IRLLIQLMTE
551 GDAKNPPVLA GLLIKIVE*

A predicted signal peptide is highlighted.

The cp6273 nucleotide sequence <SEQ ID 50> is:

1    ATGGGACTAT TCCATCCAAC TCTCTTTGGA CTTTTATTGT
     GTAGTCTTCC
51   CATTTCTCTT GTTGCTAAAT TCCCTGAGTC TGTAGGTCAT
     AAGATCCTTT
101  ATATAAGTAC GCAATCTACA CAGCAGGCCT TAGCAACATA
     TCTGGAAGCT
151  CTAGATGCCT ACGGTGATCA TGACTTCTTC GTTTTAAGAA
     AAATCGGAGA
201  AGACTATCTC AAGCAAAGCA TCCACTCCTC AGATCCGCAA
     ACTAGAAAAA
251  GCACCATCAT TGGAGCAGGC CTGGCGGGAT CTTCAGAAGC
     CTTGGACGTG
301  CTCTCCCAAG CTATGGAAAC TGCAGACCCC CTGCAGCAGC
     TACTGGTTTT
351  ATCGGCAGTC TCAGGACATC TTGGGAAAAC TTCTGACGAC
     TTACTGTTTA
401  AAGCTTTAGC ATCTCCCTAT CCTGTCATCC GCTTAGAAGC
     CGCCTATAGA
451  CTTGCTAATT TGAAGAACAC TAAAGTCATT GATCATCTAC
     ATTCTTTCAT
501  TCATAAGCTT CCCGAAGAAA TCCAATGCCT ATCTGCGGCA
     ATATTCCTAC
551  GCTTGGAGAC TGAAGAATCT GATGCTTATA TTCGGGATCT
     CTTAGCTGCC
601  AAGAAAAGCG CGATTCGGAG TGCCACAGCT TTGCAGATCG
     GAGAATACCA
651  ACAAAAACGC TTTCTTCCGA CACTTAGGAA TTTGCTAACG
     AGTGCGTCTC
701  CTCAAGATCA AGAAGCTATT CTTTATGCTT TAGGGAAGCT
     TAAGGATGGT
751  CAGAGCTACT ACAATATAAA AAAGCAATTG CAGAAGCCTG
     ATGTGGATGT
801  CACTTTAGCA GCAGCTCAAG CTTTAATTGC TTTGGGGAAA
     GAAGAGGACG
851  CTCTTCCCGT GATAAAAAAG CAAGCACTTG AGGAGCGGCC
     TCGAGCCCTG
901  TATGCCTTAC GGCATCTACC CTCTGAGATA GGGATTCCGA
     TTGCCCTGCC
951  GATAGGCCTA AAAACTAAGA ACAGCGAAGC CAAGTTGAAT
     GTAGCTTTAG
1001 CTCTCTTAGA GTTAGGGTGT GACACCCCTA AACTACTGGA
     ATACATTACC
1051 GAAAGGCTTG TCCAACCACA TTATAATGAG ACTCTAGCCT
     TGAGTTTCTC
1101 TAAGGGGCGT ACTTTACAAA ATTGGAAGCG GGTGAACATC
     ATAGTCCCTG
1151 AAGATCCCCA GGAGAGGGAA AGGTTGCTCT CCACAACCCG
     AGGTCTTGAA
1201 GAGCAGATCC TTACGTTTCT CTTCCGCCTA CCTAAAGAAG
     CTTACCTCCC
1251 CTGTATTTAT AAGCTTTTGG CGAGTCAGAA AACTCAGCTT
     GCCACTACTG
1301 CGATTTCTTT TTTAAGTCAC ACCTCACATC AGGAAGCCTT
     AGATCTACTT
1351 TTCCAAGCTG CGAAGCTTCC TGGAGAACCT ATCATCCGCG
     CCTATGCAGA
1401 TCTTGCTATT TATAATCTCA CCAAAGATCC TGAAAAAAAA
     CGTTCTCTCC
1451 ATGATTATGC AAAAAAGCTA ATTCAGGAAA CCTTGTTATT
     TGTGGACACG
1501 GAAAACCAAA GACCCCATCC CAGCATGCCC TATCTACGTT
     ATCAGGTCAC
1551 CCCAGAAAGC CGTACGAAGC TCATGTTGGA TATTCTAGAG
     ACACTAGCCA
1601 CCTCGAAGTC TTCCGAAGAT ATCCGTTTAT TGATACAACT
     GATGACGGAA
1651 GGAGATGCAA AAAATTTCCC AGTCCTTGCA GGCTTACTCA
     TAAAAATTGT
1701 GGAGTAA

The PSORT algorithm predicts a periplasmic location (0.922).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product, as shown in FIG. 25A. The recombinant GST-fusion was used to immunise mice, whose sera were used in a Western blot (FIG. 25B) and for FACS analysis (FIG. 25C).

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6273 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 26

The following C. pneumoniae protein (PID 4376735) was expressed <SEQ ID 51; cp6735>:

1   MTILRNFLTC SALFLALPAA AQVVYLHESD GYNGAINNKS
    LEPKITCYPE
51  GTSYIFLDDV RISNVKHDQE DAGVFINRSG NLFFMGNRCN
    FTFHNLMTEG
101 FGAAISNRVG DTTLTLSNFS YLAFTSAPLL PQGQGAIYSL
    GSVMIENSEE
151 VTFCGNYSSW SGAAIYTPYL LGSKASRPSV NLSGNRYLVF
    RDNVSQGYGG
201 AISTHNLTLT TRGPSCFENN HAYHDVNSNG GAIAIAPGGS
    ISISVKSGDL
251 IFKGNTASQD GNTIHNSIHL QSGAQFKNLR AVSESGVYFY
    DPISHSESHK
301 ITDLVINAPE GKETYEGTIS FSGLCLDDHE VCAENLTSTI
    LQDVTLAGGT
351 LSLSDGVTLQ LHSFKQEASS TLTMSPGTTL LCSGDARVQN
    LHILIEDTDN
401 FVPVRIRAED KDALVSLEKL KVAFEAYWSV YDFPQFKEAF
    TIPLLELLGP
451 SFDSLLLGET TLERTQVTTE NDAVRGFWSL SWEEYPPSLD
    KDRRITPTKK
501 TVFLTWNPEI TSTP*

A predicted signal peptide is highlighted.

The cp6735 nucleotide sequence <SEQ ID 52> is:

1    ATGACCATAC TTCGAAATTT TCTTACCTGC TCGGCTTTAT
     TCCTCGCTCT
51   CCCTGCAGCA GCACAAGTTG TATATCTTCA TGAAAGTGAT
     GGTTATAACG
101  GTGCTATCAA TAATAAAAGC TTAGAACCTA AAATTACCTG
     TTATCCAGAA
151  GGAACTTCTT ACATCTTTCT AGATGACGTG AGGATTTCCA
     ACGTTAAGCA
201  TGATCAAGAA GATGCTGGGG TTTTTATAAA TCGATCTGGG
     AATCTTTTTT
251  TCATGGGCAA CCGTTGCAAC TTCACTTTTC ACAACCTTAT
     GACCGAGGGT
301  TTTGGCGCTG CCATTTCGAA CCGCGTTGGA GACACCACTC
     TCACTCTCTC
351  TAATTTTTCT TACTTAGCGT TCACCTCAGC ACCTCTACTA
     CCTCAAGGAC
401  AAGGAGCGAT TTATAGTCTT GGTTCCGTGA TGATCGAAAA
     TAGTGAGGAA
451  GTGACTTTCT GTGGGAACTA CTCTTCGTGG AGTGGAGCTG
     CGATTTATAC
501  TCCCTACCTT TTAGGTTCTA AGGCGAGTCG TCCTTCAGTA
     AATCTCAGCG
551  GGAACCGCTA CCTGGTGTTT AGAGACAATG TGAGCCAAGG
     TTATGGCGGC
601  GCCATATCTA CCCACAATCT CACACTCACG ACTCGAGGAC
     CTTCGTGTTT
651  TGAAAATAAT CATGCTTATC ATGACGTGAA TAGTAATGGA
     GGAGCCATTG
701  CCATTGCTCC TGGAGGATCG ATCTCTATAT CCGTGAAAAG
     CGGAGATCTC
751  ATCTTCAAAG GAAATACAGC ATCACAAGAC GGAAATACAA
     TACACAACTC
801  CATCCATCTG CAATCTGGAG CACAGTTTAA GAACCTACGT
     GCTGTTTCAG
851  AATCCGGAGT TTATTTCTAT GATCCTATAA GCCATAGCGA
     GTCGCATAAA
901  ATTACAGATC TTGTAATCAA TGCTCCTGAA GGAAAGGAAA
     CTTATGAAGG
951  AACAATTAGC TTCTCAGGAC TATGCCTGGA TGATCATGAA
     GTTTGTGCGG
1001 AAAATCTTAC TTCCACAATC CTACAAGATG TCACATTAGC
     AGGAGGAACT
1051 CTCTCTCTAT CGGATGGGGT TACCTTGCAA CGTCATTCTT
     TTAAGCAGGA
1101 ACGAAGCTCT ACGCTTACTA TGTCTCCAGG AACCACTCTG
     CTCTGCTCAG
1151 GAGATGCTCT GGTTCAGAAT CTGCACATCC TGATTGAAGA
     TACCGACAAC
1201 TTTGTTCCTG TAAGGATTCG CGCCGAGGAC AAGGATGCTC
     TTGTCTCATT
1251 AGAAAAACTT AAAGTTGCCT TTGAGGCTTA TTGGTCCGTC
     TATGACTTTC
1301 CTCAATTTAA GGAAGCCTTT ACGATTCCTC TTCTTGAACT
     TCTAGGGCCT
1351 TCTTTTGACA GTCTTCTCCT AGGGGAGACC ACTTTGGAGA
     GAACCCAAGT
1401 CACAACAGAG AATGACGCCG TTCGAGGTTT CTGGTCCCTA
     AGCTGGGAAG
1451 AGTACCCCCC TTCTCTGGAT AAAGACAGAA GGATCACACC
     AACTAAGAAA
1501 ACTGTTTTCC TCACTTGGAA TCCTGAGATC ACTTCTACGC
     CATAA

The PSORT algorithm predicts an outer membrane location (0.922).

The protein was expressed in E. coli and purified as a as a his-tag product and as a GST-fusion product, as shown in FIG. 26A. The recombinant GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 26B).

These experiments show that cp6735 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 27

The following C. pneumoniae protein (PID 4376784) was expressed <SEQ ID 53; cp6784>:

1   MNRRKARWVV ALFAMTALIS VGCCPWSQAK SRCSIDKYIP
    VVNRLLEVCG
51  LPEAENVEDL IESSSAWVLT PEERFSGDLV SICQVKDEHA
    FYNDLSLLHM
101 TQAVPSYSAT YDCAVVFGGP LPALRQRLDF LVREWQRGVR
    FKKIVFLCGE
151 RGRYQSIEEQ EHFFDSRYNP SFTEENWESG NRVTPSSEEE
    IAKFVWMQML
201 LPRAWRDSTS GVRVTFLLAK PEENRVVANR KDTLLLFRSY
    QEAFPGRVLF
251 VSSQPFIGLD ACRVGQFFKG ESYDLAGPGF AQGVLKYHWA
    PRICLHTLAE
301 WLKETNGCLN ISEGCFG*

A predicted signal peptide is highlighted.

The cp6784 nucleotide sequence <SEQ ID 54> is:

1   ATGAATAGAA GAAAAGCAAG ATGGGTAGTG GCATTGTTCG
    CAATGACGGC
51  GCTCATTTCT GTTGGGTGTT GTCCTTGGTC ACAAGCGAAA
    TCAAGATGTT
101 CTATTGATAA GTATATTCCT GTAGTCAATC GTTTACTAGA
    AGTTTGTGGA
151 CTTCCTGAAG CTGAGAATGT TGAGGATTTA ATCGAGTCCT
    CGTCTGCTTG
201 GGTACTGACT CCTGAAGAAC GTTTTTCTGG AGAGTTAGTC
    TCTATCTGTC
251 AGGTTAAAGA TGAGCATGCT TTCTATAACG ATTTGTCTTT
    ATTACATATG
301 ACTCAGGCTG TGCCTTCGTA TTCTGCAACG TATGATTGTG
    CTGTAGTTTT
351 TGGCGGGCCT TTGCCAGCGC TACGTCAGCG CTTAGATTTT
    TTGGTGCGAG
401 AGTGGCAGCG TGGCGTGCGC TTTAAGAAAA TCGTTTTTCT
    ATGTGGAGAG
451 CGAGGGCGCT ATCAGTCTAT TGAAGAACAA GAGCATTTCT
    TTGATTCTCG
501 GTACAATCCT TTCCCTACTG AAGAGAACTG GGAATCTGGT
    AACCGAGTTA
551 CTCCCTCTTC TGAAGAAGAG ATTGCCAAAT TTGTTTGGAT
    GCAAATGCTT
601 TTACCTAGAG CATGGCGAGA TAGTACTTCA GGAGTCAGAG
    TGACATTTCT
651 TCTAGCAAAG CCAGAGGAAA ATCGTGTGGT TGCGAATCGT
    AAGGACACCT
701 TACTTTTATT CCGTTCTTAT CAAGAAGCGT TTCCGGGACG
    CGTGTTATTT
751 GTAAGTAGTC AACCCTTTAT CGGTTTAGAT GCTTGCAGGG
    TCGGGCAGTT
801 TTTCAAAGGG GAAAGCTATG ATCTTGCTGG ACCTGGATTT
    GCTCAAGGAG
851 TCTTGAAGTA TCATTGGGCT CCAAGGATTT GTCTACATAC
    TTTAGCGGAA
901 TGGTTAAAGG AAACGAACGG CTGCTTAAAT ATTTCAGAGG
    GTTGTTTTGG
951 ATGA

The PSORT algorithm predicts a periplasmic location (0.894).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product, as shown in FIG. 27A. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 27B). The GST-fusion product was used for FACS analysis (FIG. 27C).

The cp6784 protein was also identified in the 2D-PAGE experiment (Cpn0498).

These experiments show that cp6784 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 28

The following C. pneumoniae protein (PID 4376960) was expressed <SEQ ID 55; cp6960>:

1   MNRRWNLVLA TVALALSVAS CDVRSKDKDK DQGSLVEYKD
    NKDTNDIELS
51  DNQKLSRTFG HLLARQLRKS EDMFFDIAEV AKGLQAELVC
    KSAPLTETEY
101 EEKMAEVQKL VFEKKSKENL SLAEKFLKEN SKNAGVVEVQ
    PSKLQYKIIK
151 EGAGKAISGK PSALLHYKGS FINGQVFSSS EGNNEPILLP
    LGQTIPGFAL
201 GMQGMKEGET RVLYIHPDLA YGTAGQLPPN SLLIFEINLI
    QASADEVAAV
251 RQEGNQGE*

A predicted signal peptide is highlighted.

The cp6960 nucleotide sequence <SEQ ID 56> is:

1   ATGAACAGAC GGTGGAATTT AGTTTTAGCA ACAGTAGCTC
    TGGCACTCTC
51  CGTCGCTTCT TGTGACGTAC GGTCTAAGGA TAAAGACAAG
    GATCAGGGGT
101 CGTTAGTGGA ATATAAAGAT AACAAAGATA CCAATGACAT
    AGAATTATCC
151 GATAATCAAA AGTTATCCAG AACATTTGGT CATTTATTAG
    CACGCCAATT
201 ACGCAAGTCA GAAGATATGT TTTTTGATAT TGCAGAAGTG
    GCTAAGGGGT
251 TGCAGGCGGA ATTGGTTTGT AAAAGTGCTC CTTTAACAGA
    AACAGAGTAT
301 GAAGAAAAAA TGGCTGAAGT ACAGAAGTTG GTTTTTGAAA
    AAAAATCAAA
351 AGAAAATCTT TCATTGGCAG AAAAATTCTT AAAAGAAAAT
    AGCAAGAACG
401 CTGGTGTTGT TGAAGTGCAA CCAAGTAAAT TGCAATACAA
    AATTATTAAA
451 GAAGGTGCAG GGAAAGCAAT TTCAGGTAAA CCTTCAGCTC
    TATTGCACTA
501 CAAGGGTTCC TTCATCAATG GCCAAGTATT TAGCAGTTCA
    GAAGGCAACA
551 ATGAGCCTAT CTTGCTTCCT CTAGGCCAAA CAGTTCCTGG
    TTTTGCTTTA
601 GGTATGCAGG GCATGAAAGA AGGAGAAACT CGAGTTCTCT
    ACATCCATCC
651 TGATCTTGCT TACGGAACCG CAGGACAACT TCCTCCAAGC
    TCTTTATTAA
701 TTTTTGAAAT TAACTTGATT CAGGCTTCAG CAGATGAAGT
    TGCTGCTGTA
751 CCCCAAGAAG GAAATCAAGG TGAATGA

The PSORT algorithm predicts periplasmic space location (0.930).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product, as shown in FIG. 28A. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 28B) and for FACS analysis (FIG. 28C).

The cp6960 protein was also identified in the 2D-PAGE experiment.

These experiments show that cp6960 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 29

The following C. pneumoniae protein (PID 4376968) was expressed <SEQ ID 57; cp6968>:

1   MKFLLYVPLL LVLVSTGCDA KPVSFEPFSG KLSTQRFEPQ
    HSAEEYFSQG
51  QEFLKKGNFR KALLCFGIIT HHFPRDILRN QAQYLIGVCY
    FTQDHPDLAD
101 KAFASYLQLP DAEYSEELFQ MKYAIAQRFA QGKRKRICRL
    EGFPKLMNAD
151 EDALRIYDEI LTAFPSKDLG AQALYSKAAL LIVKNDLTEA
    TKTLKKLTLQ
201 FPLHILSSEA FVRLSEIYLQ QAKKEPHNLQ YLHFAKLNEE
    AMKKQHPNHP
251 LNEVVSANVG AMREHYARGL YATGRFYEKK KKAEAANIYY
    RTAITNYPDT
301 LLVAKCQKRL DRISKHTS*

A predicted signal peptide is highlighted.

The cp6968 nucleotide sequence <SEQ ID 58> is:

1   ATGAAATTTC TATTATACGT TCCACTTCTT CTTGTTCTCG
    TATCTACGGG
51  GTGCGATGCA AAACCTGTTT CTTTTGAGCC CTTTTCAGGA
    AAGCTTTCCA
101 CCCAGCGTTT TGAGCCTCAG CACTCTGCTG AAGAATATTT
    TTCTCAGGGA
151 CAGGAATTCT TAAAAAAAGG AAATTTCAGA AAAGCTTTAC
    TATGCTTTGG
201 AATCATTACG CATCACTTCC CTAGGGACAT CTTGCGTAAT
    CAAGCACAGT
251 ATCTTATAGG AGTCTGTTAC TTCACGCAGG ATCACCCAGA
    TTTAGCAGAC
301 AAGGCATTTG CATCTTACTT ACAACTTCCT GATGCGGAGT
    ACTCTGAAGA
351 GTTGTTCCAG ATGAAATATG CGATTGCTCA AAGATTTGCT
    CAAGGGAAGC
401 GTAAACGGAT TTGTCGATTA GAGGGCTTCC CAAAACTAAT
    GAATGCTGAT
451 GAAGATGCGC TACGCATTTA TGACGAGATT CTAACAGCGT
    TTCCTAGTAA
501 AGACTTAGGA GCTCAGGCCC TCTATAGTAA AGCTGCGTTA
    CTTATTGTAA
551 AAAACGATCT TACAGAAGCC ACCAAAACCT TAAAAAAACT
    CACGTTACAA
601 TTTCCTCTAC ATATTTTATC TTCAGAGGCC TTTGTACGTT
    TATCGGAAAT
651 CTATTTACAG CAAGCTAAGA AAGAGCCTCA CAATCTTCAA
    TATCTTCATT
701 TTGCAAAGCT TAATGAAGAG GCAATGAAAA AGCAGCATCC
    TAACCATCCT
751 CTGAATGAGG TTGTTTCTGC TAATGTTGGA GCTATGCGGG
    AACATTATGC
801 TCGAGGTTTG TATGCCACAG GTCGTTTCTA TGAGAAGAAG
    AAAAAGCCCG
851 AGGCTGCGAA TATCTATTAC CGCACTGCGA TTACAAACTA
    CCCAGACACT
901 TTATTAGTGG CTAAATGTCA AAAGCGTCTA GATAGAATAT
    CTAAGCATAC
951 TTCCTAA

The PSORT algorithm predicts an inner membrane location (0.790).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product, as shown in FIG. 29A. The recombinant GST-fusion was used to immunise mice, whose sera were used in a Western blot (FIG. 29B) and for FACS analysis (FIG. 29C).

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6968 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 30

The following C. pneumoniae protein (PID 4376998) was expressed <SEQ ID 59; cp6998>:

1   MKKLLKSALL SAAFAGSVGS LQALPVGNPS DPSLLIDGTI
    WEGAAGDPCD
51  PCATWCDAIS IRAGFYGDYV FDRILKVDAP KTFSMGAKPT
    GSAAANYTTA
101 VDRPNPAYNK HLHDAEWFTN AGFIALNIWD RFDVFCTLGA
    SNGYIRGNST
151 AFNLVGLFGV KGTTVNANEL PNVSLSNGVV ELYTDTSFSW
    SVGARGALWE
201 CGCATLGAEF QYAQSKPKVE ELNVICNVSQ SFVNKPKGYK
    GVAFPLPTDA
251 GVATATGTKS ATINYHEWQV GASLSYRLNS LVPYIGVQWS
    RATFDADNIR
301 IAQPKLPTAV LNLTAWNPSL LGNATALSTT DSFSDFMQIV
    SCQINKFKSR
351 KACGVTVGAT LVDADKWSLT AEARLINERA AHVSGQFRF*

A predicted signal peptide is highlighted.

The cp6998 nucleotide sequence <SEQ ID 60> is:

1    ATGAAAAAAC TCTTAAAGTC GGCGTTATTA TCCGCCGCAT
     TTGCTGGTTC
51   TGTTGGCTCC TTACAAGCCT TGCCTGTAGG GAACCCTTCT
     GATCCAAGCT
101  TATTAATTGA TGGTACAATA TGGGAAGGTG CTGCAGGAGA
     TCCTTGCGAT
151  CCTTGCGCTA CTTGGTGCGA CGCTATTAGC TTACGTGCTG
     GATTTTACGG
201  AGACTATGTT TTCGACCGTA TCTTAAAAGT AGATGCACCT
     AAAACATTTT
251  CTATGGGAGC CAAGCCTACT GGATCCGCTG CTGCAAACTA
     TACTACTGCC
301  GTAGATAGAC CTAACCCGGC CTACAATAAG CATTTACACG
     ATGCAGAGTG
351  GTTCACTAAT GCAGGCTTCA TTGCCTTAAA CATTTGGGAT
     CGCTTTGATG
401  TTTTCTGTAC TTTAGGAGCT TCTAATGGTT ACATTAGAGG
     AAACTCTACA
451  GCGTTCAATC TCGTTGGTTT ATTCGGAGTT AAAGGTACTA
     CTGTAAATGC
501  AAATGAACTA CCAAACGTTT CTTTAAGTAA CGGAGTTGTT
     GAACTTTACA
551  CAGACACCTC TTTCTCTTGG AGCGTAGGCG CTCGTGGAGC
     CTTATGGGAA
601  TGCGGTTGTG CAACTTTGGG AGCTGAATTC CAATATGCAC
     AGTCCAAACC
651  TAAAGTTGAA GAACTTAATG TGATCTGTAA CGTATCGCAA
     TTCTCTGTAA
701  ACAAACCCAA GGGCTATAAA GGCGTTGCTT TCCCCTTGCC
     AACAGACGCT
751  GGCGTAGCAA CAGCTACTGG AACAAAGTCT GCGACCATCA
     ATTATCATGA
801  ATGGCAAGTA GGAGCCTCTC TATCTTACAG ACTAAACTCT
     TTAGTGCCAT
851  ACATTGGAGT ACAATGGTCT CGAGCAACTT TTGATGCTGA
     TAACATCCGC
901  ATTGCTCAGC CAAAACTACC TACAGCTGTT TTAAACTTAA
     CTGCATGGAA
951  CCCTTCTTTA CTAGGAAATG CCACAGCATT GTCTACTACT
     GATTCGTTCT
1001 CAGACTTCAT GCAAATTGTT TCCTGTCAGA TCAACAAGTT
     TAAATCTAGA
1051 AAAGCTTGTG GAGTTACTGT AGGAGCTACT TTAGTTGATG
     CTGATAAATG
1101 GTCACTTACT GCAGAAGCTC GTTTAATTAA CGAGAGAGCT
     GCTCACGTAT
1151 CTGGTCAGTT CAGATTCTAA

The PSORT algorithm predicts an outer membrane location (0.707).

The protein was expressed in E. coli and purified as a GST-fusion (FIG. 30A) and as a his-tag product. The recombinant GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 30B) and for FACS analysis (FIG. 30C).

The cp6998 protein was also identified in the 2D-PAGE experiment (Cpn0695) and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6998 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 31

The following C. pneumoniae protein (PID 4377102) was expressed <SEQ ID 61; cp7102>:

1   MKHTFTKRVL FFFFLVIPIP LLLNLMVVGF FSFSAAKANL
    VQVLHTRATN
51  LSIEFEKKLT IHKLFLDRLA NTLALKSYAS PSAEPYAQAY
    NEMMALSNTD
101 FSLCLIDPFD GSVRTKNPGD PFIRYLKQHP EMKKKLSAAV
    GKAFLLTIPG
151 KPLLHYLILV EDVASWDSTT TSGLLVSFYP MSFLQKDLFQ
    SLHITKGNIC
201 LVNKYGEVLF CAQDSESSFV FSLDLPNLPQ FQARSPSAIE
    IEKASGILGG
251 ENLITVSINK KRYLGLVLNK IPIQGTYTLS LVPVSDLIQS
    ALKVPLNICF
301 FYVLAFLLMW WIFSKINTKL NKPLQELTFC MEAAWRGNHN
    VRFEPQPYGY
351 EFNELGNIFN CTLLLLLNSI EKADIDYHSG EKLQKELGIL
    SSLQSALLSP
401 DFPTFPKVTF SSQHLRRRQL SGHFNGWTVQ DGGDTLLGII
    GLAGDIGLPS
451 YLYALSARSL FLAYASSDVS LQKISKDTAD SFSKTTEGNE
    AVVAMTFIKY
501 VEKDRSLELL SLSEGAPTMF LQRGESFVRL PLETHQALQP
    GDRLICLTGG
551 EDILKYFSQL PIEELLKDPL NPLNTENLID SLTMMLNNET
    EHSADGTLTI
601 LSFS*

A predicted signal peptide is highlighted.

The cp7102 nucleotide sequence <SEQ ID 62> is:

1    ATGAAACATA CCTTTACCAA GCGTGTTCTA TTTTTTTTCT
     TTTTAGTGAT
51   TCCCATTCCC CTACTCCTCA ATCTTATGGT CGTAGGTTTT
     TTCTCATTTT
101  CTGCCGCTAA AGCAAATTTA GTACAGGTCC TCCATACCCG
     TGCTACGAAC
151  TTAAGTATAG AATTCGAAAA AAAACTGACG ATACACAAGC
     TTTTCCTCGA
201  TAGACTTGCC AACACATTAG CCTTAAAATC CTATGCATCT
     CCTTCTGCAG
251  AGCCCTATGC ACAGGCATAC AATGAGATGA TGGCACTCTC
     CAATACAGAC
301  TTTTCCTTAT GCCTTATAGA TCCCTTTGAT GGATCTGTAA
     GGACGAAAAA
351  TCCTGGAGAC CCTTTCATTC GCTATCTAAA ACAGCATCCT
     GAAATGAAGA
401  AAAAGCTATC CGCAGCTGTA GGGAAAGCCT TTTTATTGAC
     CATTCCAGGT
451  AAACCACTTT TACATTATCT TATTCTAGTT GAAGATGTCG
     CATCTTGGGA
501  TTCTACAACG ACTTCAGGAC TGCTTGTAAG TTTCTATCCC
     ATGTCTTTTT
551  TACAGAAAGA TTTATTCCAA TCCTTACACA TCACCAAAGG
     AAATATCTGC
601  CTTGTAAATA AGTATGGCGA GGTCCTCTTC TGTGCTCAGG
     ACAGTGAATC
651  TTCTTTTGTA TTTTCTCTAG ATCTCCCTAA TTTACCGCAA
     TTCCAAGCAA
701  GAAGCCCCTC TGCCATAGAA ATTGAGAAAG CTTCTGGAAT
     TCTTGGTGGG
751  GAGAACCTAA TCACAGTGAG TATCAACAAG AAACGCTACC
     TAGGATTGGT
801  ACTGAATAAA ATTCCTATCC AAGGGACCTA CACTCTATCT
     TTAGTTCCAG
851  TTTCTGATCT CATCCAATCC GCCTTGAAAG TTCCTCTCAA
     TATTTGTTTT
901  TTCTATGTAC TTGCTTTCCT CCTCATGTGG TGGATTTTCT
     CTAAGATCAA
951  CACCAAACTT AACAAGCCTC TTCAAGAACT GACCTTCTGT
     ATGGAAGCTG
1001 CCTGGCGAGG AAACCATAAC GTGAGGTTTG AACCCCAGCC
     TTACGGTTAT
1051 GAATTCAATG AACTAGGAAA TATTTTCAAT TGCACTCTCC
     TACTCTTATT
1101 GAATTCCATT GAGAAAGCAG ATATCGATTA CCATTCAGGC
     GAAAAATTAC
1151 AAAAAGAATT AGGGATTTTA TCTTCACTAC AAAGTGCGTT
     ACTAAGTCCG
1201 GATTTCCCTA CGTTCCCTAA AGTTACCTTT AGTTCCCAAC
     ATCTCCGGAG
1251 AAGGCAACTT TCCGGTCATT TTAATGGTTG GACAGTTCAA
     GATGGTGGCG
1301 ATACCCTTTT AGGGATCATA GGGCTCGCTG GCGATATTGG
     TCTTCCTTCC
1351 TATCTCTATG CTTTATCCGC ACGGAGTCTT TTTCTTGCCT
     ATGCTTCCTC
1401 GGACGTTTCG TTACAAAAAA TCAGCAAGGA TACTGCCGAC
     AGCTTCTCAA
1451 AAACAACAGA AGGCAATGAG GCTGTAGTTG CTATGACTTT
     CATTAAATAT
1501 GTAGAAAAAG ATCGATCTCT AGAGCTCCTC TCGTTAAGCG
     AGGGAGCTCC
1551 TACCATGTTT CTACAACGAG GAGAATCTTT CGTACGTCTC
     CCCTTAGAGA
1601 CTCACCAAGC TCTACAGCCT GGAGATCGGT TGATCTGCCT
     CACTGGAGGA
1651 GAAGACATCC TCAAGTACTT TTCTCAGCTT CCTATTGAAG
     AGCTCTTAAA
1701 AGATCCTTTA AACCCTCTAA ATACAGAGAA TCTTATTGAT
     TCTCTAACCA
1751 TGATGTTAAA CAACGAAACC GAACATTGTG CAGATGGAAC
     TCTGACCATC
1801 CTTTCATTT CATAA

The PSORT algorithm predicts an inner membrane location (0.338).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product. The purified GST-fusion product is shown in FIG. 31A. The recombinant GST-fusion protein was used to immunise mice, whose sera were used in a Western blot and for FACS analysis (FIG. 31B).

These experiments show that cp7102 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 32

The following C. pneumoniae protein (PID 4377106) was expressed <SEQ ID 63; cp7106>:

1   MKDLGTLGGT SSTAKTVSPD GKVIMGRSQI ADGSWHAFMC
    HTDFSSNNVL
51  FDLDNTYKTL RENGRQLNSI FNLQNMMLQR ASDHEFTEFG
    RSNIALGAGL
101 YVNALQNLPS NLAAQYFGIA YKIRPKYRLG VFLDHNFSSH
    VPNNFNVSHN
151 RLWMGAFIGW CDSDALGSSV KVSFGYGKQK ATITREQLEN
    TEAGSGESHF
201 EGVAAQIEGR YGKSLGGHVR VQPFLGLQFV HITRKEYTEN
    AVQFPVHYDP
251 IDYSTGVVYL GIGSHIALVD SLHVGTRMGM EQNFAAHTDR
    FSGSIASIGN
301 FVFEKLDVTH TRAFAEMRVN YELPYLQSLN LILRVNQQPL
    QGVMGFSSDL
351 RYALGF*

The cp7106 nucleotide sequence <SEQ ID 64> is:

1    ATGAAAGATT TGGGGACTCT TGGGGGTACC TCTTCTACAG
     CAAAAACAGT
51   GTCCCCAGAT GGTAAAGTGA TCATGGGTAG ATCACAAATT
     GCTGATGGCA
101  GTTGGCACGC ATTTATGTGT CATACGGATT TCTCCTCTAA
     TAATGTACTC
151  TTTGATCTCG ATAATACGTA TAAAACTCTA AGAGAAAATG
     GCCGTCAGCT
201  AAATTCCATA TTCAACCTAC AAAATATGAT GTTACAGAGA
     GCCTCAGATC
251  ATGAGTTCAC AGAGTTTGGA AGGAGTAACA TCGCTCTTGG
     TGCCGGGCTT
301  TATGTGAATG CCTTGCAGAA TCTCCCTAGC AATTTAGCAG
     CACAATATTT
351  TGGAATCGCA TACAAAATAC GTCCTAAATA TCGTTTGGGG
     GTGTTTTTGG
401  ACCATAATTT CAGCTCCCAC GTTCCTAATA ATTTTAACGT
     AAGCCACAAT
451  AGACTCTGGA TGGGAGCCTT TATTGGATGG CAGGATTCTG
     ATGCTCTAGG
501  ATCTAGTGTC AAGGTGTCTT TCGGATATGG AAAACAAAAA
     GCCACGATTA
551  CAAGAGAGCA ATTAGAGAAT ACAGAAGCCG GGAGTGGGGA
     GAGCCATTTT
601  GAAGGGGTCG CTGCTCAGAT AGAAGGGCGG TATGGTAAGA
     GCCTCGGAGG
651  ACATGTCAGG GTCCAGCCTT TCCTAGGACT GCAGTTTGTC
     CACATTACAA
701  GGAAAGAATA TACCGAAAAT GCAGTGCAAT TTCCTGTACA
     CTATGATCCT
751  ATAGACTATT CTACAGGTGT AGTGTATTTA GGAATTGGAT
     CTCATATTGC
801  ACTTGTAGAT TCTTTACATG TAGGCACACG CATGGGAATG
     GAGCAAAACT
851  TTGCAGCCCA TACGGACAGG TTCTCAGGAT CTATAGCGTC
     TATTGGAAAC
901  TTTGTGTTTG AAAAGCTTGA TGTGACTCAC ACAAGGGCAT
     TTGCGGAAAT
951  GCGTGTCAAC TATGAGCTTC CCTATCTACA GTCTCTGAAT
     CTTATTCTAC
1001 GAGTTAATCA ACAGCCCCTA CAAGGGGTTA TGGGATTTTC
     CAGTGATCTT
1051 AGGTATGCCT TAGGATTCTA A

The PSORT algorithm predicts a cytoplasmic location (0.224).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product. The purified GST-fusion product is shown in FIG. 32A. The recombinant GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 32B) and for FACS analysis (FIG. 32C).

This protein also showed very good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp7106 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 33

The following C. pneumoniae protein (PID 4377228) was expressed <SEQ ID 65; cp7228>:

1   MTAVLILTSF PSEESARSLA RHLITERLAS CVHVFPKGTS
    TYLWFGKLCE
51  SEEHHIQIKS IDIRFSEICL AIQEFSGYEV PEVLLFPIEN
    GDPRYLNWLT
101 ILSYPEKPPL SD*

The cp7228 nucleotide sequence <SEQ ID 66> is:

1   ATGACTGCTG TTCTTATTCT TACATCTTTC CCTTCGGAGG
    AAAGTGCTCG
51  CTCCTTAGCT AGACATCTGA TTACAGAGCG TCTTGCTTCC
    TGTGTGCATG
101 TATTCCCTAA AGGCACATCG ACATATCTAT GGGAAGGCAA
    GCTATGTGAG
151 TCTGAAGAAC ATCATATACA AATCAAATCG ATAGACATAC
    GCTTCTCGGA
201 AATTTGTCTT GCTATTCAGG AGTTCTCTGG CTATGAGGTT
    CCTGAAGTCT
251 TACTATTTCC TATTGAAAAT GGGGATCCGA GGTACTTGAA
    TTGGTTAACG
301 ATTCTCAGCT ATCCAGAGAA GCCTCCGCTT TCAGATTAG

The PSORT algorithm predicts an inner membrane location (0.040).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product, as shown in FIG. 33A (his-tag=left-hand arrow, GST=right-hand arrow). The proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 33B) and FACS analysis.

These experiments show that cp7228 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 34

The following C. pneumoniae protein (PID 4377170) was expressed <SEQ ID 67; cp7170>:

1   MNSKMLKHLR LATLSFSMFF GIVSSPAVYA LGAGNPAAPV
    LPGVNPEQTG
51  WCAFQLCNSY DLFAALAGSL KFGFYGDYVF SESAHITNVP
    VITSVTTSGT
101 GTTPTITSTT KNVDFDLNNS SISSSCVFAT IALQETSPAA
    IPLLDIAFTA
151 RVGGLKQYYR LPLNAYRDFT SNPLNAESEV TDGLIEVQSD
    YGIVWGLSLQ
201 KVLWKDGVSF VGVSADYRHG SSPINYIIVY NKANPEIYFD
    ATDGNLSYKE
251 WSASIGISTY LNDYVLPYAS VSIGNTSRKA PSDSFTELEK
    QFTNFKFKIR
301 KITNFDRVNF CFGTTCCISN NFYYSVEGRW GYQRAINITS
    GLQF*

A predicted signal peptide is highlighted.

The cp7170 nucleotide sequence <SEQ ID 68> is:

1    ATGAATAGCA AGATGCTAAA ACATTTACGT TTAGCAACCC
     TTTCCTTCTC
51   TATGTTCTTC GGGATTGTAT CTTCTCCCGC AGTATATGCC
     CTAGGGGCTG
101  GAAACCCTGC AGCTCCAGTA CTCCCAGGTG TGAATCCTGA
     GCAAACGGGA
151  TGGTGTGCCT TCCAACTTTG TAATAGTTAC GATCTTTTTG
     CTGCTCTTGC
201  AGGAAGCCTC AAATTTGGGT TCTATGGAGA TTATGTCTTC
     TCAGAAAGTG
251  CCCATATTAC CAATGTCCCT GTCATTACCT CCGTTACGAC
     TTCAGGCACA
301  GGAACAACGC CAACCATTAC CTCTACAACT AAAAACGTAG
     ACTTTGATCT
351  TAACAACAGC TCCATCAGCT CGAGCTGTGT TTTTGCAACC
     ATAGCTCTAC
401  AGGAAACATC CCCAGCTGCC ATTCCCCTTT TAGATATAGC
     CTTCACTGCA
451  CGTGTCGGAG GACTTAAGCA GTACTACCGC CTCCCTCTCA
     ATGCTTACAG
501  AGACTTCACT TCAAATCCTT TAAATGCAGA ATCTGAAGTT
     ACAGATGGTC
551  TCATTGAAGT CCAGTCAGAC TATGGAATTG TCTGGGGTCT
     GAGTTTACAA
601  AAAGTATTGT GGAAAGATGG AGTGTCTTTT GTAGGGGTGA
     GCGCTGACTA
651  CCGTCACGGT TCCAGTCCCA TCAACTATAT CATCGTTTAC
     AACAAGGCCA
701  ACCCCGAGAT CTATTTCGAT GCTACTGATG GAAACCTAAG
     CTATAAAGAA
751  TGGTCTGCAA GCATCGGCAT CTCTACGTAT CTTAATGACT
     ATGTGCTTCC
801  CTATGCATCC GTATCTATAG GAAATACTTC AAGAAAAGCT
     CCTTCTGATA
851  GCTTCACAGA ACTCGAAAAG CAATTTACGA ATTTTAAATT
     TAAAATTCGT
901  AAAATCACAA ACTTCGACAG AGTAAACTTC TGCTTCGGAA
     CTACCTGCTG
951  CATCTCAAAT AACTTCTACT ATAGTGTAGA AGGCCGTTGG
     GGATATCAGC
1001 GTGCTATCAA CATTACGTCA GGTCTGCAGT TTTAG

The PSORT algorithm predicts a bacterial outer membrane location (0.936).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product. The purified GST-fusion product is shown in FIG. 34A. The GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (34B) and for FACS analysis (34C).

The cp7170 protein was also identified in the 2D-PAGE experiment (Cpn0854).

These experiments show that cp7170 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 35

The following C. pneumoniae protein (PID 4377072) was expressed <SEQ ID 69; cp7072>:

1   MDIKKLFCLF LCSSLIAMSP IYGKTGDYEK LTLTGINIID
    RNGLSETICS
51  KEKLKKYTKV DFLAPQPYQK VMRMYKNKRG DNVSCLTAYH
    TNGQIKQYLE
101 CLNNRAYGRY REWHVNGNIK IQAEVIGGIA DLHPSAESGW
    LFDQTTFAYN
151 DEGILEAAIV YEKGLLEGSS VYYHTNGNIW KECPYHKGVP
    QGKELTYTSS
201 GKLLKEQNYQ QGKRHGLSIR YSEDSEEDVL AWEEYHEGRL
    LKAEYLDPQT
251 HEIYATIHEG NGIQAIYGKY AVIETRAFYR GEPYGKVTRF
    DNSGTQIVQT
301 YNLLQGAKHG EEFFFYPETG KPKLLLNWHE GILNGIVKTW
    YPGGTLESCK
351 ELVNNKKSGL LTIYYPEGQI MATEEYDNDL LIKGEYFRPG
    DRHPYSKIDR
401 GCGTAVFFSS AGTITKKIPY QDGKPLLN*

A predicted signal peptide is highlighted.

The cp7072 nucleotide sequence <SEQ ID 70> is:

1    ATGGATATAA AAAAACTCTT TTGCTTATTT CTATGTTCTT
     CTCTAATTGC
51   CATGAGTCCC ATTTATGGGA AAACAGGTGA CTATGAGAAA
     CTCACCCTTA
101  CAGGGATCAA TATCATTGAT AGAAACGGCC TGTCAGAAAC
     TATTTGCTCT
151  AAAGAGAAGC TAAAGAAATA CACCAAGGTA GACTTTCTTG
     CTCCCCAGCC
201  CTATCAAAAG GTCATGAGGA TGTATAAAAA CAAACGCGGA
     GATAACGTTT
251  CTTGTTTAAC AGCCTATCAC ACTAACGGGC AAATTAAGCA
     GTACCTGGAG
301  TGTCTCAATA ATCGTGCTTA TGGAAGATAT CTGTAATGGC
     ACGTCAACGG
351  GAATATCAAA ATCCAAGCTG AGGTTATCGG AGGTATTGCG
     GATCTTCATC
401  CCTCAGCAGA GTCTGGCTGG CTATTTGATC AAACTACATT
     TGCCTATAAT
451  GATGAAGGTA TCTTAGAAGC CGCTATCGTC GATGAAAAAG
     GGCTGCTCGA
501  AGGATCTTCG GTGTATTACC ATACTAATGG GAATATTTGG
     AAAGAGTGTC
551  CCTATCATAA GGGAGTTCCT CAAGGTAAAT TCCTGACATA
     CACATCTTCG
601  GGGAAACTGC TCAAAGAACA GAATTACCAA CAAGGCAAAA
     GACACGGTCT
651  TTCGATTCGC TACAGCGAAG ATTCCGAAGA AGATGTTTTA
     GCCTGGGAAG
701  AATATCATGA GGGACGACTC CTAAAAGCAG AGTACTTAGA
     TCCTCAAACT
751  CACGAAATCT ATGCGACTAT ACACGAAGGG AACGGCATTC
     AAGCAATCTA
801  CGGCAAGTAT GCCGTTATAG AAACTAGGGC ATTTTACCGA
     GGGGAACCTT
851  ATGGAAAAGT TACCAGATTC GACAACTCCG GAACACAGAT
     TGTCCAAACG
901  TATAACCTTT TCGAAGGCGC GAAGCACGGA GAAGAATTTT
     TCTTTTATCC
951  TGAGACAGGG AAACCCAAGC TGCTTCTTAA TTGGCATGAA
     GGAATTTTAA
1001 ATGGGATAGT AAAAACTTGG TATCCCGGAG GAACCTTAGA
     AAGTTGTAAA
1051 GAACTCGTAA ATAACAAAAA ATCCGGGTTA CTGACCATTT
     ACTACCCTGA
1101 AGGACAGATC ATGGCGACCG AAGAGTATGA TAATGATCTT
     CTAATTAAAG
1151 GAGAGTACTT CCGCCCTGGA GACCGTCATC CCTACTCTAA
     AATAGATCGT
1201 GGTTGTGGGA CTGCAGTATT TTTCTCGTCG GCGGGAACTA
     TTACTAAAAA
1251 AATCCCCTAT CAGGACGGCA AACCTTTGCT CAACTAG

The PSORT algorithm predicts a periplasmic location (0.688).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 35A) and as a GST-fusion product (FIG. 35B). The recombinant his-tag protein was used to immunise mice, whose sera were used in a Western blot (FIG. 35C) and for FACS analysis.

These experiments show that cp7072 is a useful immunogen. These properties are not evident from the sequence alone.

Example 36

The following C. pneumoniae protein (PID 4376879) was expressed <SEQ ID 71; cp6879>:

1   MATPAQKSPT FQDPSFVREL GSNHPVFSPL TLEERGEMAI
    ARVQQCGWNH
51  TIVKVSLIIL ALLTILGGGL LVGLLPAVPM FIGTGLIALG
    AVIFALALIL
101 CLYDSQGLPE ELPPVPEPQQ IQIEDLRNET REVLEGTLLE
    VLLKDRDAKD
151 PAVPQVVVDC EKRLGMLDRK LRREEEILYR STAELKDEER
    YEFLLELLEM
201 RSLVADRLEF NRRSYERFVQ GIMTVRSEEG EKEISRLQDL
    ISLQQQTVQD
251 LRSRIDDEQK RCWTALQRIN QSQKDIQRAH DREASQRACE
    GTEMDCAERQ
301 QLEKDLRRQL KSMQEWIEMR GTIHQQEKAW RKQNAKLERL
    QEDLRLTGIA
351 FDEQSLFYRE YKEKYLSQKL DMQKILQEVN AEKSEKACLE
    SLVHDYEKQL
401 EQKDANLKKA AAVWEEELGK QQQEDYEQTQ EIRRLSTFIL
    EYQDSLREAE
451 KVEKDFQELQ QRYSRLQEEK QVKEKILEES MNHFADLFEK
    AQKENMAYKK
501 KLADLEGAAA PTEIGEDDDW VLTDSASLSQ KKIRELVEEN
    QELLKALAFK
551 SNELTQLVAD AVEAEKEISK LREHIEEQKE GLRALDKMHA
    QAIKDCEAAQ
601 RKCCDLESLL SPVREDAGMR FELEVELQRL QEENAQLRAE
    VERLEQEQFQ
651 G*

The cp6879 nucleotide sequence <SEQ ID 72> is:

1    ATGGCAACAC CCGCTCAAAA ATCCCCTACA TTTCAAGATC
     CTAGTTTTGT
51   AAGAGAGCTA GGCAGTAACC ACCCTGTCTT TTCCCCGCTA
     ACGCTTGAGG
101  AAAGAGGGGA GATGGCAATA GCTCGAGTCC AGCAGTGTGG
     ATGGAATCAT
151  ACAATTGTTA AGGTAAGTCT TATTATTCTT GCTCTTCTTA
     CTATTTTAGG
201  GGGAGGATTA CTCGTAGGAT TGCTGCCAGC AGTTCCTATG
     TTTATTGGAA
251  CAGGTCTGAT TGCTTTGGGA GCCGTTATAT TTGCTTTGGC
     TTTGATTTTA
301  TGTCTTTATG ATTCTCAGGG CCTTCCTGAG GAACTCCCTC
     CGGTTCCTGA
351  ACCACAACAA ATTCAGATTG AAGATTTAAG AAACGAGACC
     AGAGAAGTTC
401  TTGAAGGGAC TCTTTTAGAG GTTCTCTTAA AGGATAGAGA
     CGCTAAGGAC
451  CCTGCGGTGC CCCAGGTGGT TGTAGACTGT GAAAAGCGTC
     TTGGAATGTT
501  GGATCGTAAG CTGCGACGTG AAGAGGAGAT TCTGTATCGC
     TCGACGGCCC
551  ATCTTAAAGA CGAGGAAAGG TATGAGTTCT TGCTGGAGCT
     CTTGGAAATG
601  CGTAGTCTGG TTGCCGATCG GCTAGAATTT AACCGTAGAA
     GTTATGAGCG
651  ATTTGTTCAA GGAATTATGA CAGTTAGATC AGAGGAGGGG
     GAAAAAGAGA
701  TTTCTCGTCT ACAAGATCTA ATCAGTTTGC AGCAGCAGAC
     GGTGCAAGAT
751  TTAAGGAGTC GGATCGATGA CGAGCAGAAG AGATGCTGGA
     CGGCTTTACA
801  ACGTATTAAC CAATCTCAGA AGGATATACA ACGGGCTCAT
     GATCGCGAGG
851  CTTCGCAGCG TGCCTGTGAG GGCACAGAGA TGGATTGTGC
     AGAACGCCAG
901  CAACTGGAGA AGGATTTAAG GAGACAGCTG AAATCTATGC
     AGGAGTGGAT
951  TGAGATGAGG GGCACAATCC ATCAACAAGA GAAGGCTTGG
     CGTAAGCAGA
1001 ATGCCAAATT AGAAAGATTA CAAGAGGATC TGAGACTTAC
     TGGGATTGCT
1051 TTTGACGAAC AATCTCTGTT CTATCGCGAA TATAAAGAGA
     AATATCTGAG
1101 TCAGAAACTA GATATGCAAA AGATTTTACA GGAAGTCAAC
     GCAGAGAAAA
1151 GTGAGAAGGC TTGCTTAGAG AGTCTGGTCC ATGACTATGA
     GAAGCAGCTC
1201 GAACAAAAAG ATGCTAATCT GAAGAAAGCA GCAGCTGTTT
     GGGAAGAAGA
1251 ATTAGGGAAG CAGCAACAGG AAGACTACGA ACAAACCCAA
     GAAATTAGAC
1301 GTCTGAGTAC ATTCATTCTT GAGTACCAGG ACAGTCTGCG
     TGAGGCAGAA
1351 AAAGTTGAGA AAGATTTCCA AGAGCTACAA CAAAGGTATA
     GCCGTCTTCA
1401 AGAGGAGAAA CAGGTAAAAG AAAAAATCTT AGAAGAAAGT
     ATGAATCATT
1451 TTGCCGATCT CTTTGAGAAG GCTCAAAAGG AAAACATGGC
     CTACAAGAAG
1501 AAGTTAGCGG ATTTAGAGGG TGCCGCTGCT CCTACTGAGA
     TCGGTGAGGA
1551 CGATGACTGG GTACTCACAG ATTCTGCTTC TCTCAGCCAG
     AAGAAGATCC
1601 GCGAACTCGT GGAAGAGAAT CAAGAACTCC TGAAAGCACT
     TGCATTTAAA
1651 TCTAACGAAT TGACTCAACT GGTTGCCGAT GCTGTAGAAG
     CTGAAAAAGA
1701 AATCAGCAAG CTTCGAGAAC ACATAGAAGA GCAGAAAGAA
     GGATTACGAG
1751 CTCTTGATAA GATGCATGCA CAAGCGATCA AAGATTGCGA
     AGCTGCTCAG
1801 AGAAAATGCT GTGACCTTGA GAGCCTTCTC TCTCCTGTTC
     GAGAAGATGC
1851 TGGAATGAGA TTTGAGCTAG AGGTCGAGCT TCAAAGATTG
     CAAGAAGAAA
1901 ATGCACAGCT TAGAGCGGAG GTTGAAAGAC TAGAGCAAGA
     GCAATTTCAA
1951 GGATAA

The PSORT algorithm predicts an inner membrane location (0.646).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product. The purified GST-fusion product is shown in FIG. 36A. The recombinant GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 36B) and for FACS analysis.

These experiments show that cp6879 is useful immunogen. These properties are not evident from the sequence alone.

Example 37

The following C. pneumoniae protein (PID 4376767) was expressed <SEQ ID 73; cp6767>:

1   MIKQIGRFFR AFIFIMPLSL TSCESKIDRN RIWIVGTNAT
    YPPFEYVDAQ
51  GEVVGFDIDL AKAISEKLGK QLEVREFAFD ALILNLKKHR
    IDAILAGMSI
101 TPSRQKEIAL LPYYGDEVQE LMVVSKRSLE TPVLPLTQYS
    SVAVQTGTFQ
151 EHYLLSQPGI CVRSFDSTLE VIMEVRYGKS PVAVLEPSVG
    RVVLKDFPNL
201 VATRLELPPE CWVLGCGLGV AKDRPEEIQT IQQAITDLKS
    EGVIQSLTKK
251 WQLSEVAYE*

The cp6767 nucleotide sequence <SEQ ID 74> is:

1   ATGATAAAAC AAATAGGCCG TTTTTTTAGA GCATTTATTT
    TTATAATGCC
51  TTTATCTTTA ACAAGTTGTG AGTCTAAAAT CGATCGAAAT
    CGCATCTGGA
101 TTGTAGGTAC GAATGCTACA TATCCTCCTT TTGAGTATGT
    GGATGCTCAG
151 GGGGAAGTTG TAGGTTTCGA TATAGATTTG GCAAAGGCAA
    TTAGTGAAAA
201 ACTTGGCAAG CAATTGGAAG TTAGAGAATT CGCTTTCGAT
    GCTTTAATTT
251 TAAATTTAAA AAAACATCGT ATCGATGCAA TTTTAGCAGG
    AATGTCCATT
301 ACTCCTTCGC GTCAGAAGGA AATCGCCCTG CTTCCCTATT
    ATGGCGATGA
351 GGTTCAAGAG CTGATGGTGG TTTCTAAGCG GTCTTTAGAG
    ACCCCTGTGC
401 TTCCCCTAAC ACAGTATTCT TCTGTTGCTG TTCAGACAGG
    AACGTTTCAG
451 GAGCATTATC TTTTATCTCA GCCCGGAATT TGTGTCCGTT
    CTTTTGATAG
501 CACCTTGGAG GTGATTATGG AAGTTCGTTA TGGGAAATCT
    CCGGTTGCCG
551 TTCTAGAACC CTCGGTAGGA CGTGTCGTTC TTAAAGACTT
    CCCTAATCTT
601 GTTGCAACAA GATTAGAGCT CCCTCCTGAA TGTTGGGTGT
    TGGGCTGTGG
651 TCTCGGCGTA GCTAAAGATC GTCCTGAAGA AATACAAACG
    ATTCAACAAG
701 CGATTACAGA TTTAAAGAGC GAAGGGGTGA TTCAATCTTT
    AACCAAGAAA
751 TGGCAACTTT CTGAAGTTGC TTACGAATAG

The PSORT algorithm predicts an inner membrane location (0.083).

The protein was expressed in E. coli and purified as a his-tag product and as a GST-fusion product. The purified his-tag product is shown in FIG. 37A. The recombinant his-tag protein was used to immunise mice, whose sera were used in a Western blot (FIG. 37B) and for FACS analysis (FIG. 37C). The GST-fusion was also used in a Western blot (FIG. 37D).

The cp6767 protein was also identified in the 2D-PAGE experiment and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6767 is a useful immunogen. These properties are not evident from the sequence alone.

Example 38

The following C. pneumoniae protein (PID 4376717) was expressed <SEQ ID 75; cp6717>:

1   MMSRLRFRLA ALGIFFILLV PNSVSAKTIV ASDKEKVGVL
    VYDNSVEAFQ
51  QILDCIDHAN FYVELCPCMT GGRTLKEMVD HLEARMDLVP
    ELCSYIIIQP
101 TFTDAEDQKL LKALKERHPN RFFYVFTGCP PSTSILAPNV
    IEMHIKLSII
151 DGKYCILGGT NFEEFMCTPG DEVPEKVDNP RLFVSGVRRP
    LAFRDQDIML
201 RSTAFGLQLR EEYHKQFAMW DYYAHHMWFI DNPEQFAGAC
    PPLTLEQAEE
251 TVFPGFDKHE DLVLVDSSKI RIVLGGPHDK QPNPVTQEYL
    KLIQGARSSV
301 KLAHMYFIPK DELLNALVDV SHNHGVHLSL ITNGCHELSP
    AITGPYAWGN
351 RINYFALLYG KRYPIWKKWF CEKLKPYERV SIYEFAIWET
    QLHKKCMIID
401 DEIFVIGSYN FGKKSDAFDY ESIVVIESPE VAAKANKVFN
    KDIGLSIPVS
451 HGDIFSWYFH SVHHTLGHLQ LTYMPA*

A predicted signal peptide is highlighted.

The cp6717 nucleotide sequence <SEQ ID 76> is:

1    ATGATGAGTC GGTTGCGTTT TCGCTTGGCA GCTCTTGGAA
     TATTTTTTAT
51   TTTGCTGGTT CCTAATTCTG TTTCAGCAAA GACAATCGTA
     GCTTCAGACA
101  AGGAGAAGGT TGGAGTTCTT GTTTATGACA ATAGTGTAGA
     GGCCTTTCAA
151  CAGATATTGG ATTGCATAGA TCATGCAAAT TTTTATGTAG
     AACTGTGTCC
201  CTGCATGACA GGAGGCCGAA CGCTTAAAGA GATGGTAGAT
     CACCTCGAGG
251  CTCGTATGGA TCTGGTTCCA GAGCTCTGTA GCTATATCAT
     TATCCAACCC
301  ACGTTTACCG ATGCTGAAGA CCAAAAATTA CTCAAAGCTC
     TCAAAGAACG
351  TCATCCCAAC CGGTTTTTCT ACGTTTTTAC AGGGTGCCCA
     CCCTCAACAA
401  GCATCCTCGC TCCTAATGTC ATTGAAATGC ATATCAAACT
     TTCTATCATC
451  GATGGGAAAT ATTGTATTTT AGGTGGTACC AATTTTGAAG
     AGTTTATGTG
501  CACTCCAGGG GATGAGGTTC CTGAGAAAGT GGATAACCCA
     CGTTTATTTG
551  TCAGTGGAGT GCGTCGGCCC CTAGCATTTC GTGATCAGGA
     TATCATGTTG
601  CGTTCTACAG CATTCGGTTT GCAGCTCAGA GAAGAATATC
     ATAAGCAATT
651  TGCTATGTGG GACTACTATG CACATCATAT GTGGTTCATT
     GATAATCCTG
701  AACAGTTTGC AGGCGCCTGT CCTCCACTGA CTTTAGAACA
     AGCCGAGGAG
751  ACAGTATTTC CTGGATTTGA CAAACATGAA GATCTTGTTC
     TTGTCGACTC
801  TTCCAAGATC AGGATAGTTT TAGGTGGTCC CCACGATAAG
     CAACCCAATC
851  CTGTGACTCA AGAATATTTG AAACTTATCC AGGGAGCTAG
     ATCTTCTGTG
901  AAGCTTGCTC ACATGTATTT CATCCCTAAG GACGAGCTTT
     TAAATGCTCT
951  TGTCGACGTT TCTCATAATC ACGGTGTTCA TCTGAGTTTA
     ATTACGAACG
1001 GCTGTCATGA ATTAAGTCCT GCAATTACAG GACCCTATGC
     TTGGGGAAAC
1051 CGTATTAACT ATTTCGCCTT GCTCTATGGG AAACGGTATC
     CTCTTTGGAA
1101 AAAATGGTTT TGCGAAAAGC TAAAACCTTA TGAGCGGGTT
     TCTATTTATG
1151 AGTTTGCTAT TTGGGAAACG CAGTTGCACA AGAAGTGTAT
     GATTATCGAT
1201 GATGAAATTT TTGTGATCGG AAGTTATAAT TTTGGAAAGA
     AAAGTGATGC
1251 CTTTGATTAC GAAAGTATTG TAGTTATCGA ATCTCCAGAA
     GTCGCTGCAA
1301 AAGCTAACAA AGTCTTCAAT AAAGATATCG GATTGTCGAT
     TCCTGTAAGT
1351 CATGGCGACA TTTTCTCTTG GTATTTCCAT TCCGTACACC
     ACACTTTGGG
1401 ACATTTGCAG CTGACCTATA TGCCAGCCTA G

The PSORT algorithm predicts a periplasmic location (0.939).

The protein was expressed in E. coli and purified as a GST-fusion (FIG. 38A), as a his-tagged protein, and as a GST/his fusion product. The proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 38B) and for FACS analysis.

These experiments show that cp6717 is a useful immunogen. These properties are not evident from the sequence alone.

Example 39

The following C. pneumoniae protein (PID 4376577) was expressed <SEQ ID 77; cp6577>:

1   MKKLLFSTFL LVLGSTSAAH ANLGYVNLKR CLEESDLGKK
    ETEELEAMKQ
51  QFVKNAEKIE EELTSIYNKL QDEDYMESLS DSASEELRKK
    FEDLSGEYNA
101 YQSQYYQSIN QSNVKRIQKL IQEVKIAAES VRSKEKLEAI
    LNEEAVLAIA
151 PGTDKTTEII AILNESFKKQ N*

A predicted signal peptide is highlighted.

The cp6577 nucleotide sequence <SEQ ID 78> is:

1   ATGAAAAAAT TATTATTTTC TACATTTCTT CTTGTTTTAG
    GATCAACAAG
51  CGCAGCTCAT GCAAATTTAG GCTATGTTAA TTTAAAGCGA
    TGTCTTGAAG
101 AATCCGATCT AGGTAAAAAG GAAACTGAAG AATTGGAAGC
    TATGAAACAG
151 CAGTTTGTAA AAAATGCTGA GAAAATAGAA GAAGAACTCA
    CTTCTATTTA
201 TAATAAGTTG CAAGATGAAG ATTACATGGA AAGCCTATCG
    GATTCTGCCT
251 CTGAAGAGTT GCGAAAGAAA TTCGAAGATC TTTCAGGAGA
    GTACAATGCG
301 TACCAGTCTC AGTACTATCA ATCTATCAAT CAAAGTAATG
    TAAAACGCAT
351 TCAAAAACTC ATCCAAGAAG TAAAAATAGC TGCAGAATCA
    GTGCGGTCCA
401 AAGAAAAACT AGAAGCTATC CTTAATGAAG AAGCTGTCTT
    AGCAATAGCA
451 CCTGGGACTG ATAAAACAAC CGAAATTATT GCTATTCTTA
    ACGAATCTTT
501 CAAAAAACAA AACTAG

The PSORT algorithm predicts a periplasmic space location (0.932).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 39A) and as a GST-fusion product (FIG. 39B). The recombinant GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 39C) and for FACS analysis.

The cp6577 protein was also identified in the 2D-PAGE experiment.

These experiments show that cp6577 is a useful immunogen. These properties are not evident from the sequence alone.

Example 40

The following C. pneumoniae protein (PID 4376446) was expressed <SEQ ID 79; cp6446>:

1   MKQPMSLIFS SVCLGLGLGS LSSCNQKPSW NYHNTSTSEE
    FFVHGNKSVS
51  QLPHYPSAFR TTQIFSEEHN DPYVVAKTDE ESRKIWREIH
    KNLKIKGSYI
101 PISTYGSLMH PKSAALTLKT YRPHPIWING YERSFNIDTG
    KYLKNGSRRR
151 TSHDGPKNRA VLNLIKSSGR RCNAIGLEMT EEDFVIARRR
    EGVYSLYPVE
201 VCSYPQGNPF VIAYAWIADE SACSKEVLPV KGYYSLVWES
    VSSSDSLNAF
251 GDSFAEDYLR STFLANGTSI LCVHESYKKV PPQP*

A predicted signal peptide is highlighted.

The cp6446 nucleotide sequence <SEQ ID 80> is:

1   ATGAAACAGC CCATGTCTCT TATCTTTTCA AGTGTATGTT
    TAGGATTAGG
51  TCTTGGATCT CTTTCCTCCT GTAATCAAAA GCCCTCTTGG
    AATTATCACA
101 ACACTTCAAC GAGCGAAGAA TTCTTTGTTC ATGGAAATAA
    GAGTGTTTCG
151 CAACTGCCTC ATTATCCTTC TGCATTTCGT ACGACTCAAA
    TCTTTTCTGA
201 AGAGCACAAT GATCCTTATG TCGTAGCTAA GACTGATGAA
    GAGTCTCGTA
251 AAATTTGGAG AGAAATCCAT AAAAATCTCA AAATCAAAGG
    TTCTTACATT
301 CCCATATCGA CTTATGGAAG TCTGATGCAC CCAAAATCAG
    CAGCTCTTAC
351 ATTAAAAACG TATCGTCCAC ATCCTATTTG GATAAATGGA
    TACGAGCGTT
401 CTTTTAATAT AGACACAGGA AAGTACTTAA AAAACGGAAG
    TCGCCGTAGA
451 ACTTCTCACG ATGGTCCGAA AAATCGAGCT GTACTGAATC
    TCATTAAATC
501 TTCGGGACGA CGCTGTAATG CTATAGGCCT TGAGATGACA
    GAAGAAGACT
551 TTGTAATAGC TAGAAGGCGA GAAGGTGTTT ATAGCCTGTA
    TCCCGTTGAA
601 GTGTGCTCGT ATCCTCAGGG GAATCCTTTT GTCATTGCTT
    ATGCCTGGAT
651 TGCAGATGAG AGTGCTTGCT CAAAAGAGGT CCTACCTGTA
    AAAGGGTACT
701 ATTCTTTAGT CTGGGAAAGC GTTTCTTCCT CTGATTCTCT
    GAATGCTTTT
751 GGAGATTCCT TTGCAGAGGA CTACCTCAGA AGCACGTTTT
    TAGCAAACGG
801 AACTTCTATA CTCTGTGTTC ATGAAAGCTA TAAGAAAGTT
    CCTCCTCAGC
851 CCTAA

The PSORT algorithm predicts an inner membrane location (0.177).

The protein was expressed in E. coli and purified as a his-tag product and a GST-fusion product. The GST-fusion product is shown in FIG. 40A. The recombinant his-tag protein was used to immunise mice, whose sera were used in a Western blot (FIG. 40B) and for FACS analysis.

These experiments show that cp6446 is a useful immunogen. These properties are not evident from the sequence alone.

Example 41

The following C. pneumoniae protein (PID 4377108) was expressed <SEQ ID 81; cp7108>:

1   MSKKIKVLGH LTLCTLFRGV LCAAALSNIG YASTSQESPY
    QKSIEDWKGY
51  TFTDLELLSK EGWSEAHAVS GNGSRIVGAS GAGQGSVTAV
    IWESHLIKHL
101 GTLGGEASSA EGISKDGEVV VGWSDTREGY THAFVFDGRD
    MKDLGTLGAT
151 YSVARGVSGD GSIIVGVSAT ARGEDYGWQV GVKWEKGKIK
    QLKLLPQGLW
201 SEANAISEDG TVIVGRGEIS RNHIVAVKWN KNAVYSLGTL
    GGSVASAEAI
251 SANGKVIVGW STTNNGETHA FMHKDETMHD LGTLGGGFSV
    ATGVSADGRA
301 IVGFSAVKTG EIHAFYYAEG EMEDLTTLGG EEARVFDISS
    EGNDIIGSIK
351 TDAGAERAYL FHIHK*

A predicted signal peptide is highlighted.

The cp7108 nucleotide sequence <SEQ ID 82> is:

1    ATGAGTAAGA AGATAAAGGT TCTAGGTCAT TTGACGCTCT
     GCACTCTGTT
51   TAGAGGAGTG CTGTGTGCAG CGGCCCTTTC CAACATAGGA
     TATGCGAGTA
101  CTTCTCAGGA ATCACCATAT CAGAAGTCTA TAGAAGACTG
     GAAAGGGTAT
151  ACCTTTACAG ATCTTGAGTT ACTGAGTAAG GAAGGGTGGT
     CTGAAGCTCA
201  TGCAGTTTCT GGAAATGGCA GTAGAATTGT AGGAGCTTCG
     GGAGCTGGCC
251  AAGGTAGTGT GACTGCTGTC ATATGGGAAA GTCACCTGAT
     AAAACATCTC
301  GGCACTTTAG GTGGCGAGGC TTCATCTGCA GAGGGAATTT
     CAAAGGATGG
351  AGAGGTGGTC GTTGGGTGGT CAGATACTAG AGAGGGATAT
     ACTCATGCCT
401  TTGTCTTCGA CGGTAGAGAT ATGAAAGATC TCGGTACTCT
     AGGAGCTACC
451  TATTCTGTAG CAAGGGGTGT TTCTGGAGAT GGTAGTATCA
     TCGTAGGAGT
501  CTCTGCAACT GCTCGTGGAG AGGATTACGG ATGGCAAGTT
     GGTGTCAAGT
551  GGGAAAAAGG GAAAATCAAA CAATTGAAGT TGTTGCCTCA
     AGGTCTCTGG
601  TCTGAGGCGA ATGCAATCTC TGAGGATGGT ACGGTGATTG
     TCGGGAGAGG
651  GGAAATCTCT CGCAATCACA TCGTTGCTGT AAAATGGAAT
     AAAAATGCTG
701  TGTATAGTTT GGGGACTCTC GGAGGTAGTG TCGCTTCAGC
     AGAGGCTATA
751  TCGGCAAATG GGAAAGTAAT TGTAGGATGG TCCACGACTA
     ATAATGGTGA
801  GACTCATGCC TTTATGCACA AAGATGAGAC AATGCACGAT
     CTCGGCACTC
851  TAGGAGGAGG TTTTTCTGTC GCAACTGGAG TTTCTGCTGA
     TGGGAGAGCC
901  ATCGTAGGAT TTTCAGCAGT GAAGACCGGA GAAATTCATG
     CTTTTTACTA
951  TGCAGAAGGA GAAATGGAGG ATTTAACAAC TTTGGGAGGG
     GAAGAAGCTC
1001 GAGTGTTCGA CATATCTAGC GAAGGAAACG ATATCATTGG
     CTCTATAAAA
1051 ACTGACGCTG GAGCTGAACG CGCCTATCTG TTCCATATAC
     ATAAATAA

The PSORT algorithm predicts an outer membrane location (0.921).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 41A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 41B) and for FACS analysis (FIG. 41C). A his-tagged protein was also expressed.

The cp7108 protein was also identified in the 2D-PAGE experiment.

These experiments show that cp7108 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 42

The following C. pneumoniae protein (PID 4377287) was expressed <SEQ ID 83; cp7287>:

1    MVAKKTVRSY RSSFSHSVIV AILSAGAIFE AHSLHSSELD
     LGVFNKQFEE
51   HSAHVEEAQT SVLKGSDPVN PSQKESEKVL YTQVPLTQGS
     SGESLDLADA
101  NFLEHFQHLF EETTVFGIDQ KLVWSDLDTR NFSQPTQEPD
     TSNAVSEKIS
151  SDTKENRKDL ETEDPSKKSG LDEVSSDLPK SPETAVAAIS
     EDLEISENIS
201  ARDPLQGLAF FYKNTSSQSI SEKDSSFQGI IFSGSGANSG
     LGFENLKAPK
251  SGAAVYSDRD IVFENLVKGL SFISCESLED GSAAGVNIVV
     THCGDVTLTD
301  CATGLDLEAL RLVKDFSRGG AVFTARNHEV QNNLAGGILS
     VVGNKGAIVV
351  EKNSAEKSNG GAFACGSFVY SNNENTALWK ENQALSGGAI
     SSASDIDIQG
401  NCSAIEFSGN QSLIALGEHI GLTDFVGGGA LAAQGTLTLR
     NNAVVQCVKN
451  TSKTHGGAIL AGTVDLNETI SEVAFKQNTA ALTGGALSAN
     DKVIIANNFG
501  EILFEQNEVR NHGGAIYCGC RSNPKLEQKD SGENINIIGN
     SGAITFLKNK
551  ASVLEVMTQA EDYAGGGALW GHNVLLDSNS GNIQFIGNIG
     GSTFWIGEYV
601  GGGAILSTDR VTISNNSGDV VFKGNKGQCL AQKYVAPQET
     APVESDASST
651  NKDEDSLNAC SHGDHYPPKT VEEEVPPSLL EEHPVVSSTD
     IRGGGAILAQ
701  HIFITDNTGN LRFSGNLGGG EESSTVGDLA IVGGGALLST
     NEVNVCSNQN
751  VVFSDNVTSN GCDSGGAILA KKVDISANHS VEFVSNGSGK
     FGGAVCALNE
801  SVNITDNGSA VSFSKNRTRL GGAGVAAPQG SVTICGNQGN
     IAFKENFVFG
851  SENQRSGGGA IIANSSVNIQ DNAGDILFVS NSTGSYGGAI
     FVGSLVASEG
901  SNPRTLTITG NSGDILFAKN STQTAASLSE KDSFGGGAIY
     TQNLKIVKNA
951  GNVSFYGNRA PSGAGVQIAD GSTVCLEAFG GDILFEGNIN
     FDGSFNAIHL
1001 CGNDSKIVEL SAVQDKNIIF QDAITYEENT IRGLPDKDVS
     PLSAPSLIFN
1051 SKPQDDSAQH HEGTIRFSRG VSKIPQIAAI QEGTLALSQN
     AELWLAGLKQ
1101 ETGSSIVLSA GLILRIFDSQ VDSSAPLPTE NKEETLVSAG
     VQINMSSPTP
1151 NKDKAVDTPV LADIISITVD LSSFVPEQDG TLPLPPEIII
     PKGTKLHSNA
1201 IDLKIIDPTN VGYENHALLS SHKDIPLISL KTAEGMTGTP
     TADASLSNIK
1251 IDVSLPSITP ATYGHTGVWS ESKMEDGRLV VGWQPTGYKL
     NPEKQGALVL
1301 NNLWSHYTDL RALKQEIFAH HTIAQRMELD FSTNVWGSGL
     GVVEDCQNIG
1351 EFDGFKHHLT GYALGLDTQL VEDFLIGGCF SQFFGKTESQ
     SYKAKNDVKS
1401 YMGAAYAGIL AGPWLIKGAF VYGNINNDLT TDYGTLGIST
     GSWIGKGFIA
1451 GTSIDYRYIV NPRRFISAIV STVVPFVEAE YVRIDLPEIS
     EQGKEVRTFQ
1501 KTRFENVAIP FGFAIEHAYS RGSRAEVNSV QLAYVFDVYR
     KGPVSLITLK
1551 DAAYSWKSYG VDIPCKAWKA RLSNNTEWNS YLSTYLAFNY
     EWREDLIAYD
1601 FNGGIRIIF*

A predicted signal peptide is highlighted.

The cp7287 nucleotide sequence <SEQ ID 84> is:

1    ATGGTAGCGA AAAAAACAGT ACGATCTTAT AGGTCTTCAT
     TTTCTCATTC
51   CGTAATAGTA GCAATATTGT CAGCAGGCAT TGCTTTTGAA
     GCACATTCCT
101  TACACAGCTC AGAACTAGAT TTAGGTGTAT TCAATAAACA
     GTTTGAGGAA
151  CATTCTGCTC ATGTTGAAGA GGCTCAAACA TCTGTTTTAA
     AGGGATCAGA
201  TCCTGTAAAT CCCTCTCAGA AAGAATCCGA GAAGGTTTTG
     TACACTCAAG
251  TGCCTCTTAC CCAAGGAAGC TCTGGAGAGA GTTTGGATCT
     CGCCGATGCT
301  AATTTCTTAG AGCATTTTCA GCATCTTTTT GAAGAGACTA
     CAGTATTTGG
351  TATCGATCAA AAGCTGGTTT GGTCAGATTT AGATACTAGG
     AATTTTTCCC
401  AACCCACTCA AGAACCTGAT ACAAGTAATG CTGTAAGTGA
     GAAAATCTCC
451  TCAGATACCA AAGAGAATAG AAAAGACCTA GAGACTGAAG
     ATCCTTCAAA
501  AAAAAGTGGC CTTAAAGAAG TTTCATCAGA TCTCCCTAAA
     AGTCCTGAAA
551  CTGCAGTAGC AGCTATTTCT GAAGATCTTG AAATCTCAGA
     AAACATTTCA
601  GCAAGAGATC CTCTTCAGGG TTTAGCATTT TTTTATAAAA
     ATACATCTTC
651  TCAGTCTATC TCTGAAAAGG ATTCTTCATT TCAAGGAATT
     ATCTTTTCTG
701  GTTCAGGAGC TAATTCAGGG CTAGGTTTTG AAAATCTTAA
     GGCGCCGAAA
751  TCTGGGGCTG CAGTTTATTC TGATCGAGAT ATTGTTTTTG
     AAAATCTTGT
801  TAAAGGATTG AGTTTTATAT CTTGTGAATC TTTAGAAGAT
     GGCTCTGCCG
851  CAGGTGTAAA CATTGTTGTG ACCCATTGTG GTGATGTAAC
     TCTCACTGAT
901  TGTGCCACTG GTTTAGACCT TGAAGCTTTA CGTCTGGTTA
     AAGATTTTTC
951  TCGTGGAGGA GCTGTTTTCA CTGCTCGCAA CCATGAAGTG
     CAAAATAACC
1001 TTGCAGGTGG AATTCTATCC GTTGTAGGCA ATAAAGGAGC
     TATTGTTGTA
1051 GAGAAAAATA GTGCTGAGAA GTCCAATGGA GGAGCTTTTG
     CTTGCGGAAG
1101 TTTTGTTTAC AGTAACAACG AAAACACCGC CTTGTGGAAA
     GAAAATCAAG
1151 CATTATCAGG AGGAGCCATA TCCTCAGCAA GTGATATTGA
     TATTCAAGGG
1201 AACTGTAGCG CTATTGAATT TTCAGGAAAC CAGTCTCTAA
     TTGCTCTTGG
1251 AGAGCATATA GGGCTTACAG ATTTTGTAGG TGGAGGAGCT
     TTAGCTGCTC
1301 AAGGGACGCT TACCTTAAGA AATAATGCAG TAGTGCAATG
     TGTTAAAAAC
1351 ACTTCTAAAA CACATGGTGG AGCTATTTTA GCAGGTACTG
     TTGATCTCAA
1401 CGAAACAATT AGCGAAGTTG CCTTTAAGCA GAATACAGCA
     GCTCTAACTG
1451 GAGGTGCTTT AAGTGCAAAT GATAAGGTTA TAATTGCAAA
     TAACTTTGGA
1501 GAAATTCTTT TTGAGCAAAA CGAAGTGAGG AATCACGGAG
     GAGCCATTTA
1551 TTGTGGATGT CGATCTAATC CTAAGTTAGA ACAAAAGGAT
     TCTGGAGAGA
1601 ACATCAATAT TATTGGAAAC TCCGGAGCTA TCACTTTTTT
     AAAAAATAAG
1651 GCTTCTGTTT TAGAAGTGAT GACACAAGCT GAAGATTATG
     CTGGTGGAGG
1701 CGCTTTATGG GGGCATAATG TTCTTCTAGA TTCCAATAGT
     GGGAATATTC
1751 AATTTATAGG AAATATAGGT GGAAGTACCT TCTGGATAGG
     AGAATATGTC
1801 GGTGGTGGTG CGATTCTCTC TACTGATAGA GTGACAATTT
     CTAATAACTC
1851 TGGAGATGTT GTTTTTAAAG GAAACAAAGG CCAATGTCTT
     GCTCAAAAAT
1901 ATGTAGCTCC TCAAGAAACA GCTCCCGTGG AATCAGATGC
     TTCATCTACA
1951 AATAAAGACG AGAAGAGCCT TAATGCTTGT AGTCATGGAG
     ATCATTATCC
2001 TCCTAAAACT GTAGAAGAGG AAGTGCCACC TTCATTGTTA
     GAAGAACATC
2051 CTGTTGTTTC TTCGACAGAT ATTCGTGGTG GTGGGGCCAT
     TCTAGCTCAA
2101 CATATCTTTA TTACAGATAA TACAGGAAAT CTGAGATTCT
     CTGGGAACCT
2151 TGGTGGTGGT GAAGAGTCTT CTACTGTCGG TGATTTAGCT
     ATCGTAGGAG
2201 GAGGTGCTTT GCTTTCTACT AATGAAGTTA ATGTTTGCAG
     TAACCAAAAT
2251 GTTGTTTTTT CTGATAACGT GACTTCAAAT GGTTGTGATT
     CAGGGGGAGC
2301 TATTTTAGCT AAAAAAGTAG ATATCTCCGC GAACCACTCG
     GTTGAATTTG
2351 TCTCTAATGG TTCAGGGAAA TTCGGTGGTG CCGTTTGCGC
     TTTAAACGAA
2401 TCAGTAAACA TTACGGACAA TGGCTCGGCA GTATCATTCT
     CTAAAAATAG
2451 AACACGTCTT GGCGGTGCTG GAGTTGCAGC TCCTCAAGGC
     TCTGTAACGA
2501 TTTGTGGAAA TCAGGGAAAC ATAGCATTTA AAGAGAACTT
     TGTTTTTGGC
2551 TCTGAAAATC AAAGATCAGG TGGAGGAGCT ATCATTGCTA
     ACTCTTCTGT
2601 AAATATTCAG GATAACGCAG GAGATATCCT ATTTGTAAGT
     AACTCTACGG
2651 GATCTTATGG AGGTGCTATT TTTGTAGGAT CTTTGGTTGC
     TTCTGAAGGC
2701 AGCAACCCAC GAACGCTTAC AATTACAGGC AACAGTGGGG
     ATATCCTATT
2751 TGCTAAAAAT AGCACGCAAA CAGCCGCTTC TTTATCAGAA
     AAAGATTCCT
2801 TTGGTGGAGG GGCCATCTAT ACACAAAACC TCAAAATTGT
     AAAGAATGCA
2851 GGGAACGTTT CTTTCTATGG CAACAGAGCT CCTAGTGGTG
     CTGGTGTCCA
2901 AATTGCAGAC GGAGGAACTG TTTGTTTAGA GGCTTTTGGA
     GGAGATATCT
2951 TATTTGAAGG GAATATCAAT TTTGATGGGA GTTTCAATGC
     GATTCACTTA
3001 TGCGGGAATG ACTCAAAAAT CGTAGAGCTT TCTGCTGTTC
     AAGATAAAAA
3051 TATTATTTTC CAAGATGCAA TTACTTATGA AGAGAACACA
     ATTCGTGGCT
3101 TGCCAGATAA AGATGTCAGT CCTTTAAGTG CCCCTTCATT
     AATTTTTAAC
3151 TCCAAGCCAC AAGATGACAG CGCTCAACAT CATGAAGGGA
     CGATACGGTT
3201 TTCTCGAGGG GTATCTAAAA TTCCTCAGAT TGCTGCTATA
     CAAGAGGGAA
3251 CCTTAGCTTT ATCACAAAAC GCAGAGCTTT GGTTGGCAGG
     ACTTAAACAG
3301 GAAACAGGAA GTTCTATCGT ATTGTCTGCG GGATCTATTC
     TCCGTATTTT
3351 TGATTCCCAG GTTGATAGCA GTGCGCCTCT TCCTACAGAA
     AATAAAGAGG
3401 AGACTCTTGT TTCTGCCGGA GTTCAAATTA ACATGAGCTC
     TCCTACACCC
3451 AATAAAGATA AAGCTGTAGA TACTCCAGTA CTTGCAGATA
     TCATAAGTAT
3501 TACTGTAGAT TTGTCTTCAT TTGTTCCTGA GCAAGACGGA
     ACTCTTCCTC
3551 TTCCTCCTGA AATTATCATT CCTAAGGGAA CAAAATTACA
     TTCTAATGCC
3601 ATAGATCTTA AGATTATAGA TCCTACCAAT GTGGGATATG
     AAAATCATGC
3651 TCTTCTAAGT TCTCATAAAG ATATTCCATT AATTTCTCTT
     AAGACAGCGG
3701 AAGGAATGAC AGGGACGCCT ACAGCAGATG CTTCTCTATC
     TAATATAAAA
3751 ATAGATGTAT CTTTACCTTC GATCACACCA GCAACGTATG
     GTCACACAGG
3801 AGTTTGGTCT GAAAGTAAAA TGGAAGATGG AAGACTTGTA
     GTCGGTTGGC
3851 AACCTACGGG ATATAAGTTA AATCCTGAGA AGCAAGGGGC
     TCTAGTTTTG
3901 AATAATCTCT GGAGTCATTA TACAGATCTT AGAGCTCTTA
     AGCAGGAGAT
3951 CTTTGCTCAT CATACGATAG CTCAAAGAAT GGAGTTAGAT
     TTCTCGACAA
4001 ATGTCTGGGG ATCAGGATTA GGTGTTGTTG AAGATTGTCA
     GAACATCGGA
4051 GAGTTTGATG GGTTCAAACA TCATCTCACA GGGTATGCCC
     TAGGCTTGGA
4101 TACACAACTA GTTGAAGACT TCTTAATTGG AGGATGTTTC
     TCACAGTTCT
4151 TTGGTAAAAC TGAAAGCCAA TCCTACAAAG CTAAGAACGA
     TGTGAAGAGT
4201 TATATGGGAG CTGCTTATGC GGGGATTTTA GCAGGTCCTT
     GGTTAATAAA
4251 AGGAGCTTTT GTTTACGGTA ATATAAACAA CGATTTGACT
     ACAGATTACG
4301 GTACTTTAGG TATTTCAACA GGTTCATGGA TAGGAAAAGG
     GTTTATCGCA
4351 GGCACAAGCA TTGATTACCG CTATATTGTA AATCCTCGAC
     GGTTTATATC
4401 GGCAATCGTA TCCACAGTGG TTCCTTTTGT AGAAGCCGAG
     TATGTCCGTA
4451 TAGATCTTCC AGAAATTAGC GAACAGGGTA AAGAGGTTAG
     AACGTTCCAA
4501 AAAACTCGTT TTGAGAATGT CGCCATTCCT TTTGGATTTG
     CTTTAGAACA
4551 TGCTTATTCG CGTGGCTCAC GTGCTGAAGT GAACAGTGTA
     CAGCTTGCTT
4601 ACGTCTTTGA TGTATATCGT AAGGGACCTG TCTCTTTGAT
     TACACTCAAG
4651 GATGCTGCTT ATTCTTGGAA GAGTTATGGG GTAGATATTC
     CTTGTAAAGC
4701 TTGGAAGGCT CGCTTGAGCA ATAATACGGA ATGGAATTCA
     TATTTAAGTA
4751 CGTATTTAGC GTTTAATTAT GAATGGAGAG AAGATCTGAT
     AGCTTATGAC
4801 TTCAATGGTG GTATCCGTAT TATTTTCTAG

The PSORT algorithm predicts an inner membrane location (0.106).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 42A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 42B) and for FACS analysis (FIG. 42C). A his-tagged protein was also expressed.

The cp7287 protein was also identified in the 2D-PAGE experiment and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp7287 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 43

The following C. pneumoniae protein (PID 4377105) was expressed <SEQ ID 85; cp7105>:

1   MSLYQKWWNS QLKKSLCYST VAALIFMIPS QESFADSLID
    LNLGLDPSVE
51  CLSGDGAFSV GYFTKAGSTP VEYQPFKYDV SKKTFTILSV
    ETANQSGYAY
101 GISYDGTITV GTCSLGAGKY NSAKWSADGT LTPLTGITGG
    TSHTEARAIS
151 KDTQVIEGFS YDASGQPKAV QWASGATTVT QLADISGGSR
    SSYAYAISDD
201 GTIIVGSMES TITRKTTAVK WVNNVPTYLG TLGGDASTGL
    YISGDGTVIV
251 GAANTATVTN GNQESHAYMY KDNQMKD*

The cp7105 nucleotide sequence <SEQ ID 86> is:

1   GTGAGTCTAT ATCAAAAATG GTGGAACAGT CAGTTAAAGA
    AGAGCCTCTG
51  CTATTCGACT GTTGCTGCTC TAATATTTAT GATTCCTTCT
    CAAGAATCCT
101 TTGCAGATAG TCTTATAGAT TTAAATTTAG GTTTAGATCC
    TTCGGTCGAA
151 TGTCTGTCAG GAGATGGTGC ATTTTCTGTT GGGTATTTTA
    CTAAGGCGGG
201 ATCGACTCCC GTAGAATATC AGCCGTTTAA ATACGACGTA
    TCTAAGAAGA
251 CATTCACAAT CCTTTCCGTA GAAACGGCAA ATCAGAGCGG
    CTATGCTTAC
301 GGAATCTCCT ACGATGGCAC GATCACTGTA GGAACGTGTA
    GCCTAGGTGC
351 AGGAAAATAT AACGGCGCAA AATGGAGTGC GGATGGCACT
    TTAACACCCT
401 TAACTGGAAT CACGGGGGGG ACGTCACATA CGGAAGCGCG
    TGCGATTTCT
451 AAGGATACTC AGGTGATCGA GGGTTTCTCA TATGATGCTT
    CAGGGCAACC
501 CAAGGCTGTG CAGTGGGCAA GCGGAGCGAC TACAGTAACA
    CAATTAGCAG
551 ATATTTCAGG AGGCTCTAGA AGCTCTTATG CGTATGCTAT
    ATCTGATGAT
601 GGCACGATTA TTGTTGGGTC TATGGAGAGC ACGATAACAA
    GGAAAACTAC
651 AGCTGTAAAA TGGGTAAATA ATGTTCCTAC GTATCTGGGA
    ACCTTAGGAG
701 GAGATGCTTC TACAGGTCTT TATATTTCTG GAGACGGCAC
    CGTGATTGTA
751 GGTGCGGCAA ATACAGCAAC TGTAACCAAT GGGAATCAGG
    AATCCCACGC
801 CTATATGTAT AAAGATAACC AAATGAAAGA TTGA

The PSORT algorithm predicts an inner membrane location (0.100).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 43A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 43B) and for FACS analysis (FIG. 43C). A his-tagged protein was also expressed.

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp7105 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 44

The following C. pneumoniae protein (PID 4376802) was expressed <SEQ ID 87; cp6802>:

1   MSNQLQPCIS LGCVSYINSF PLSLQLIKRN DIRCVLAPPA
    DLLNLLIEGK
51  LDVALTSSLG AISHNLGYVP GFGIAANQRI LSVNLYAAPT
    FFNSPQPRIA
101 ATLFSRSSIG LLKVLCRHLW RIPTPHILRF ITTKVLRQTP
    ENYDGLLLIG
151 DAALQHPVLP GFVTYDLASG WYDLTKLPFV FALLLHSTSW
    KEHPLPNLAM
201 EEALQQFESS PEEVLKEAHQ HTGLPPSLLQ EYYALCQYRL
    GEEHYESFEK
251 FREYYGTLYQ QARL*

A predicted signal peptide is highlighted.

The cp6802 nucleotide sequence <SEQ ID 88> is:

1   ATGTCTAACC AACTCCAGCC ATGTATAAGC TTAGGCTGCG
    TAAGTTATAT
51  TAATTCCTTT CCGCTGTCCC TACAACTCAT AAAAAGAAAC
    GATATTCGCT
101 GTGTTCTTGC TCCCCCTGCA GACCTCCTCA ACTTGCTAAT
    CGAAGGGAAA
151 CTCGATGTTG CTTTGACCTC ATCCCTAGGA GCTATCTCTC
    ATAACTTGGG
201 GTATGTCCCC GGCTTTGGAA TTGCAGCAAA CCAACGTATC
    CTCAGTGTAA
251 ACCTCTATGC AGCTCCCACT TTCTTTAACT CACCGCAACC
    TCGGATTGCC
301 GCAACTTTAG AAAGTCGCTC CTCTATAGGA CTCTTAAAAG
    TGCTTTGTCG
351 TCATCTCTGG CGCATCCCAA CTCCTCATAT CCTAAGATTC
    ATAACTACAA
401 AAGTACTCAG ACAAACCCCT GAAAATTATG ATGGCCTCCT
    CCTAATCGGA
451 GATGCAGCGC TACAACATCC TGTACTTCCT GGATTTGTAA
    CCTATGACCT
501 TGCCTCGGGG TGGTATGATC TTACAAAGCT ACCTTTTGTA
    TTTGCTCTTC
551 TTCTACACAG CACCTCTTGG AAAGAACATC CCCTACCCAA
    CCTTGCGATG
601 GAAGAAGCCC TCCAACAGTT CGAATCTTCA CCCGAAGAAG
    TCCTTAAAGA
651 AGCTCATCAA CATACAGGTC TGCCCCCTTC TCTTCTTCAA
    GAATACTATG
701 CCCTATGCCA GTACCGTCTA GGAGAAGAAC ACTACGAAAG
    CTTTGAAAAA
751 TTCCGGGAAT ATTATGGAAC CCTCTACCAA CAAGCCCGAC
    TGTAA

The PSORT algorithm predicts an inner membrane location (0.060).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 44A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 44B) and for FACS analysis (FIG. 44C). A his-tagged protein was also expressed.

These experiments show that cp6802 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 45

The following C. pneumoniae protein (PID 4376390) was expressed <SEQ ID 89; cp6390>:

1   MVFSYYCMGL FFFSGAISSC GLLVSLGVGL GLSVLGVLLL
    LLAGLLLFKI
51  QSMLREVPKA PDLLDLEDAS ERLRVKASRS LASLPKEISQ
    LESYIRSAAN
101 DLNTIKTWPH KDQRLVETVS RKLERLAAAQ NYMISELCEI
    SEILEEEEHH
151 LILAQESLEW IGKSLFSTFL DMESFLNLSH LSEVRPYLAV
    NDPRLLEITE
201 ESWEVVSHFI NVTSAEKKAQ ILFKNNEHSR MKKKLESVQE
    LLETFIYKSL
251 KRSYRELGCL SEKMRIIHDN PLFPWVQDQQ KYAHAKNEFG
    EIARCLEEFE
301 KTFFWLDEEC AISYMDCWDF LNESIQNKKS RVDRDYISTK
    KIALKDRART
351 YAKVLLEENP TTEGKIDLQD AQRAFERQSQ EFYTLEHTET
    KVRLEALQQC
401 FSDLREATNV RQVRFTNSEN ANDLKESFEK IDKERVRYQK
    EQRLYWETID
451 RNEQELREEI GESLRLQNRR KGYRAGYDAG RLKGLLRQWK
    KNLRDVEAHL
501 EDATMDFEHE VSKSELCSVR ARLEVLEEEL MDMSPKVADI
    EELLSYEERC
551 ILPIRENLER AYLQYNKCSE ILSKAKFFFP EDEQLLVSEA
    NLREVGAQLK
601 QVQGKCQERA QKFAIFEKHI QEQKSLIKEQ VRSFDLAGVG
    FLKSELLSIA
651 CNLYIKAVVK ESIPVDVPCM QLYYSYYEDN EAVVRNRLLN
    MTERYQNFKR
701 SLNSIQFNGD VLLRDPVYQP EGHETRLKER ELQETTLSCK
    KLKVAQDRLS
751 ELESRLSRR

A predicted signal peptide is highlighted.

The cp6390 nucleotide sequence <SEQ ID 90> is:

1    TTGGTATTCT CATACTATTG CATGGGATTA TTTTTTTTCT
     CTGGAGCTAT
51   TTCTAGTTGT GGTCTTTTAG TGTCTCTAGG ACTTGGTTTA
     GGACTTAGTG
101  TTTTAGGAGT ACTTTTACTT CTCTTAGCAG GTCTTTTGCT
     TTTTAAGATC
151  CAAAGTATGC TTCGAGAGGT GCCTAAGGCT CCTGATCTAT
     TAGATTTAGA
201  AGATGCAAGT GAACGGCTTA GAGTAAAGGC TAGCCGTTCT
     TTAGCAAGCC
251  TCCCGAAGGA AATCAGTCAG CTAGAGAGCT ACATTCGTTC
     TGCAGCTAAT
301  GATCTAAATA CAATTAAGAC TTGGCCGCAT AAAGATCAAA
     GACTCGTCGA
351  GACCGTGTCA CGAAAATTAG AGCGTCTGGC AGCTGCTCAA
     AACTATATGA
401  TTTCTGAACT CTGCGAGATT AGTGAGATTC TTGAGGAAGA
     GGAGCATCAT
451  CTAATTTTGG CTCAGGAATC TCTAGAATGG ATAGGTAAGA
     GTCTATTTTC
501  TACCTTTCTG GACATGGAAT CTTTTTTAAA TTTGAGCCAT
     CTATCTGAAG
551  TGCGTCCGTA CTTAGCTGTA AATGATCCTA GATTATTAGA
     AATTACCGAA
601  GAATCTTGGG AAGTAGTGAG TCATTTCATA AATGTAACGT
     CTGCTTTTAA
651  GAAAGCTCAG ATTCTTTTTA AGAACAACGA ACATTCTCGG
     ATGAAGAAGA
701  AGTTAGAAAG TGTTCAAGAG TTACTGGAAA CATTTATTTA
     TAAGAGTTTA
751  AAGAGAAGTT ATCGAGAATT AGGATGCTTA AGTGAAAAGA
     TGAGAATCAT
801  TCACGACAAT CCTCTCTTCC CTTGGGTGCA AGATCAGCAG
     AAGTATGCTC
851  ATGCTAAGAA TGAATTTGGA GAGATTGCGC GGTGTTTAGA
     GGAGTTTGAA
901  AAGACGTTCT TCTGGTTGGA TGAGGAGTGT GCTATTTCTT
     ACATGGACTG
951  TTGGGATTTT CTAAATGAGT CTATTCAGAA TAAGAAGTCC
     AGAGTAGATC
1001 GAGATTATAT ATCCACGAAG AAAATTGCAT TAAAGGATAG
     AGCCCGCACT
1051 TATGCTAAGG TTCTTTTAGA AGAGAATCCG ACTACAGAGG
     GTAAAATAGA
1101 TTTGCAAGAC GCTCAAAGAG CCTTTGAGCG TCAAAGTCAG
     GAGTTTTATA
1151 CACTAGAGCA TACGGAAACA AAGGTGAGAC TAGAAGCACT
     TCAACAGTGC
1201 TTCTCGGATC TTAGGGAGGC GACGAACGTA AGGCAAGTTA
     GGTTTACAAA
1251 TTCTGAAAAT GCGAATGATT TAAAGGAGAG TTTCGAGAAG
     ATAGATAAAG
1301 AGCGTGTGCG ATATCAAAAA GAGCAAAGGC TCTATTGGGA
     AACAATAGAT
1351 CGCAATGAGC AAGAGCTTAG GGAAGAGATT GGGGAGTCGC
     TTCGTTTACA
1401 AAATCGGAGA AAAGGGTATA GGGCTGGATA TGATGCTGGG
     CGTTTAAAAG
1451 GTTTGTTGCG TCAGTGGAAG AAAAATCTCC GCGATGTGGA
     AGCCCACCTT
1501 GAAGATGCAA CTATGGATTT TGAGCATGAA GTAAGCAAGA
     GCGAATTGTG
1551 CAGTGTTCGG GCGAGGCTCG AGGTTCTAGA AGAAGAGCTG
     ATGGATATGT
1601 CTCCTAAAGT TGCGGATATA GAAGAGTTGT TGTCCTATGA
     AGAGCGTTGT
1651 ATTCTTCCTA TTAGGGAAAA TTTAGAAAGG GCATACCTCC
     AATATAATAA
1701 GTGTTCTGAA ATTTTATCCA AGGCAAAGTT CTTCTTTCCG
     GAAGACGAGC
1751 AATTGCTAGT TTCGGAAGCG AATCTAAGAG AGGTGGGTGC
     CCAGTTAAAA
1801 CAAGTACAGG GAAAATGTCA AGAGAGGGCC CAAAAGTTCG
     CAATATTTGA
1851 AAAGCATATT CAGGAGCAGA AAAGCCTTAT TAAAGAGCAA
     GTGCGGAGTT
1901 TTGATCTAGC GGGAGTTGGG TTTTTAAAGA GTGAGCTTCT
     TAGTATTGCT
1951 TGTAACCTTT ATATAAAGGC GGTTGTTAAG GAGTCTATAC
     CAGTTGATGT
2001 GCCTTGTATG CAGTTATATT ATAGTTATTA CGAAGATAAT
     GAAGCTGTAG
2051 TGCGAAACCG CCTTTTAAAT ATGACGGAGA GGTATCAAAA
     TTTTAAAAGG
2101 AGTTTGAATT CCATACAATT TAATGGTGAC GTTCTTTTAC
     GGGATCCGGT
2151 CTATCAACCT GAAGGTCATG AGACCAGGCT AAAGGAACGG
     GAGCTACAAG
2201 AAACAACTTT GTCTTGTAAG AAATTAAAAG TGGCTCAAGA
     TCGTCTTTCT
2251 GAATTAGAGT CAAGGCTGTC TAGGAGATAG

The PSORT algorithm predicts a periplasmic location (0.932).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 45A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 45B) and for FACS analysis (FIG. 45C). A his-tagged protein was also expressed.

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6390 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 46

The following C. pneumoniae protein (PID 4376272) was expressed <SEQ ID 91; cp6272>:

1   MKRCFLFLAS FVLMGSSADA LTHQEAVKKK NSYLSHFKSV
    SGIVTIEDGV
51  LNIHNNLRIQ ANKVYVENTV GQSLKLVAHG NVMVNYRAKT
    LVCDYLEYYE
101 DTDSCLLTNG RFAMYPWFLG GSMITLTPET IVIRKGYIST
    SEGPKKDLCL
151 SGDYLEYSSD SLLSIGKTTL RVCRIPILFL PPFSIMPMEI
    PKPPINFRGG
201 TGGFLGSYLG MSYSPISRKH FSSTFFLDSF FKHGVGMGFN
    LHCSQKQVPE
251 NVFNMKSYYA HRLAIDMAEA HDRYRLHGDF CFTHKHVNFS
    GEYHLSDSWE
301 TVADIFPNNF MLKNTGPTRV DCTWNDNYFE GYLTSSVKVN
    SFQNANQELP
351 YLTLRQYPIS IYNTGVYLEN IVECGYLNFA FSDHIVGENF
    SSLRLAARPK
401 LHKTVPLPIG TLSSTLGSSL IYYSDVPEIS SRHSQLSAKL
    QLDYRFLLHK
451 SYIQRRHIIE PFVTFITETR PLAKNEDHYI FSIQDAFHSL
    NLLKAGIDTS
501 VLSKTNPRFP RIHAKLWTTH ILSNTESKPT FPKTACELSL
    PFGKKNTVSL
551 DAEWIWKKHC WDHMNIRWEW IGNDNVAMTL ESLHRSKYSL
    IKCDRENFIL
601 DVSRPIDQLL DSPLSDHRNL ILGKDFVRPH PCWNYRLSLR
    YGWHRQDTPN
651 YLEYQMILGT KIFEHWQLYG VYERREADSR FFFFLKLDKP
    KKPPF*

A predicted signal peptide is highlighted.

The cp6272 nucleotide sequence <SEQ ID 92> is:

1    ATGAAACGTT GCTTCTTATT TCTAGCTTCC TTTGTTCTTA
     TGGGTTCCTC
51   AGCTGATGCT TTGACTCATC AAGAGGCTGT GAAAAAGAAA
     AACTCCTATC
101  TTAGTCACTT TAAGAGTGTT TCTGGGATTG TGACCATCGA
     AGATGGGGTA
151  TTGAATATCC ATAACAACCT GCGGATACAA GCCAATAAAG
     TGTATGTAGA
201  AAATACTGTG GGTCAAAGCC TGAAGCTTGT CGCACATGGC
     AATGTTATGG
251  TGAACTATAG GGCAAAAACC CTAGTTTGTG ATTACCTAGA
     GTATTACGAA
301  GATACAGACT CTTGTCTTCT TACTAATGGA AGATTCGCGA
     TGTATCCTTG
351  GTTTCTAGGG GGGTCTATGA TCACTCTAAC CCCAGAAACC
     ATAGTCATTC
401  GGAAGGGATA TATCTCTACC TCCGAGGGTC CCAAAAAAGA
     CCTGTGCCTC
451  TCCGGAGATT ACCTGGAATA TTCTTCAGAT AGTCTTCTTT
     CTATAGGGAA
501  GACAACATTA AGGGTGTGTC GCATTCCGAT ACTTTTCTTA
     CCTCCATTTT
551  CTATCATGCC TATGGAGATC CCTAAGCCTC CGATAAACTT
     TCGAGGAGGA
601  ACAGGAGGAT TTCTGGGATC CTATTTGGGG ATGAGCTACT
     CGCCGATTTC
651  TAGGAAGCAT TTCTCCTCGA CATTTTTCTT GGATAGCTTT
     TTCAAGCATG
701  GCGTCGGCAT GGGATTCAAC CTCCATTGTT CTCAGAAGCA
     GGTTCCTGAG
751  AATGTCTTCA ATATGAAAAG CTATTATGCC CACCGCCTTG
     CTATCGATAT
801  GGCAGAAGCT CATGATCGCT ATCGCCTACA CGGAGATTTC
     TGCTTCACGC
851  ATAAGCATGT AAATTTTTCT GGAGAATACC ATCTCAGCGA
     TAGTTGGGAA
901  ACTGTTGCTG ACATTTTCCC CAACAACTTC ATGTTGAAAA
     ATACAGGCCC
951  CACACGTGTC GATTGCACTT GGAATGACAA CTAAAAAGAA
     GGGTATCTCA
1001 CCTCTTCTGT TAAGGTAAAC TCTTTCCAAA ATGCCAACCA
     AGAGCTCCCT
1051 TATTTAACAT TAAGGCAGTA CCCGATTTCT ATTTATAATA
     CGGGAGTGTA
1101 CCTTGAAAAC ATCGTAGAAT GTGGGTATTT AAACTTTGCT
     TTTAGCGATC
1151 ATATCGTTGG CGAGAATTTC TCTTCACTAC GTCTTGCTGC
     GCGCCCTAAG
1201 CTCCATAAAA CTGTGCCTCT ACCTATAGGA ACGCTCTCCT
     CCACCCTAGG
1251 GAGTTCTCTG ATTTACTATA GCGATGTTCC TGAGATCTCC
     TCGCGCCATA
1301 GTCAGCTTTC CGCGAAGCTA CAACTTGATT ATCGCTTTCT
     ATTACATAAG
1351 TCCTACATTC AAAGACGCCA TATTATAGAG CCGTTCGTTA
     CCTTCATTAC
1401 AGAGACTCGT CCTCTAGCTA AGAATGAAGA TCATTATATC
     TTTTCTATTC
1451 AAGATGCCTT TCACTCCTTA AACCTTCTGA AAGCGGGTAT
     AGATACCTCG
1501 GTACTGAGTA AGACTAACCC TCGATTCCCG AGAATCCATG
     CGAAGCTGTG
1551 CAGTACCCAC ATCTTGAGCA ATACAGAAAG CAAACCCACG
     TTTCCCAAAA
1601 CTGCATGCGA GCTATCTCTA CCTTTTGGAA AGAAAAATAC
     AGTCTCCTTA
1651 GATGCTGAAT GGATTTGGAA AAAGCACTGT TGGGATCACA
     TGAACATACG
1701 TTGGGAGTGG ATCGGAAATG ACAATGTGGC TATGACTCTA
     GAATCCCTGC
1751 ATAGAAGCAA ATACAGCCTG ATTAAGTGTG ACAGGGAGAA
     CTTCATTTTA
1801 GATGTCAGCC GTCCCATTGA CCAGCTTTTA GACTCCCCTC
     TCTCTGATCA
1851 TAGGAATCTC ATTTTAGGGA AATTATTTGT ACGACCTCAT
     CCCTGTTGGA
1901 ATTACCGCTT ATCCTTACGC TATGGCTGGC ATCGCCAGGA
     CACTCCGAAC
1951 TACCTAGAAT ACCAGATGAT TCTAGGGACG AAGATCTTCG
     AACATTGGCA
2001 GCTCTATGGG GTGTATGAAC GCCGAGAAGC AGATAGTCGA
     TTTTTCTTCT
2051 TCTTAAAGCT CGACAAACCT AAAAAACCTC CCTTCTAA

The PSORT algorithm predicts an outer membrane location (0.48).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 46A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot and for FACS analysis (FIG. 46B). A his-tagged protein was also expressed.

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6272 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 47

The following C. pneumoniae protein (PID 4377111) was expressed <SEQ ID 93; cp7111>:

1   MFEAVIADIQ AREILDSRGY PTLHVKVTTS TGSVGEARVP
    SGASTGKKEA
51  LEFRDTDSPR YQGKGVLQAV KNVKEILFPL VKGCSVYEQS
    LIDSLMMDSD
101 GSPNKETLGA NAILGVSLAT AHAAAATLRR PLYRYLGGCF
    ACSLPCPMMN
151 LINGGMHADN GLEFQEFMIR PIGASSIKEA VNMGADVFHT
    LKKLLHERGL
201 STGVGDEGGF APNLASNEEA LELLLLAIEK AGFTPGKDIS
    LALDCAASSF
251 YNVKTGTYDG RHYEEQIAIL SNLCDRYPID SIEDGLAEED
    YDGWALLTEV
301 LGEKVQIVGD DLFVTNPELI LEGISNGLAN SVLIKPNQIG
    TLTETVYAIK
351 LAWMAGYTTI ISHRSGETTD TTIADLAVAF NAGQIKTGSL
    SRSERVAKYN
401 RLMEIEEELG SEAIFTDSNV FSYEDSEE*

A predicted signal peptide is highlighted.

The cp7111 nucleotide sequence <SEQ ID 94> is:

1    ATGTTTGAAG CTGTCATTGC CGATATCCAG GCTAGGGAAA
     TCTTGGATTC
51   TCGCGGGTAT CCCACTTTAC ATGTTAAAGT AACCACTAGC
     ACAGGTTCTG
101  TTGGAGAAGC TCGGGTTCCT TCAGGAGCAT CCACAGGGAA
     AAAAGAAGCC
151  TTAGAGTTTC GTGATACAGA TTCTCCTCGT TATCAAGGCA
     AAGGGGTTTT
201  GCAAGCTGTA AAAAACGTAA AAGAAATTCT TTTTCCCCTC
     GTCAAGGGAT
251  GTAGTGTTTA TGAGCAATCC TTAATTGATT CTCTGATGAT
     GGATTCTGAC
301  GGCTCTCCGA ACAAAGAAAC TCTAGGGGCC AATGCTATTT
     TAGGAGTCTC
351  TCTAGCTACA GCACATGCAG CAGCAGCAAC ACTACGCAGA
     CCTCTGTATC
401  GTTATTTAGG AGGGTGTTTT GCCTGCAGTC TTCCCTGTCC
     TATGATGAAT
451  CTGATCAATG GAGGCATGCA TGCCGATAAC GGCTTGGAGT
     TCCAAGAATT
501  TATGATCCGT CCTATTGGAG CCTCTTCCAT CAAAGAAGCT
     GTCAACATGG
551  GTGCTGACGT TTTTCATACT TTGAAAAAAT TACTCCATGA
     AAGAGGCTTA
601  TCTACTGGAG TGGGTGACGA AGGAGGCTTC GCCCCGAATC
     TTGCTTCTAA
651  TGAAGAAGCT CTAGAGCTCC TATTGCTGGC TATTGAAAAA
     GCAGGCTTTA
701  CTCCAGGAAA AGATATATCG CTAGCCTTAG ACTGCGCAGC
     ATCCTCATTC
751  TATAACGTAA AAACAGGCAC GTATGATGGG AGGCACTATG
     AAGAGCAAAT
801  CGCAATCCTT TCTAATTTAT GTGATCGCTA TCCTATAGAC
     TCCATAGAAG
851  ATGGTCTTGC TGAAGAAGAC TATGACGGGT GGGCCTTGTT
     AACTGAAGTT
901  CTTGGAGAAA AAGTACAGAT TGTGGGTGAT GACCTATTTG
     TTACAAATCC
951  GGAATTAATA TTAGAGGGTA TTAGCAATGG ATTAGCGAAC
     TCTGTGTTGA
1001 TTAAACCAAA TCAGATAGGG ACGCTTACTG AAACAGTGTA
     TGCTATCAAG
1051 CTTGCGCAAA TGGCTGGCTA TACTACAATT ATTTCTCATC
     GCTCAGGAGA
1101 AACTACGGAC ACTACGATTG CAGATCTTGC TGTTGCCTTC
     AACGCCGGTC
1151 AAATCAAAAC AGGCTCTTTA TCACGTTCTG AGCGTGTTGC
     AAAATACAAT
1201 AGACTCATGG AAATTGAAGA AGAGCTTGGA TCCGAAGCAA
     TTTTCACAGA
1251 TTCTAATGTA TTTTCTTAC GAGGATTCT GAGGAATAG

The PSORT algorithm predicts an inner membrane location (0.100).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 47A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 47B) and for FACS analysis (FIG. 47C). A his-tagged protein was also expressed.

The cp7111 protein was also identified in the 2D-PAGE experiment and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp7111 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 48

The following C. pneumoniae protein (PID 4455886) was expressed <SEQ ID 95; cp0010>:

1   MKSQFSWLVL SSTLACFTSC STVFAATAEN IGPSDSFDGS
    TNTGTYTPKN
51  TTTGIDYTLT GDITLQNLGD SAALTKGCFS DTTESLSFAG
    KGYSLSFLNI
101 KSSAEGAALS VTTDKNLSLT GFSSLTFLAA PSSVITTPSG
    KGAVKCGGDL
151 TFDNNGTILF KQDYCEENGG AISTKNLSLK NSTGSISFEG
    NKSSATGKKG
201 GAICATGTVD ITNNTAPTLF SNNIAEAAGG AINSTGNCTI
    TGNTSLVFSE
251 NSVTATAGNG GALSGDADVT ISGNQSVTFS GNQAVANGGA
    IYAKKLTLAS
301 GGGGVSPFLT IIVQGTTAGN GGAISILAAG ECSLSAEAGD
    ITFNGNAIVA
351 TTPQTTKRNS IDIGSTAKIT NLRAISGHSI FFYDPITANT
    AADSTDTLNL
401 NKADAGNSTD YSGSIVFSGE KLSEDEAKVA DNLTSTLKQP
    VTLTAGNLVL
451 KRGVTLDTKG FTQTAGSSVI MDAGTTLKAS TEEVTLTGLS
    IPVDSLGEGK
501 KVVIAASAAS KNVALSGPIL LLDNQGNAYE NHDLGKTQDF
    SFVQLSALGT
551 ATTTDVPAVP TVATPTHYGY QGTWGMTWVD DTASTPKTKT
    ATLAWTNTGY
601 LPNPERQGPL VPNSLWGSFS DIQAIQGVIE RSALTLCSDR
    GFWAAGVANF
651 LDKDKKGEKR KYRHKSGGYA IGGAAQTCSE NLISFAFCQL
    FGSDKDFLVA
701 KNHTDTYAGA FYIQHITECS GFIGCLLDKL PGSWSHKPLV
    LEGQLAYSHV
751 SNDLKTKYTA YPEVKGSWGN NAFNMMLGAS SHSYPEYLHC
    FDTYAPYIKL
801 NLTYIRQDSF SEKGTEGRSF DDSNLFNLSL PIGVKFEKFS
    DCNDFSYDLT
851 LSYVPDLIRN DPKCTTALVI SGASWETYAN NLARQALQVR
    AGSHYAFSPM
901 FEVLGQFVFE VRGSSRIYNV DLGGKFQF*

A predicted signal peptide is highlighted.

The cp0010 nucleotide sequence <SEQ ID 96> is:

1    ATGAAATCGC AATTTTCCTG GTTAGTGCTC TCTTCGACAT
     TGGCATGTTT
51   TACTAGTTGT TCCACTGTTT TTGCTGCAAC TGCTGAAAAT
     ATAGGCCCCT
101  CTGATAGCTT TGACGGAAGT ACTAACACAG GCACCTATAC
     TCCTAAAAAT
151  ACGACTACTG GAATAGACTA TACTCTGACA GGAGATATAA
     CTCTGCAAAA
201  CCTTGGGGAT TCGGCAGCTT TAACGAAGGG TTGTTTTTCT
     GACACTACGG
251  AATCTTTAAG CTTTGCCGGT AAGGGGTACT CACTTTCTTT
     TTTAAATATT
301  AAGTCTAGTG CTGAAGGCGC AGCACTTTCT GTTACAACTG
     ATAAAAATCT
351  GTCGCTAACA GGATTTTCGA GTCTTACTTT CTTAGCGGCC
     CCATCATCGG
401  TAATCACAAC CCCCTCAGGA AAAGGTGCAG TTAAATGTGG
     AGGGGATCTT
451  ACATTTGATA ACAATGGAAC TATTTTATTT AAACAAGATT
     ACTGTGAGGA
501  AAATGGCGGA GCCATTTCTA CCAAGAATCT TTCTTTGAAA
     AACAGCACGG
551  GATCGATTTC TTTTGAAGGG AATAAATCGA GCGCAACAGG
     GAAAAAAGGT
601  GGGGCTATTT GTGCTACTGG TACTGTAGAT ATTACAAATA
     ATACGGCTCC
651  TACCCTCTTC TCGAACAATA TTGCTGAAGC TGCAGGTGGA
     GCTATAAATA
701  GCACAGGAAA CTGTACAATT ACAGGGAATA CGTCTCTTGT
     ATTTTCTGAA
751  AATAGTGTGA CAGCGACCGC AGGAAATGGA GGAGCTCTTT
     CTGGAGATGC
801  CGATGTTACC ATATCTGGGA ATCAGAGTGT AACTTTCTCA
     GGAAACCAAG
851  CTGTAGCTAA TGGCGGAGCC ATTTATGCTA AGAAGCTTAC
     ACTGGCTTCC
901  GGGGGGGGGG GGGTATCTCC TTTTCTAACA ATAaTAGTCC
     AAGGTACCAC
951  TGCAGGTAAT GGTGGAGCCA TTTCTATACT GGCAGCTGGA
     GAGTGTAGTC
1001 TTTCAGCAGA AGCAGGGGAC ATTACCTTCA ATGGGAATGC
     CATTGTTGCA
1051 ACTACACCAC AAACTACAAA AAGAAATTCT ATTGACATAG
     GATCTACTGC
1101 AAAGATCACG AATTTACGTG CAATATCTGG GCATAGCATC
     TTTTTCTACG
1151 ATCCGATTAC TGCTAATACG GCTGCGGATT CTACAGATAC
     TTTAAATCTC
1201 AATAAGGCTG ATGCAGGTAA TAGTACAGAT TATAGTGGGT
     CGATTGTTTT
1251 TTCTGGTGAA AAGCTCTCTG AAGATGAAGC AAAAGTTGCA
     GACAACCTCA
1301 CTTCTACGCT GAAGCAGCCT GTAACTCTAA CTGCAGGAAA
     TTTAGTACTT
1351 AAACGTGGTG TCACTCTCGA TACGAAAGGC TTTACTCAGA
     CCGCGGGTTC
1401 CTCTGTTATT ATGGATGCGG GCACAACGTT AAAAGCAAGT
     ACAGAGGAGG
1451 TCACTTTAAC AGGTCTCTCC ATTCCTGTAG ACTCTTTAGG
     CGAGGGTAAG
1501 AAAGTTGTAA TTGCTGCTTC TGCAGCAAGT AAAAATGTAG
     CCCTTAGTGG
1551 TCCGATTCTT CTTTTGGATA ACCAAGGGAA TGCTTATGAA
     AATCACGACT
1601 TAGGAAAAAC TCAAGACTTT TCATTTGTGC AGCTCTCTGC
     TCTGGGTACT
1651 GCAACAACTA CAGATGTTCC AGCGGTTCCT ACAGTAGCAA
     CTCCTACGCA
1701 CTATGGGTAT CAAGGTACTT GGGGAATGAC TTGGGTTGAT
     GATACCGCAA
1751 GCACTCCAAA GACTAAGACA GCGACATTAG CTTGGACCAA
     TACAGGCTAC
1801 CTTCCGAATC CTGAGCGTCA AGGACCTTTA GTTCCTAATA
     GCCTTTGGGG
1851 ATCTTTTTCA GACATCCAAG CGATTCAAGG TGTCATAGAG
     AGAAGTGCTT
1901 TGACTCTTTG TTCAGATCGA GGCTTCTGGG CTGCGGGAGT
     CGCCAATTTC
1951 TTAGATAAAG ATAAGAAAGG GGAAAAACGC AAATACCGTC
     ATAAATCTGG
2001 TGGATATGCT ATCGGAGGTG CAGCGCAAAC TTGTTCTGAA
     AACTTAATTA
2051 GCTTTGCCTT TTGCCAACTC TTTGGTAGCG ATAAAGATTT
     CTTAGTCGCT
2101 AAAAATCATA CTGATACCTA TGCAGGAGCC TTCTATATCC
     AACACATTAC
2151 AGAATGTAGT GGGTTCATAG GTTGTCTCTT AGATAAACTT
     CCTGGCTCTT
2201 GGAGTCATAA ACCCCTCGTT TTAGAAGGGC AGCTCGCTTA
     TAGCCACGTC
2251 AGTAATGATC TGAAGACAAA GTATACTGCG TATCCTGAGG
     TGAAAGGTTC
2301 TTGGGGGAAT AATGCTTTTA ACATGATGTT GGGAGCTTCT
     TCTCATTCTT
2351 ATCCTGAATA CCTGCATTGT TTTGATACCT ATGCTCCATA
     CATCAAACTG
2401 AATCTGACCT ATATACGTCA GGACAGCTTC TCGGAGAAAG
     GTACAGAAGG
2451 AAGATCTTTT GATGACAGCA ACCTCTTCAA TTTATCTTTG
     CCTATAGGGG
2501 TGAAGTTTGA GAAGTTCTCT GATTGTAATG ACTTTTCTTA
     TGATCTGACT
2551 TTATCCTATG TTCCTGATCT TATCCGCAAT GATCCCAAAT
     GCACTACAGC
2601 ACTTGTAATC AGCGGAGCCT CTTGGGAAAC TTATGCCAAT
     AACTTAGCAC
2651 GACAGGCCTT GCAAGTGCGT GCAGGCAGTC ACTACGCCTT
     CTCTCCTATG
2701 TTTGAAGTGC TCGGCCAGTT TGTCTTTGAA GTTCGTGGAT
     CCTCACGGAT
2751 TTATAATGTA GATCTTGGGG GTAAGTTCCA ATTCTAG

The PSORT algorithm predicts an outer membrane location (0.922).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 48A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 48B) and for FACS analysis (FIG. 48C). A his-tagged protein was also expressed.

The cp0010 protein was also identified in the 2D-PAGE experiment and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp0010 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 49

The following C. pneumoniae protein (PID 4376296) was expressed <SEQ ID 97; cp6296>:

1   MEEVSEYLQQ VENQLESCSK RLTKMETFAL GVRLEAKEEI
    ESIILSDVVN
51  RFEVLCRDIE DMLSRVEEIE RMLRMAELPL LPIKEALTKA
    FVQHNSCKEK
101 LTKVEPYFKE SPAYLTSEER LQSLNQTLQR AYKESQKVSG
    LESEVRACRE
151 QLKDQVRQFE TQGVSLIKEE ILFVTSTFRT KFSYHSFRLH
    VPCMRLYEEY
201 YDDIDLERTR ARWMAMSERY RDAFQAFQEM LKEGLVEEAQ
    ALRETEYWLY
251 REERKSKKKH*

The cp6296 nucleotide sequence <SEQ ID 98> is:

1   ATGGAGGAGG TGTCTGAGTA TCTTCAGCAA GTAGAAAATC
    AGTTGGAATC
51  CTGTTCCAAG CGATTAACCA AGATGGAAAC TTTTGCCTTA
    GGTGTGAGGT
101 TGGAAGCTAA AGAAGAGATA GAGTCTATCA TACTTTCTGA
    TGTAGTGAAC
151 CGTTTTGAGG TTTTATGTAG AGATATTGAA GATATGCTAT
    CTCGAGTCGA
201 GGAGATAGAG CGGATGTTAC GTATGGCGGA GCTTCCTCTA
    CTTCCTATAA
251 AAGAAGCGCT TACCAAGGCT TTTGTACAAC ATAACAGCTG
    TAAAGAGAAG
301 TTAACCAAGG TAGAGCCTTA CTTTAAAGAG AGCCCTGCAT
    ATCTAACTAG
351 TGAAGAGCGA TTGCAGAGTT TGAATCAGAC TTTACAACGT
    GCGTACAAAG
401 AGTCCCAAAA GGTTTCAGGT TTAGAATCGG AAGTGAGAGC
    CTGTCGAGAG
451 CAGCTTAAAG ATCAAGTAAG ACAGTTTGAA ACTCAAGGAG
    TGAGCTTGAT
501 AAAAGAAGAG ATTCTCTTTG TGACTAGTAC CTTTAGAACT
    AAATTTAGCT
551 ATCATTCATT TCGATTACAT GTTCCTTGCA TGAGGTTGTA
    TGAGGAGTAT
601 TATGATGACA TTGATCTAGA GAGAACTCGA GCTCGATGGA
    TGGCGATGTC
651 TGAGAGGTAT AGAGATGCTT TTCAGGCATT CCAGGAGATG
    TTGAAGGAAG
701 GCCTAGTTGA AGAAGCTCAG GCTCTTAGAG AAACCGAGTA
    CTGGTTATAT
751 CGAGAGGAGA GAAAGAGTAA AAAGAAACAT TGA

The PSORT algorithm predicts a cytoplasmic location (0.523).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 49A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 49B) and for FACS analysis (FIG. 49C). A his-tagged protein was also expressed.

These experiments show that cp6296 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 50

The following C. pneumoniae protein (PID 4376664) was expressed <SEQ ID 99; cp6664>:

1   MVLFHAQASG RNRVKADAIV LPFWHFKDAK NAASFEAEFE
    PSYLPALENF
51  QGKIGEIELL YSSPKAKEKR IVLLGLGKNE ELTSDVVFQT
    YATLTRVLRK
101 AKCSTVNIIL PTISELRLSA EEFLVGLSSG ILSLNYDYPR
    YNKVDRNLET
151 PLSKVTVIGI VPKMADAIFR KEAAIFEGVY LTRDLVNRNA
    DEITPKKLAE
201 VALNLGKEFP SIDTKVLGKD AIAKEKMGLL LAVSKGSCVD
    PHFIVVRYQG
251 RPKSKDHTVL IGKGVTFDSG GLDLKPGKSM LTMKEDMAGG
    ATVLGILSAL
301 AVLELPINVT GIIPATENAI DGASYKMGDV YVGMSGLSVE
    ICSTDAEGRL
351 ILADAITYAL KYCKPTRIID FATLTGAMVV SLGEEVAGFF
    SNNDVLAEDL
401 LEASAETSEP LWRLPLVKKY DKTLHSDIAD MKNLGSNRAG
    AITAALFLQR
451 FLEESSVAWA HLDIAGTAYH EKEEDRYPKY ASGFGVRSIL
    YYLENSLSK*

The cp6664 nucleotide sequence <SEQ ID 100> is:

1    GTGGTTTTAT TTCATGCTCA AGCCTCTGGG GCTAATCGTG
     TTAAGGCAGA
51   TGCTATACTC CTGCCCTTTT GGCATTTTAA GGATGCAAAA
     AATGCAGCTT
101  CTTTTGAAGC CGAGTTTGAA CCCTCGTATC TCCCCGCTTT
     AGAAAACTTT
151  CAAGGAAAAA CCGGGGAGAT TGAACTCCTT TATAGTAGTC
     CTAAAGCTAA
201  GGAAAAACGC ATTGTCCTCT TAGCTTAAGG GAAAAATGAA
     GAGCTCACCT
251  CTGATGTTGT TTTCCAAACC TATGCGACAC TAACTCGTGT
     CTTACGTAAA
301  GCAAAGTGTT CCACAGTCAA TATCATCTTA CCTACAATTT
     CTGAATTGCG
351  GCTTTCTGCC GAAGAATTCT TAGTGGGGTT GTCCTCAGGA
     ATTTTGTCAT
401  TAAACTATGA CTACCCACGT TATAATAAGG TAGATCGTAA
     TCTTGAAACT
451  CCTCTTTCTA AAGTCACGGT TATCGGTATC GTTCCCAAAA
     TGGCGGATGC
501  TATCTTTAGG AAAGAAGCAG CCATTTTCGA AGGCGTATAT
     CTCACTCGAG
551  ATCTTGTGAA CAGGAATGCT GATGAAATTA CCCCTAAGAA
     ATTGGCAGAG
601  GTTGCTCTGA ATCTGGGAAA AGAGTTCCCT AGTATTGATA
     CTAAGGTCTT
651  GGGAAAAGAT GCCATCGCCA AAGAGAAAAT GGGACTCCTA
     TTGGCTGTTT
701  CCAAGGGTTC TTGTGTGGAT CCACACTTTA TCGTTGTCCG
     TTATCAAGGA
751  CGTCCTAAGT CTAAAGATCA CACCGTCTTG ATAGGGAAAG
     GGGTCACTTT
801  TGACTCTGGA GGTTTAGACC TCAAGCCTGG AAAATCCATG
     CTTACTATGA
851  AAGAAGACAT GGCAGGTGGG GCTACAGTCC TCGGGATTCT
     CTCGGCGTTA
901  GCAGTTTTAG AGCTTCCTAT AAATGTCACG GGGATCATTC
     CTGCTACAGA
951  GAATGCTATC GATGGCGCCT CCTATAAAAT GGGAGATGTC
     TATGTAGGAA
1001 TGTCGGGGCT TTCTGTTGAG ATTTGTAGTA CCGATGCTGA
     GGGACGTCTT
1051 ATCCTCGCTG ATGCGATTAC ATATGCTTTA AAATATTGTA
     AACCGACACG
1101 TATTATAGAT TTTGCAACTC TAACAGGAGC TATGGTAGTC
     TCTCTAGGAG
1151 AAGAGGTTGC AGGTTTCTTT TCCAATAACG ATGTTTTAGC
     TGAAGATCTT
1201 TTAGAGGCGT CAGCCGAAAC CTCCGAGCCG TTATGGAGAC
     TTCCTCTAGT
1251 TAAGAAGTAT GATAAAACAT TGCATTCTGA TATTGCTGAT
     ATGAAAAATC
1301 TAGGCAGTAA CCGTGCAGGG GCTATTACAG CAGCATTATT
     CTTGCAGAGA
1351 TTTTTGGAAG AATCTTCGGT AGCTTGGGCA CATCTTGATA
     TTGCAGGTAC
1401 TGCATATCAT GAAAAAGAAG AAGACCGTTA TCCAAAATAT
     GCTTCAGGTT
1451 TTGGTGTTCG TTCTATTCTT TATTACTTAG AAAATAGTCT
     TTCTAAGTAG

The PSORT algorithm predicts an inner membrane location (0.268).

The protein was expressed in E. coli and purified as a GST-fusion (FIG. 50A), as a his-tagged protein, and as a GST/His fusion. The proteins were used to immunise mice, whose sera were used in Western blot Western blot (50B) and FACS (50C) analyses.

The cp6664 protein was also identified in the 2D-PAGE experiment (Cpn0385) and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6664 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 51

The following C. pneumoniae protein (PID 4376696) was expressed <SEQ ID 101; cp6696>:

1   MTLIFVIIIV WCNAFLIKLC VIMGLQSRLQ HCIEVSQNSN
    FDSQVKQFIY
51  ACQDKTLRQS VLKIFRYHPL LKIHDIARAV YLLMALEEGE
    DLGLSFLNVQ
101 QYPSGAVELF SCGGFPWKGL PYPAEHAEFG LLLLQIAEFY
    EESQAYVSKM
151 SHFQQALFDH QGSVFPSLWS QENSRLLKEK TTLSQSFLFQ
    LGMQIHPEYS
201 LEDPALGFWM QRTRSSSAFV AASGCQSSLG AYSSGDVGVI
    AYGPCSGDIS
251 DCYYFGCCGI AKEFVCQKSH QTTEISFLTS TGKPHPRNTG
    FSYLRDSYVH
301 LPIRCKITIS DKQYRVHAAL AEATSAMTFS IFCKGKNCQV
    VDGPRLRSCS
351 LDSYKGPGND IMILGENDAI NIVSASPYME IFALQGKEKF
    WNADFLINIP
401 YKEEGVMIIF EKKVTSEKSR FFTKMN*

A predicted signal peptide is highlighted.

The cp6696 nucleotide sequence <SEQ ID 102> is:

1    TTGACTCTAA TTTTTGTTAT TATTATCGTT TGGTGCAATG
     CTTTTCTGAT
51   CAAATTGTGC GTGATAATGG GGCTGCAATC CAGGTTACAA
     CATTGTATAG
101  AAGTGTCCCA GAATTCGAAC TTTGATTCAC AAGTAAAACA
     GTTTATCTAT
151  GCGTGCCAAG ATAAGACATT AAGGCAGTCT GTACTCAAGA
     TTTTCCGCTA
201  CCATCCTTTA CTAAAAATTC ATGATATTGC TCGGGCCGTC
     TATCTTTTGA
251  TGGCCTTAGA AGAAGGCGAG GATTTAGGCT TAAGCTTTTT
     AAATGTACAG
301  CAGTACCCTT CAGGTGCTGT AGAACTGTTT TCTTGTGGGG
     GATTTCCTTG
351  GAAAGGATTA CCTTATCCTG CAGAACATGC GGAATTTGGC
     CTACTCCTGT
401  TACAGATCGC AGAGTTTTAT GAAGAGAGTC AGGCATACGT
     CTCTAAAATG
451  AGTCATTTTC AACAGGCACT CTTTGATCAC CAAGGGAGCG
     TCTTTCCCTC
501  TCTCTGGAGC CAGGAGAACT CTCGACTCCT AAAAGAAAAG
     ACAACTCTTA
551  GCCAATCGTT TCTCTTCCAA TTAGGAATGC AAATTCACCC
     AGAATACAGT
601  CTTGAGGATC CTGCACTAGG GTTCTGGATG CAAAGAACGC
     GTTCTTCATC
651  CGCTTTTGTA GCCGCTTCAG GATGTCAAAG TAGCTTGGGA
     GCGTATTCCT
701  CAGGGGATGT CGGTGTTATC GCTTATGGAC CTTGCTCTGG
     AGACATTAGT
751  GATTGTTATT ATTTTGGATG TTGTGGAATC GCTAAAGAGT
     TCGTGTGCCA
801  AAAATCTCAC CAAACTACAG AGATTTCTTT TCTCACCTCT
     ACAGGAAAGC
851  CTCATCCCAG AAATACGGGA TTTTCCTACC TTCGAGATTC
     CTATGTACAT
901  CTGCCGATCC GCTGTAAGAT CACTATTTCC GACAAGCAAT
     ATCGCGTGCA
951  CGCTGCGTTG GCTGAGGCCA CCTCTGCCAT GACGTTTTCT
     ATTTTCTGTA
1001 AGGGGAAGAA TTGTCAGGTT GTTGACGGCC CTCGCTTGCG
     CTCCTGTTCC
1051 CTAGATTCTT ATAAAGGTCC CGGAAACGAC ATTATGATTC
     TTGGGGAAAA
1101 TGACGCAATC AACATTGTTT CTGCAAGTCC CTATATGGAA
     ATTTTTGCTT
1151 TGCAAGGCAA AGAAAAATTT TGGAATGCAG ACTTTTTGAT
     TAATATTCCT
1201 TACAAAGAAG AGGGCGTCAT GTTAATTTTT GAAAAAAAAG
     TGACCTCTGA
1251 GAAAGGAAGA TTCTTTACGA AGATGAATTA A

The PSORT algorithm predicts an inner membrane location (0.463).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 51A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 51B) and for FACS analysis (FIG. 51C). A his-tagged protein was also expressed.

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6696 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 52

The following C. pneumoniae protein (PID 4376790) was expressed <SEQ ID 103; cp6790>:

1   MSEHKKSSKI IGIDIGTTNS CVSVMEGGQA KVITSSEGTR
    TTPSIVAFKG
51  NEKLVGIPAK RQAVTNPEKT LGSTKRFIGR KYSEVASEIQ
    TVPYTVTSGS
101 KGDAVFEVDG KQYTPEEIGA QILMKMKETA EAYLGETVTE
    AVITVPAYFN
151 DSQRASTKDA GRIAGLDVKR IIPEPTAAAL AYGIDKVGDK
    KIAVFDLGGG
201 TFDISILEIG DGVEEVLSTN GDTLLGGDDF DEVIIKWMIE
    EFKKQEGIDL
251 SKDNMALQRL KDAAEKAKIE LSGVSSTEIN QPFITMDAQG
    PKHLALTLTR
301 AQFEKLAASL IERTKSPCIK ALSDAKLSAK DIDDVLLVGG
    MSRMPAVQET
351 VKELFGKEPN KGVNPDEVVA IGAAIQGGVL GGEVKDVLLL
    DVIPLSLGIE
401 TLGGVMTTLV ERNTTIPTQK KQIFSTAADN QPAVTIVVLQ
    GERPMAKDNK
451 EIGRFDLTDI PPAPRGHPQI EVSFDIDANG IFHVSAKDVA
    SGKEQKIRIE
501 ASSGLQEDEI QRMVRDAEIN KEEDKKRREA SDAKNEADSM
    IFRAFKAIKD
551 YKEQIPETLV KEIEERIENV RNALKDDAPI EKIKEVTEDL
    SKHMQKIGES
601 MQSQSASAAA SSAANAKGGP NINTEDLKKH SFSTKPPSNN
    GSSEDHIEEA
651 DVEIIDNDDK*

The cp6790 nucleotide sequence <SEQ ID 104> is:

1    ATGAGTGAAC ACAAAAAATC AAGCAAAATT ATAGGTATAG
     ACTTAGGCAC
51   AACAAACTCC TGCGTATCTG TTATGGAAGG AGGACAAGCT
     AAAGTAATTA
101  CATCATCCGA AGGAACAAGA ACCACGCCAT CGATCGTTGC
     CTTCAAAGGT
151  AATGAGAAAT TAGTGGGGAT TCCAGCAAAA CGTCAAGCAG
     TGACAAATCC
201  AGAAAAAACT CTCGGCTCTA CAAAACGCTT TATTGGCCGT
     AAGTACTCTG
251  AAGTAGCTTC GGAAATCCAA ACCGTTCCTT ATACAGTCAC
     CTCCGGATCT
301  AAAGGTGATG CCGTTTTCGA AGTTGATGGC AAACAATACA
     CTCCAGAAGA
351  AATTGGCGCA CAAATCTTAA TGAAAATGAA AGAGACAGCA
     GAAGCTTATC
401  TAGGCGAAAC TGTCACAGAA GCAGTGATCA CCGTCCCCGC
     ATACTTCAAT
451  GATTCTCAAC GAGCATCCAC AAAAGATGCT GGACGCATTG
     CAGGTCTAGA
501  TGTAAAACGT ATCATTCCAG AACCTACCGC AGCAGCTCTT
     GCCTACGGAA
551  TCGATAAAGT CGGTGATAAA AAAATCGCTG TCTTCGACCT
     TGGTGGAGGA
601  ACTTTTGATA TCTCCATCCT AGAAATCGGT GATGGCGTCT
     TCGAAGTTCT
651  ATCTACAAAT GGAGATACTC TCCTCGGTGG AGACGACTTT
     GATGAAGTCA
701  TTATCAAATG GATGATCGAA GAATTCAAAA AACAAGAAGG
     CATTGATCTT
751  AGCAAAGATA ATATGGCCTT ACAAAGACTT AAAGATGCTG
     CTGAGAAAGC
801  AAAAATAGAA CTTTCAGGAG TCTCTTCCAC AGAAATCAAT
     CAGCCATTCA
851  TCACAATGGA TGCACAAGGA CCTAAACACC TTGCATTGAC
     ACTCACACGT
901  GCGCAATTCG AGAAACTCGC AGCCTCTCTA ATCGAAAGAA
     CAAAATCTCC
951  ATGCATCAAA GCACTCAGTG ACGCAAAACT TTCCGCTAAG
     GATATCGATG
1001 ATGTTCTCTT AGTTGGAGGT ATGTCAAGAA TGCCCGCAGT
     GCAAGAAACT
1051 GTAAAAGAAC TCTTCGGCAA AGAGCCTAAT AAAGGAGTCA
     ACCCCGACGA
1101 AGTTGTTGCT ATTGGAGCCG CAATTCAAGG TGGTGTTCTT
     GGCGGAGAAG
1151 TTAAGGATGT TCTACTCCTA GACGTTATCC CCCTATCTCT
     GGGTATCGAA
1201 ACTCTAGGAG GCGTCATGAC GACTCTGGTA GAGAGAAATA
     CTACAATCCC
1251 TACACAGAAA AAACAAATCT TCTCCACAGC TGCTGATAAC
     CAGCCTGCGG
1301 TTACCATCGT AGTTCTCCAA GGAGAGCGTC CCATGGCCAA
     AGATAACAAG
1351 GAAATCGGAA GATTCGATCT TACAGATATC CCTCCGGCTC
     CTCGAGGCCA
1401 TCCTCAAATC GAAGTCTCCT TCGATATCGA TGCAAACGGA
     ATTTTCCATG
1451 TCTCAGCTAA AGATGTTGCC AGCGGTAAAG AACAGAAAAT
     TCGTATCGAA
1501 GCAAGCTCAG GACTTCAAGA AGATGAAATC CAAAGAATGG
     TTCGAGATGC
1551 CGAAATTAAT AAGGAAGAAG ATAAAAAACG TCGTGAAGCT
     TCAGATGCTA
1601 AAAATGAAGC CGATAGCATG ATCTTCAGAG CCGAAAAAGC
     TATTAAAGAT
1651 TATAAGGAGC AAATTCCTGA AACTTTAGTT AAAGAAATCG
     AAGAGCGAAT
1701 CGAAAACGTG CGCAACGCAC TCAAAGATGA CGCTCCTATT
     GAAAAAATTA
1751 AAGAGGTTAC TGAAGACCTA AGCAAGCATA TGCAAAAAAT
     TGGAGAGTCT
1801 ATGCAATCGC AGTCTGCATC AGCAGCAGCA TCATCGGCAG
     CCAATGCTAA
1851 AGGTGGACCT AACATCAATA CAGAAGATTT GAAAAAACAT
     AGTTTCAGTA
1901 CGAAGCCTCC TTCAAATAAC GGTTCTTCAG AAGACCATAT
     CGAAGAAGCT
1951 GATGTAGAAA TTATTGATAA CGACGATAAG TAA

The PSORT algorithm predicts an inner membrane location (0.151).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 52A) and a his-tagged product. The proteins were used to immunise mice, whose sera were used in Western blot (FIG. 52B) and FACS (FIG. 52C) analyses.

The cp6790 protein was also identified in the 2D-PAGE experiment (Cpn0503).

These experiments show that cp6790 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 53

The following C. pneumoniae protein (PID 4376878) was expressed <SEQ ID 105; cp6878>:

1   MNVPDSKNLH PPAYELLEIK ARITQSYKEA SAILTAIPDG
    ILLLSFTGHF
51  LICNSQAREI LGIDENLEII NRSFTDVLPD TCLGFSIQEA
    LESLKVPKTL
101 RLSLCKESKE KEVELFIRKN EISGYLFIQI RDRSDYKQLE
    NAIERYKNIA
151 ELGKMTATLA HEIRNPLSGI VGFASILKKE ISSPRHQRML
    SSIISGTRSL
201 NNLVSSMLFY TKSQPLNLKI INLQDFFSSL IPLLSVSFPN
    CKFVREGAQP
251 LFRSIDPDRM NSVVWNLVKN AVETGNSPIT LTLHTSGDIS
    VTNPGTIPSE
301 IMDKLFTPFF TTKREGNGLG LAEAQKIIRL HGGDIQLKTS
    DSAVSFFIII
351 PELLAALPKE RAAS*

The cp6878 nucleotide sequence <SEQ ID 106> is:

1    ATGAACGTCC CTGATTCCAA GAACCTCCAT CCTCCTGCAT
     ACGAACTCCT
51   AGAGATCAAG GCTCGCATCA CACAATCTTA TAAAGAAGCG
     AGTGCTATAC
101  TGACAGCGAT TCCTGATGGT ATCCTATTAC TTTCTGAAAC
     AGGACACTTT
151  CTTATCTGCA ATTCACAAGC ACGTGAAATT CTAGGAATTG
     ATGAAAATCT
201  AGAAATTCTT AATAGATCCT TTACCGATGT TCTCCCCGAT
     ACGTGTCTTG
251  GATTTTCTAT TCAAGAGGCT CTTGAATCTC TAAAAGTCCC
     TAAAACTCTT
301  AGACTCTCTC TCTGTAAAGA ATCTAAAGAA AAAGAAGTGG
     AACTCTTCAT
351  CCGTAAAAAC GAGATCAGTG GATACCTGTT TATCCAAATC
     CGCGATCGGT
401  CCGACTATAA ACAACTAGAA AACGCTATAG AAAGATATAA
     AAATATCGCA
451  GAACTTGGGA AAATGACGGC TACCCTAGCT CACGAAATCC
     GCAATCCGCT
501  AAGTGGAATC GTTGGATTTG CCTCTATCCT AAAGAAAGAG
     ATTTCCTCTC
551  CTCGCCACCA ACGAATGCTC TCCTCAATCA TCTCCGGCAC
     AAGGTCTCTA
601  AATAACCTTG TCTCTTCTAT GTTAGAATAT ACAAAATCAC
     AACCGTTGAA
651  CCTAAAGATT ATAAATTTAC AAGACTTCTT CTCTTCTCTT
     ATCCCTCTGC
701  TCTCCGTCTC TTTCCCGAAT TGCAAGTTTG TAAGAGAGGG
     CGCACAACCT
751  CTATTCAGAT CTATAGATCC TGATCGGATG AACAGTGTCG
     TTTGGAACCT
801  AGTGAAAAAT GCTGTAGAAA CAGGGAACTC TCCGATCACT
     CTGACCCTGC
851  ATACATCGGG AGACATCTCG GTAACGAACC CCGGAACGAT
     TCCTTCCGAG
901  ATCATGGACA AGCTCTTCAC TCCATTCTTC ACAACAAAGA
     GAGAGGGAAA
951  TGGTTTGGGA CTTGCTGAAG CTCAAAAAAT TATAAGACTC
     CATGGAGGAG
1001 ATATCCAATT AAAAACAAGC GACTCCGCCG TTAGCTTCTT
     CATAATCATC
1051 CCCGAACTTC TAGCGGCCCT ACCCAAAGAA AGAGCCGCTA
     G

The PSORT algorithm predicts an inner membrane location (0.204).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 53A) and as a GST-fusion product. The recombinant GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 53B) and for FACS analysis.

These experiments show that cp6878 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 54

The following C. pneumoniae protein (PID 4377224) was expressed <SEQ ID 107; cp7224>:

1   MMKKIRKVAL AVGGSGGHIV PALSVKEAFS REGIDVLLLG
    KGLKNHPSLQ
51  QGISYREIPS GLPTVLNPIK IMSRTLSLCS GYLKARKELK
    IFDPDLVIGF
101 GSYHSLPVLL AGLSHKIPLF LHEQNLVPGK VNQLFSRYAR
    GIGVNFSPVT
151 KHFRCPAEEV FLPKRSFSLG SPMMKRCTNH TPTICVVGGS
    QGAQILNTCV
201 PQALVKLVNK YPNMYVHHIV GPKSDVMKVQ HVYNRGEVLC
    CVKPFEEQLL
251 DVLLAADIVI SRAGATILEE ILWAKVPGIL IPYPGAYGHQ
    EVNAKFFVDV
301 LEGGTMILEK ELTEKLLVEK VTFALDSHNR EKQRNSLAAY
    SQQRSTKTFH
351 AFICECL

The cp7224 nucleotide sequence <SEQ ID 108> is:

1    ATGATGAAGA AAATTCGAAA AGTAGCCTTG GCTGTAGGAG
     GTTCAGGAGG
51   CCACATTGTC CCAGCTCTCT CGGTAAAGGA AGCTTTTTCT
     CGTGAAGGAA
101  TAGACGTATT ACTACTAGGG AAAGGTCTCA AGAACCATCC
     TTCTTTGCAA
151  CAGGGAATCA GCTATCGGGA AATCCCCTCA GGACTTCCTA
     CAGTCCTTAA
201  TCCCATAAAG ATCATGAGCA GGACCCTTTC TCTATGTTCA
     GGATACCTGA
251  AAGCAAGAAA GGAACTTAAA ATTTTTGACC CTGACCTGGT
     CATAGGATTT
301  GGGAGCTACC ACTCTCTTCC CGTGTTGCTC GCAGGACTGT
     CCCATAAAAT
351  TCCCTTATTT CTACACGAAC AAAATCTAGT TCCTGGAAAA
     GTAAATCAAT
401  TGTTTTCCCG CTATGCTCGA GGTATTGGAG TGAATTTCTC
     CCCCGTTACT
451  AAACACTTCC GCTGCCCCGC AGAAGAGGTC TTCCTTCCTA
     AACGAAGCTT
501  CTCCTTAGGA AGCCCTATGA TGAAGCGATG TACAAATCAT
     ACCCCTACAA
551  TCTGTGTTGT TGGAGGTTCT CAGGGAGCAC AGATATTAAA
     TACTTGTGTT
601  CCCCAAGCTC TTGTCAAGCT AGTCAATAAG TACCCAAATA
     TGTACGTCCA
651  TCATATTGTA GGACCTAAAA GTGATGTTAT GAAGGTGCAA
     CATGTTTACA
701  ATCGTGGAGA GGTCCTCTGC TGTGTGAAGC CCTTCGAAGA
     GCAACTCCTA
751  GATGTCTTGC TTGCCGCAGA TTTGGTCATC AGTAGGGCAG
     GAGCCACAAT
801  TTTAGAAGAA ATTCTTTGGG CAAAAGTTCC CGGAATTTTA
     ATTCCCTATC
851  CAGGAGCTTA TGGACATCAG GAAGTTAATG CTAAATTCTT
     TGTAGACGTC
901  TTAGAAGGGG GAACTATGAT CCTAGAAAAA GAATTAACAG
     AGAAGCTATT
951  AGTAGAAAAA GTAACGTTTG CTTTAGACTC CCATAACAGA
     GAAAAACAAC
1001 GCAATTCCCT AGCGGCGTAT AGTCAGCAAA GGTCAACAAA
     AACATTCCAT
1051 GCATTCATTT GTGAATGCTT ATAG

The PSORT algorithm predicts an inner membrane location (0.164).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 54A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 54B) and for FACS analysis (FIG. 54C). A his-tagged protein was also expressed.

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp7224 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 55

The following C. pneumoniae protein (PID 4377140) was expressed <SEQ ID 109; cp7140>:

1   MVRRSISFCL FFLMTLLCCT SCNSRSLIVH GLPGREANEI
    VVLLVSKGVA
51  AQKLPQAAAA TAGAATEQMW DIAVRSAQIT EALAILNQAG
    LPRMKGTSLL
101 DLFAKQGLVP SELQEKIRYQ EGLSEQMAST IRKMDGVVDA
    SVQISFTTEN
151 EDNLRLTASV YIKHRGVLDN PNSIMVSKIK RLIASAVPGL
    VPENVSVVSD
201 RAAYSDITIN GPWGLTEEID YVSVWGIILA KSSLTKFRLI
    FYVLILILFV
251 ISCGLLWVIW KTHTLIMTMG GTKGFFNPTP YTKNALEAKK
    AEGAAADKEK
301 KEDADEQGES KNAETSDKDS SDKDAPEGSN EIEGA*

A predicted signal peptide is highlighted.

The cp7140 nucleotide sequence <SEQ ID 110> is:

1    ATGGTTCGTC GATCTATTTC TTTTTGCTTG TTCTTTCTAA
     TGACATTGCT
51   GTGCTGTACA AGCTGTAACA GCAGGTCTCT AATTGTGCAC
     GGTCTTCCTG
101  GCAGAGAAGC GAATGAGATT GTGGTGCTTT TGGTAAGCAA
     AGGGGTGGCT
151  GCACAAAAAT TGCCTCAAGC TGCAGCGGCT ACAGCCGGAG
     CAGCTACTGA
201  GCAAATGTGG GATATCGCGG TTCCGTCAGC ACAAATCACA
     GAGGCCCTTG
251  CCATTCTAAA TCAAGCGGGT CTTCCACGTA TGAAAGGGAC
     AAGCCTGTTA
301  GATCTTTTTG CAAAACAAGG TCTTGTTCCT TCCGAGCTTC
     AGGAAAAAAT
351  CCGTTATCAA GAAGGCTTAT CAGAACAGAT GGCCTCTACG
     ATTAGAAAAA
401  TGGATGGCGT TGTCGATGCC TCAGTACAGA TTTCCTTCAC
     TACAGAAAAT
451  GAAGATAATC TTCCTTTAAC AGCCTCTGTG TATATTAAGC
     ATCGAGGGGT
501  TTTGGACAAT CCGAACAGCA TTATGGTTTC CAAAATTAAG
     CGCCTTATTG
551  CAAGTGCTGT TCCAGGACTT GTGCCAGAGA ACGTCTCTGT
     AGTGAGCGAT
601  CGCGCAGCTT ATAGTGATAT TACAATTAAT GGTCCTTGGG
     GATTAACAGA
651  AGAAATCGAT TATGTTTCTG TTTGGGGTAT TATTCTTGCG
     AAGTCTTCGC
701  TCACCAAATT CCGTCTCATT TTTTATGTCT TGATTCTCAT
     TTTATTTGTT
751  ATTTCTTGTG GTCTCCTTTG GGTCATTTGG AAAACTCATA
     CTCTCATTAT
801  GACTATGGGA GGTACAAAAG GGTTCTTCAA CCCTACACCA
     TATACAAAGA
851  ATGCCTTGGA AGCCAAGAAA GCCGAGGGAG CAGCTGCTGA
     CAAAGAGAAA
901  AAAGAAGATG CAGATTCACA GGGGGAAAGC AAAAATGCGG
     AAACCAGTGA
951  TAAAGACTCT AGTGATAAAG ATGCTCCAGA AGGAAGCAAT
     GAAATTGAGG
1001 GTGCTTAG

The PSORT algorithm predicts an inner membrane location (0.650).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 55A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 55B) and for FACS analysis (FIG. 55C). A his-tagged protein was also expressed.

These experiments show that cp7140 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 56

The following C. pneumoniae protein (PID 4377306) was expressed <SEQ ID 111; cp7306>:

1   MITKQLRSWL AVLVGSSLLA LPLSGQAVGK KESRVSELPQ
    DVLLKEISGG
51  FSKVATKATP AVVYIESFPK SQAVTHPSPG RRGPYENPFD
    YFNDEFFNRF
101 FGLPSQREKP QSKEAVRGTG FLVSPDGYIV TNNHVVEDTG
    KIHVTLHDGQ
151 KYPATVIGLD PKTDLAVIKI KSQNLPYLSF GNSDHLKVGD
    WAIAIGNPFG
201 LQAIVIVGVI SAKGRNQLHI ADFEDFIQTD AAINPGNSGG
    PLLNIDGQVI
251 GVNTAIVSGS GGYIGIGFAI PSLMANRIID QLIRDGQVTR
    GFLGVTLQPI
301 DAELAACYKL ENVYGALVTD VVKGSPADKA GLEQEDVIIA
    YNGKEVDSLS
351 MFRNAVSLMN PDTRIVLKVV REGKVIEIPV TVSQAPKEDG
    MSALQRVGIR
401 VQNLTPETAK KLGIAPETKG ILIISVEPGS VAASSGIAPG
    QLILAVNRQK
451 VSSIEDLNRT IKDSNNENIL LMVSQGDVIR FIALKPEE*

A predicted signal peptide is highlighted.

The cp7306 nucleotide sequence <SEQ ID 112> is:

1    ATGATAACTA AGCAATTGCG TTCGTGGCTA GCTGTACTTG
     TTGGTTCAAG
51   TCTGCTAGCT CTTCCTTTAT CAGGGCAAGC TGTCGGGAAA
     AAAGAATCTC
101  GAGTTTCCGA GCTGCCTCAA GACGTTCTTC TTAAAGAGAT
     CTCGGGAGGG
151  TTTTCTAAGG TCGCTACCAA GGCGACTCCC GCTGTTGTGT
     ACATAGAAAG
201  TTTCCCAAAG AGCCAGGCTG TAACACATCC TTCTCCTGGA
     CGCCGTGGGC
251  CTTATGAAAA TCCTTTTGAT TATTTTAATG ATGAGTTTTT
     CAATCGTTTT
301  TTTGGTCTAC CTTCACAGAG GGAAAAACCT CAAAGTAAAG
     AGGCGGTTCG
351  AGGAACAGGT TTCCTAGTAT CTCCAGATGG CTATATTGTG
     ACTAATAACC
401  ATGTTGTCGA AGATACAGGT AAGATTCACG TAACTCTTCA
     TGATGGGCAA
451  AAGTACCCAG CAACTGTAAT CGGACTCGAT CCTAAAACAG
     ACCTTGCAGT
501  CATTAAAATT AAATCCCAAA ACCTCCCGTA TCTTTCTTTT
     GGAAACTCCG
551  ACCACTTAAA AGTCGGAGAT TGGGCAATTG CAATTGGAAA
     TCCCTTCGGT
601  CTTCAAGCTA CGGTCACCGT AGGTGTCATC AGTGCTAAAG
     GAAGAAATCA
651  ACTCCACATT GCAGATTTTG AAGATTTTAT TCAGACAGAT
     GCTGCGATTA
701  ATCCAGGCAA CTCTGGAGGC CCTCTTCTAA ATATTGATGG
     ACAGGTCATC
751  GGTGTTAATA CTGCCATTGT CAGTGGTAGT GGTGGCTATA
     TTGGAATCGG
801  GTTTGCGATT CCTAGCCTTA TGGCAAATAG AATCATAGAT
     CAGCTGATTC
851  GTGATGGTCA AGTTACCCGA GGATTCTTAG GAGTGACTTT
     ACAACCTATA
901  GATGCGGAAC TCGCTGCTTG CTACAAACTC GAAAAGGTTT
     ATGGCGCTTT
951  AGTCACAGAT GTTGTTAAAG GATCTCCAGC AGATAAAGCA
     GGGCTAAAAC
1001 AAGAAGATGT GATCATTGCT TATAATGGGA AAGAAGTCGA
     TTCACTGAGT
1051 ATGTTCCGTA ATGCTGTTTC TTTAATGAAT CCAGATACAC
     GTATTGTTCT
1101 AAAGGTAGTT CGTGAAGGAA AGGTTATCGA AATACCCGTG
     ACAGTTTCTC
1151 AAGCTCCAAA AGAAGATGGA ATGTCGGCTT TACAGCGTGT
     GGGAATCCGT
1201 GTGCAAAACC TAACTCCTGA AACTGCTAAG AAGCTGGGAA
     TTGCTCCAGA
1251 GACTAAAGGC ATTTTGATTA TAAGTGTTGA ACCAGGGTCT
     GTAGCAGCTT
1301 CTTCAGGAAT TGCTCCTGGT CAGCTGATCC TTGCTGTGAA
     TAGACAAAAA
1351 GTATCTTCGA TTGAAGATCT GAATAGAACG TTAAAAGATT
     CTAACAATGA
1401 GAATATTCTT CTTATGGTTT CTCAAGGAGA TGTTATTCGC
     TTCATTGCCC
1451 TGAAACCTGA AGAATAA

The PSORT algorithm predicts a periplasmic location (0.923).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 56A) and as a GST-fusion product (FIG. 56B). The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 56C) and for FACS (FIG. 56D) analyses.

The cp7306 protein was also identified in the 2D-PAGE experiment (Cpn0979) and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp7306 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 57

The following C. pneumoniae protein (PID 4377132) was expressed <SEQ ID 113; cp7132>:

1   MCNSIAMKKQ KRGFVLMELL MSFTLIALLL GTLGFWYRKI
    YTVQKQKERI
51  YNFYIEESRA YKQLRTLFSM SLSSSYEEPG SLFSLIFDRG
    VYRDPKLAGA
101 VRASLHHDTK DQRLELRICN IKDQSYFETQ RLLSHVTHVV
    LSFQRNPDPE
151 KLPETIALTI TREPKAYPPR TLTYQFAVGK*

A predicted signal peptide is highlighted.

The cp7132 nucleotide sequence <SEQ ID 114> is:

1   ATGTGTAACT CTATAGCTAT GAAAAAGCAA AAGCGTGGCT
    TTGTGCTTAT
51  GGAATTACTC ATGTCGTTCA CTCTAATTGC TTTGTTATTA
    GGGACTTTAG
101 GATTTTGGTA TCGGAAAATT TATACTGTAC AAAAGCAAAA
    AGAACGTATT
151 TATAACTTTT ATATCGAAGA AAGCCGAGCC TACAAGCAGC
    TCAGAACCCT
201 GTTTAGCATG TCCTTGTCTT CATCTTACGA GGAGCCTGGA
    TCATTATTTT
251 CTTTAATCTT TGATCGGGGT GTTTATCGAG ATCCTAAGCT
    GGCAGGTGCG
301 GTACGAGCTT CTCTCCATCA TGACACCAAG GATCAGAGAT
    TGGAACTTCG
351 TATTTGTAAT ATTAAGGATC AGTCTTACTT TGAAACACAG
    CGACTGCTCT
401 CCCACGTGAC CCATGTTGTA CTTTCCTTCC AGAGAAATCC
    TGATCCTGAA
451 AAACTTCCTG AAACAATTGC TTTAACTATA ACACGGGAAC
    CTAAAGCATA
501 TCCTCCAAGG ACGTTAACAT ACCAATTTGC GGTTGGGAAA
    TAA

The PSORT algorithm predicts a periplasmic location (0.915).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 57A) or as a GST-fusion. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 57B) and FACS (FIG. 57C) analyses.

These experiments show that cp7132 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 58

The following C. pneumoniae protein (PID 4376733) was expressed <SEQ ID 115; cp6733>:

1   MKTSIPWVLV SSVLAFSCHL QSLANEELLS PDDSFNGNID
    SGTFTPKTSA
51  TTYSLTGDVF FYEPGKGTPL SDSCFKQTTD NLTFLGNGHS
    LTFGFIDAGT
101 HAGAAASTTA NKNLTFSGFS LLSFDSSPST TVTTGQGTLS
    SAGGVNLENI
151 RKLVVAGNFS TADGGAIKGA SFLLTGTSGD ALFSNNSSST
    KGGAIATTAG
201 ARIANNTGYV RFLSNIASTS GGAIDDEGTS ILSNNKFLYF
    EGNAAKTTGG
251 AICNTKASGS PELIISNNKT LIFASNVAET SGGAIHAKKL
    ALSSGGFTEF
301 LRNNVSSATP KGGAISIDAS GELSLSAETG NITFVRNTLT
    TTGTDTPRKR
351 NAINIGSNGK FTELRAAKNH TIFFYDPITS EGTSSDVLKI
    NNGSAGALNP
401 YQGTILFSGE TLTADELKVA DNLKSSFTQP VSLSGGKLLL
    QKGVTLESTS
451 FSQEAGSLLG MDSGTTLSTT AGSITITNLG INVDSLGLKQ
    PVSLTAKGAS
501 NKVIVSGKLN IIDIEGNIYE SHMFSHDQLF SLLKITVDAD
    VDTNVDISSL
551 IPVPAEDPNS EYGFQGQWNV MWTTDTATNT KEATATWTKT
    GFVPSPERKS
601 ALVCNTLWGV FTDIRSLQQL VEIGATGMEH KWGFWVSSMT
    NFLHKTGDEN
651 RKGFRHTSGG YVIGGSAHTP KDDLFTFAFC HLFARDKDCF
    IAHNNSRTYG
701 GTLFFKHSHT LQPQNYLRLG RAKFSESAIE KFPREIPLAL
    DVQVSFSHSD
751 NRMETHYTSL PESEGSWSNE CIAGGIGLDL PFVLSNPHPL
    FKTFIPQMKV
801 EMVYVSQNSF FESSSDGRGF SIGRLLNLSI PVGAKFVQGD
    IGDSYTYDLS
851 GFFVSDVYRN NPQSTATLVM SPDSWKIRGG NLSRQAFLLR
    GSNNYVYNSN
901 CELFGHYAME LRGSSRNYNV DVGTKLRF*

A predicted signal peptide is highlighted.

The cp6733 nucleotide sequence <SEQ ID 116> is:

1    ATGAAGACTT CGATTCCTTG GGTTTTAGTT TCCTCCGTGT
     TAGCTTTCTC
51   ATGTCACCTA CAGTCACTAG CTAACGAGGA ACTTTTATCA
     CCTGATGATA
101  GCTTTAATGG AAATATCGAT TCAGGAACGT TTACTCCAAA
     AACTTCAGCC
151  ACAACATATT CTCTAACAGG AGATGTCTTC TTTTACGAGC
     CTGGAAAAGG
201  CACTCCCTTA TCTGACAGTT GTTTTAAGCA AACCACGGAC
     AATCTTACCT
251  TCTTGGGGAA CGGTCATAGC TTAACGTTTG GCTTTATAGA
     TGCTGGCACT
301  CATGCAGGTG CTGCTGCATC TACAACAGCA AATAAGAATC
     TTACCTTCTC
351  AGGGTTTTCC TTACTGAGTT TTGATTCCTC TCCTAGCACA
     ACGGTTACTA
401  CAGGTCAGGG AACGCTTTCC TCAGCAGGAG GCGTAAATTT
     AGAAAATATT
451  CGTAAACTTG TAGTTGCTGG GAATTTTTCT ACTGCAGATG
     GTGGAGCTAT
501  CAAAGGAGCG TCTTTCCTTT TAACTGGCAC TTCTGGAGAT
     GCTCTTTTTA
551  GTAACAACTC TTCATCAACA AAGGGAGGAG CAATTGCTAC
     TACAGCAGGC
601  GCTCGCATAG CAAATAACAC AGGTTATGTT AGATTCCTAT
     CTAACATAGC
651  GTCTACGTCA GGAGGCGCTA TCGATGATGA AGGCACGTCG
     ATACTATCGA
701  ACAACAAATT TCTATATTTT GAAGGGAATG CAGCGAAAAC
     TACTGGCGGT
751  GCGATCTGCA ACACCAAGGC GAGTGGATCT CCTGAACTGA
     TAATCTCTAA
801  CAATAAGACT CTGATCTTTG CTTCAAACGT AGCAGAAACA
     AGCGGTGGCG
851  CCATCCATGC TAAAAAGCTA GCCCTTTCCT CTGGAGGCTT
     TACAGAGTTT
901  CTACGAAATA ATGTCTCATC AGCAACTCCT AAGGGGGGTG
     CTATCAGCAT
951  CGATGCCTCA GGAGAGCTCA GTCTTTCTGC AGAGACAGGA
     AACATTACCT
1001 TTGTAAGAAA TACCCTTACA ACAACCGGAA GTACCGATAC
     TCCTAAACGT
1051 AATGCGATCA ACATAGGAAG TAACGGGAAA TTCACGGAAT
     TACGGGCTGC
1101 TAAAAATCAT ACAATTTTCT TCTATGATCC CATCACTTCA
     GAAGGAACCT
1151 CATCAGACGT ATTGAAGATA AATAACGGCT CTGCGGGAGC
     TCTCAATCCA
1201 TATCAAGGAA CGATTCTATT TTCTGGAGAA ACCCTAACAG
     CAGATGAACT
1251 TAAAGTTGCT GACAATTTAA AATCTTCATT CACGCAGCCA
     GTCTCCCTAT
1301 CCGGAGGAAA GTTATTGCTA CAAAAGGGAG TCACTTTAGA
     GAGCACGAGC
1351 TTCTCTCAAG AGGCCGGTTC TCTCCTCGGC ATGGATTCAG
     GAACGACATT
1401 ATCAACTACA GCTGGGAGTA TTACAATCAC GAACCTAGGA
     ATCAATGTTG
1451 ACTCCTTAGG TCTTAAGCAG CCCGTCAGCC TAACAGCAAA
     AGGTGCTTCA
1501 AATAAAGTGA TCGTATCTGG GAAGCTCAAC CTGATTGATA
     TTGAAGGGAA
1551 CATTTATGAA AGTCATATGT TCAGCCATGA CCAGCTCTTC
     TCTCTATTAA
1601 AAATCACGGT TGATGCTGAT GTTGATACTA ACGTTGACAT
     CAGCAGCCTT
1651 ATCCCTGTTC CTGCTGAGGA TCCTAATTCA GAATACGGAT
     TCCAAGGACA
1701 ATGGAATGTT AATTGGACTA CGGATACAGC TACAAATACA
     AAAGAGGCCA
1751 CGGCAACTTG GACCAAAACA GGATTTGTTC CCAGCCCCGA
     AAGAAAATCT
1801 GCGTTAGTAT GCAATACCCT ATGGGGAGTC TTTACTGACA
     TTCGCTCTCT
1851 GCAACAGCTT GTAGAGATCG GCGCAACTGG TATGGAACAC
     AAACAAGGTT
1901 TCTGGGTTTC CTCCATGACG AACTTCCTGC ATAAGACTGG
     AGATGAAAAT
1951 CGCAAAGGCT TCCGTCATAC CTCTGGAGGC TACGTCATCG
     GTGGAAGTGC
2001 TCACACTCCT AAAGACGACC TATTTACCTT TGCGTTCTGC
     CATCTCTTTG
2051 CTAGAGACAA AGATTGTTTT ATCGCTCACA ACAACTCTAG
     AACCTACGGT
2101 GGAACTTTAT TCTTCAAGCA CTCTCATACC CTACAACCCC
     AAAACTATTT
2151 GAGATTAGGA AGAGCAAAGT TTTCTGAATC AGCTATAGAA
     AAATTCCCTA
2201 GGGAAATTCC CCTAGCCTTG GATGTCCAAG TTTCGTTCAG
     CCATTCAGAC
2251 AACCGTATGG AAACGCACTA TACCTCATTG CCAGAATCCG
     AAGGTTCTTG
2301 GAGCAACGAG TGTATAGCTG GTGGTATCGG CCTAGACCTT
     CCTTTTGTTC
2351 TTTCCAACCC ACATCCTCTT TTCAAGACCT TCATTCCACA
     GATGAAAGTC
2401 GAAATGGTTT ATGTATCACA AAATAGCTTC TTCGAAAGCT
     CTAGTGATGG
2451 CCGTGGTTTT AGTATTGGAA GGCTGCTTAA CCTCTCGATT
     CCTGTGGGTG
2501 CGAAATTCGT GCAGGGGGAT ATCGGAGATT CCTACACCTA
     TGATCTCTCA
2551 GGATTCTTTG TTTCCGATGT CTATCGTAAC AATCCCCAAT
     CTACAGCGAC
2601 TCTTGTGATG AGCCCAGACT CTTGGAAAAT TCGCGGTGGC
     AATCTTTCAA
2651 GACAGGCATT TTTACTGAGG GGTAGCAACA ACTACGTCTA
     CAACTCCAAT
2701 TGTGAGCTCT TCGGACATTA CGCTATGGAA CTCCGTGGAT
     CTTCAAGGAA
2751 CTACAATGTA GATGTTGGTA CCAAACTCCG ATTCTAG

The PSORT algorithm predicts an outer membrane location (0.924).

The protein was expressed in E. coli and purified as a his-tag product, as shown in FIG. 58A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 58B) and for FACS (FIG. 58C) analyses. A GST-fusion protein was also expressed.

The cp6733 protein was also identified in the 2D-PAGE experiment (Cpn0451).

These experiments show that cp6733 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 59

The following C. pneumoniae protein (PID 4376814) was expressed <SEQ ID 117; cp6814>:

1   MHDALLSILA IQELDIKMIR LMRVKKEHQK ELAKVQSLKS
    DIRRKVQEKE
51  LEMENLKTQI RDGENRIQEI SEQINKLENQ QAAVKKMDEF
    NALTQEMTTA
101 NKERRSLEHQ LSDLMDKQAG GEDLIVSLKE SLASTENSSS
    VIEKEIFESI
151 KKINEEGKAL LEQRTELKHA TNPELLSIYE RLLNNKKDRV
    VVPIENRVCS
201 GCHIVLTPQH ENLVRKKDRL IFCEHCSRIL YWQESQVKAQ
    ENSTAKRRRR
251 RAAV*

The cp6814 nucleotide sequence <SEQ ID 118> is:

1   ATGCATGACG CACTTCTAAG CATTTTGGCT ATTCAAGAGC
    TTGATATTAA
51  AATGATTCGC CTTATGCGCG TAAAGAAAGA ACATCAGAAA
    GAATTGGCTA
101 AAGTCCAATC TTTAAAAAGT GATATTCGTA GAAAAGTTCA
    GGAAAAAGAA
151 CTCGAAATGG AGAATTTGAA AACTCAAATT CGAGATGGAG
    AGAATCGCAT
201 CCAAGAGATT TCTGAACAAA TCAATAAATT AGAAAATCAG
    CAAGCTGCTG
251 TAAAAAAAAT GGATGAGTTT AACGCTCTTA CCCAAGAAAT
    GACTACAGCA
301 AACAAAGAAC GTCGCTCTTT AGAGCACCAG CTTAGCGATC
    TCATGGATAA
351 GCAAGCTGGA GGCGAAGACC TTATTGTCTC TCTAAAAGAA
    AGCTTAGCTT
401 CTACAGAAAA TAGTAGCAGT GTCATTGAAA AAGAAATTTT
    TGAAAGCATC
451 AAAAAGATTA ATGAAGAAGG CAAAGCTTTG CTTGAACAAC
    GGACAGAGTT
501 AAAGCATGCG ACGAATCCCG AACTACTCAG CATCTATGAG
    CGTCTATTAA
551 ACAATAAAAA AGATCGCGTT GTTGTTCCTA TTGAAAATCG
    TGTCTGCAGT
601 GGTTGTCATA TTGTTCTAAC TCCTCAACAC GAAAATCTTG
    TAAGAAAGAA
651 AGACCGACTC ATTTTTTGCG AACATTGCTC TCGAATTCTC
    TATTGGCAAG
701 AATCCCAAGT CAATGCTCAG GAAAATTCCA CAGCAAAACG
    TCGTCGTCGT
751 CGCGCAGCTG TATAA

The PSORT algorithm predicts an inner membrane location (0.070).

The protein was expressed in E. coli and purified as a GST-fusion (FIG. 59A) or his-tagged product. The recombinant proteins were used to immunise mice, whose sera were used in Western blot (FIG. 59B) and FACS (FIG. 59C) analyses.

These experiments show that cp6814 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 60

The following C. pneumoniae protein (PID 4376830) was expressed <SEQ ID 119; cp6830>:

1    MKWLPATAVF AAVLPALTAF GDPASVEIST SHTGSGDPTS
     DAALTGFTQS
51   STETDGTTYT IVGDITFSTF TNIPVPVVTP DANDSSSNSS
     KGGSSSSGAT
101  SLIRSSNLHS DFDFTKDSVL DLYHLFFPSA SNTLNPALLS
     SSSSGGSSSS
151  SSSSSSGSAS AVVAADPKGG AAFYSNEANG TLIFTTDSGN
     PGSLTLQNLK
201  MTGDGAAIYS KGPLVFTGLK NLTFTGNESQ KSGGAAYTEG
     ALTTQAIVEA
251  VTFTGNTSAG QGGAIYVKEA TLFNALDSLK FEKNTSGQAG
     GGIYTESTLT
301  ISNITKSIEF ISNKASVPAP APEPTSPAPS SLINSTTIDT
     STLQTRAASA
351  TPAVAPVAAV TPTPISTQET AGNGGAIYAK QGISISTFKD
     LTFKSNSASV
401  DATLTVDSST IGESGGAIFA ADSIQIGGCT GTTLFSGNTA
     NKSGGGIYAV
451  GQVTLEDIAN LKMTNNTCKG EGGAIYTKKA LTINNGAILT
     TFSGNTSTDN
501  GGAIFAVGGI TLSDLVEVRF SKNKTGNYSA PITKAASNTA
     PVVSSSTTAA
551  SPAVPAAAAA FVTNAAKGGA LYSTEGLTVS GITSILSFEN
     NECQNQGGGA
601  YVTKTFQCSD SHRLQFTSNK AADEGGGLYC GDDVTLTNLT
     GKTLFQENSS
651  EKHGGGLSLA SGKSLTMTSL ESFCLNANTA KENGGGANVP
     ENIVLTFTYT
701  PTPNEPAPVQ QPVYGEALVT GNTATKSGGG IYTKNAAFSN
     LSSVTFDQNT
751  SSENGGALLT QKAADKTDCS FTYITNVNIT NNTATGNGGG
     IAGGKAHFDR
801  IDNLTVQSNQ AKKGGGVYLE DALILEKVIT GSVSQNTATE
     SGGGIYAKDI
851  QLQALPGSFT ITDNKVETSL TTSTNLYGGG IYSSGAVTLT
     NISGTFGITG
901  NSVINTATSQ DADIQGGGIY ATTSLSINQC NTPILFSNNS
     AATKKTSTTK
951  QIAGGAIFSA AVTIENNSQP IIFLNNSAKS EATTAATAGN
     KDSCGGAIAA
1001 NSVTLTNNPE ITFKGNYAET GGAIGCIDDT NGSPPRKVSI
     ADNGSVLFQD
1051 NSALNRGGAI YGETIDISRT GATFIGNSSK HDGSAICCST
     ALTLAPNSQL
1101 IFENNKVTET TATTKASINN LGAAIYGNNE TSDVTISLSA
     ENGSIFFKNN
1151 LCTATNKYCS IAGNVKFTAI EASAGKAISF YDAVNVSTKE
     TNAQELKLNE
1201 KATSTGTILF SGELHENKSY IPQKVTFAHG NLILGKVAEL
     SVVSFTQSPG
1251 TTITMGPGSV LSNHSKEAGG IAINNVIIDF SEIVPTKDNA
     TVAPPTLKLV
1301 SRTNADSKDK IDITGTVTLL DPNGNLYQNS YLGEDRDITL
     FNIDNSASGA
1351 VTATNVTLQG NLGAKKGYLG TWNLDPNSSG SKIILKWTFD
     KYLRWPYIPR
1401 DNHFYINSIW GAWNSLVTVK QSILGNMLNN ARFEDPAFNN
     FWASAIGSFL
1451 RKEVSRNSDS FTYHGRGYTA AVDAKPRQEF ILGAAFSQVF
     GHAESEYHLD
1501 NYKHKGSGHS TQASIYAGNI FYFPAIRSRP ILFQGVATYG
     YMQHDTTTYY
1551 PSIEEKNMAN WDSIAWLFDL RFSVDLKEPQ PHSTARLTFY
     TEAEYTRIRQ
1601 EKFTELDYDP RSFSACSYGN LAIPTGFSVD GALAWREIIL
     YNKVSAAYLP
1651 VILRNNPKAT YEVLSTKEKG NVVNVLPTRN AARAEVSSQI
     YLGSYWTLYG
1701 TYTIDASMNT LVQMANGGIR FVF*

A predicted signal peptide is highlighted.

The cp6830 nucleotide sequence <SEQ ID 120> is:

1    ATGAAGTGGC TACCAGCTAC AGCTGTTTTT GCTGCCGTAC
     TCCCCGCACT
51   AACAGCCTTC GGAGATCCCG CGTCTGTTGA AATAAGTACC
     AGCCATACAG
101  GATCCGGGGA TCCTACAAGC GACGCTGCCT TAACAGGATT
     TACACAAAGT
151  TCCACAGAAA CTGACGGTAC TACCTATACC ATTGTCGGTG
     ATATCACCTT
201  CTCTACTTTT ACGAATATTC CTGTTCCCGT AGTAACTCCA
     GACGCCAACG
251  ATAGTTCCAG CAATAGCTCT AAAGGAGGAA GTAGCAGTAG
     TGGAGCTACA
301  TCTCTAATCC GATCCTCAAA CCTACACTCC GATTTTGATT
     TTACAAAAGA
351  TAGCGTGTTA GACCTCTATC ACCTTTTCTT TCCTTCAGCT
     TCAAATACTC
401  TCAATCCTGC ACTCCTTTCT TCCAGTAGCA GCGGTGGATC
     CTCGAGCAGC
451  AGTAGCTCCT CATCATCTGG AAGTGCATCT GCTGTTGTTG
     CTGCGGACCC
501  AAAAGGAGGC GCTGCCTTTT ATAGTAACGA GGCTAACGGA
     ACTTTAACCT
551  TCACTACAGA CTCTGGAAAT CCCGGCTCCC TGACTCTTCA
     GAATCTTAAA
601  ATGACCGGAG ATGGAGCCGC CATCTACTCG AAGGGTCCTC
     TAGTATTTAC
651  TGGTTTAAAA AATCTAACCT TTACAGGAAA TGAATCTCAG
     AAATCTGGAG
701  GTGCTGCCTA TACTGAAGGC GCACTCACAA CACAAGCAAT
     CGTTGAAGCC
751  GTAACTTTTA CTGGCAACAC CTCGGCAGGG CAAGGAGGCG
     CTATCTATGT
801  TAAAGAAGCT ACCCTATTCA ATGCTCTAGA CAGCCTCAAA
     TTTGAAAAAA
851  ACACTTCTGG GCAAGCTGGT GGTGGAATCT ATACAGAGTC
     TACGCTCACA
901  ATCTCGAACA TCACAAAATC TATTGAATTT ATCTCTAATA
     AAGCTTCTGT
951  CCCTGCCCCC GCTCCTGAGC CCACCTCTCC GGCTCCAAGT
     AGCTTAATAA
1001 ATTCTACAAC GATCGATACC TCGACTCTCC AAACCCGAGC
     AGCATCCGCA
1051 ACTCCAGCAG TGGCTCCTGT TGCTGCCGTA ACTCCAACAC
     CAATCTCTAC
1101 TCAAGAGACC GCAGGAAATG GAGGCGCTAT CTATGCTAAA
     CAAGGTATTT
1151 CGATATCCAC GTTTAAAGAT CTGACCTTCA AGTCTAACTC
     TGCATCGGTA
1201 GATGCCACCC TTACTGTCGA TTCTAGCACT ATTGGAGAAT
     CTGGAGGTGC
1251 TATCTTTGCA GCAGACTCTA TACAAATCCA ACAGTGCACG
     GGAACCACCT
1301 TATTCAGTGG CAATACTGCC AATAAGTCTG GTGGGGGTAT
     TTACGCTGTA
1351 GGACAAGTCA CCCTAGAAGA TATAGCGAAT CTGAAGATGA
     CCAACAACAC
1401 CTGTAAAGGT GAAGGTGGAG CCATCTACAC TAAAAAGGCT
     TTAACTATCA
1451 ACAACGGTGC CATTCTCACT ACATTTTCTG GAAATACATC
     GACAGATAAT
1501 GGTGGGGCTA TTTTTGCTGT AGGTGGCATC ACTCTCTCTG
     ATCTTGTAGA
1551 AGTCCGCTTT AGTAAAAATA AGACCGGAAA TTATTCCGCT
     CCTATTACCA
1601 AAGCGGCTAG CAACACAGCT CCTGTAGTTT CTAGCTCTAC
     AACTGCTGCA
1651 TCTCCTGCGG TCCCTGCTGC CGCTGCAGCA CCTGTTACAA
     ACGCAGCAAA
1701 AGGAGGGGCT TTATATAGTA CAGAAGGACT GACTGTATCT
     GGAATCACAT
1751 CGATATTGTC GTTTGAAAAC AACGAATGCC AGAATCAAGG
     AGGTGGGGCT
1801 TACGTTACTA AAACCTTCCA GTGTTCCGAT TCTCATCGCC
     TCCAGTTTAC
1851 TAGTAATAAA GCAGCAGATG AAGGCGGGGG CCTGTATTGT
     CGTGACGATG
1901 TCACGCTAAC GAACCTGACA GGGAAAACAC TATTTCAAGA
     GAATAGCAGT
1951 GAGAAACATG GAGGTGGGCT CTCTCTCGCC TCAGGAAAAT
     CTCTGACTAT
2001 GACATCGTTA GAGAGCTTCT GCTTAAATGC AAATACAGCA
     AAGGAAAACG
2051 GAGGCGGTGC GAATGTCCCT GAAAATATTG TACTCACCTT
     CACCTATACT
2101 CCCACTCCAA ATGAACCTGC GCCTGTGCAG CAGCCCGTGT
     ATGAGGAAGC
2151 TCTTGTTACT GGAAATACAG CCACAAAAAG TGGTGGGGGC
     ATTTACACGA
2201 AAAATGCGGC CTTCTCAAAT TTATCTTCTG TAACTTTTGA
     TCAAAATACC
2251 TCTTCAGAAA ATGGTGGTGC CTTACTTACC CAAAAAGCTG
     CAGATAAAAC
2301 GGACTGTTCT TTCACCTATA TTACAAATGT CAATATCACC
     AACAATACAG
2351 CTACAGGAAA TGGTGGGGGC ATTGCTGGGG GAAAAGCACA
     TTTCGATCGC
2401 ATTGATAATC TTACAGTCCA AAGCAACCAA GCAAAGAAAG
     GTGGTGGGGT
2451 TTATCTTGAA GATGCCCTCA TCCTGGAAAA GGTTATTACA
     GGTTCTGTCT
2501 CACAAAATAC AGCTACAGAA AGTGGTGGGG GTATCTACGC
     TAAGGATATT
2551 CAACTACAAG CTCTACCTGG AAGCTTCACA ATTACCGATA
     ATAAAGTCGA
2601 AACTAGTCTT ACTACTAGCA CTAATTTATA TGGTGGGGGC
     ATCTATTCCA
2651 GTGGAGCTGT CACGCTAACC AATATATCTG GAACCTTTGG
     CATTACAGGA
2701 AACTCTGTTA TCAATACAGC GACATCCCAG GATGCAGATA
     TACAAGGTGG
2751 GGGCATTTAT GCAACCACGT CTCTCTCAAT AAATCAATGT
     AATACACCCA
2801 TTCTATTTAG CAACAACTCT GCTGCCACTA AAAAAACATC
     AACAACAAAG
2851 CAAATTGCTG GTGGGGCTAT CTTCTCCGCT GCAGTAACTA
     TCGAGAATAA
2901 CTCTCAGCCC ATTATTTTCT TAAATAATTC CGCAAAGTCG
     GAAGCAACTA
2951 CAGCAGCAAC TGCAGGAAAT AAAGATAGCT GTGGAGGAGC
     CATTGCAGCT
3001 AACTCTGTTA CTTTAACAAA TAACCCTGAA ATAACCTTTA
     AAGGAAATTA
3051 TGCAGAAACT GGAGGAGCGA TTGGCTGTAT TGATCTTACT
     AATGGCTCAC
3101 CTCCCCGTAA AGTCTCTATT GCAGACAACG GTTCTGTCCT
     TTTTCAAGAC
3151 AACTCTGCGT TAAATCGCGG AGGCGCTATC TATGGAGAGA
     CTATCGATAT
3201 CTCCAGGACA GGTGCGACTT TCATCGGTAA CTCTTCAAAA
     CATGATGGAA
3251 GTGCAATTTG CTGTTCAACA GCCCTAACTC TTGCGCCAAA
     CTCCCAACTT
3301 ATCTTTGAAA ACAATAAGGT TACGGAAACC ACAGCCACTA
     CAAAAGCTTC
3351 CATAAATAAT TTAGGAGCTG CAATTTATGG AAATAATGAG
     ACTAGTGACG
3401 TCACTATCTC TTTATCAGCT GAGAATGGAA GTATTTTCTT
     TAAAAACAAT
3451 CTATGCACAG CAACAAACAA ATACTGCAGT ATTGCTGGAA
     ACGTAAAATT
3501 TACAGCAATA GAAGCTTCAG CAGGGAAAGC TATATCTTTC
     TATGATGCAG
3551 TTAACGTTTC CACCAAAGAA ACAAATGCTC AAGAGCTAAA
     ATTAAATGAA
3601 AAAGCGACAA GTACAGGAAC GATTCTATTT TCTGGGGAAC
     TTCACGAAAA
3651 TAAATCCTAT ATTCCACAGA AAGTCACTTT CGCACATGGG
     AATCTCATTC
3701 TAGGTAAAAA TCGAGAACTT AGCGTAGTTT CCTTTACCCA
     ATCTCCAGGC
3751 ACCACAATCA CTATGGGCCC AGGATCGGTT CTTTCCAACC
     ATAGCAAAGA
3801 AGCAGGAGGA ATCGCTATAA ACAATGTCAT CATTGATTTT
     AGTGAAATCG
3851 TTCCTACTAA AGATAATGCA ACAGTAGCTC CACCCACTCT
     TAAATTAGTA
3901 TCGAGAACTA ATGCAGATAG TAAAGATAAG ATTGATATTA
     CAGGAACTGT
3951 GACTCTTCTA GATCCTAATG GCAACTTATA TCAAAATTCT
     TATCTTGGTG
4001 AAGACCGCGA TATCACTCTT TTCAATATAG ACAATTCTGC
     AAGTGGGGCA
4051 GTTACAGCCA CGAATCTCAC CCTTCAAGGG AATTTAGGAG
     CTAAAAAAGG
4101 ATATTTAGGA ACCTGGAATT TGGATCCAAA TTCCTCGGGT
     TCAAAAATTA
4151 TTCTAAAATG GACCTTTGAC AAATACCTGC GCTGGCCCTA
     CATCCCTAGA
4201 GACAACCACT TCTACATCAA CTCTATTTGG GGAGCACAAA
     ACTCTTTAGT
4251 GACTGTGAAA CAAGGGATCT TAGGGAACAT GTTGAACAAT
     GCAAGGTTTG
4301 AAGATCCTGC TTTCAACAAC TTCTGGGCTT CGGCTATAGG
     ATCTTTCCTT
4351 AGGAAAGAAG TATCTCGAAA TTCTGACTCA TTCACCTATC
     ATGGCAGAGG
4401 CTATACCGCT GCTGTGGATG CCAAACCTCG CCAAGAATTT
     ATTTTAGGAG
4451 CTGCCTTCAG TCAGGTTTTT GGTCACGCCG AGTCTGAATA
     TCACCTTGAC
4501 AACTATAAGC ATAAAGGCTC AGGTCACTCT ACACAAGCAT
     CTCTTTATGC
4551 TGGCAATATC TTCTATTTTC CTGCGATACG GTCTCGGCCT
     ATTCTATTCC
4601 AAGGTGTGGC GACCTATGGT TATATGCAAC ATGACACCAC
     AACCTACTAT
4651 CCTTCTATTG AAGAAAAAAA TATGGCAAAC TGGGATAGCA
     TTGCTTGGTT
4701 ATTTGATCTG CGTTTCAGTG TGGATCTTAA AGAACCTCAA
     CCTCACTCTA
4751 CAGCAAGGCT TACCTTCTAT ACAGAAGCTG AGTATACCAG
     AATTCGCCAG
4801 GAGAAATTCA CAGAGCTAGA CTATGATCCT AGATCTTTCT
     CTGCATGCTC
4851 TTATGGGAAC TTAGCAATTC CTACTGGATT CTCTGTAGAC
     GGAGCATTAG
4901 CTTGGCGTGA GATTATTCTA TATAATAAAG TATCAGCTGC
     GTACCTCCCT
4951 GTGATTCTCA GGAATAATCC AAAAGCGACC TATGAAGTTC
     TCTCTACAAA
5001 AGAAAAGGGC AACGTAGTCA ACGTTCTCCC TACAAGAAAC
     GCAGCTCGTG
5051 CAGAGGTGAG CTCTCAAATT TATCTTGGAA GTTACTGGAC
     ACTCTACGGC
5101 ACGTATACTA TTGATGCTTC AATGAATACT TTAGTGCAAA
     TGGCCAACGG
5151 AGGGATCCGG TTTGTATTCT AG

The PSORT algorithm predicts an outer membrane location (0.926).

The protein was expressed in E. coli and purified as a GST-fusion (FIG. 60A) or his-tagged product. The recombinant proteins were used to immunise mice, whose sera were used in Western blot (FIG. 60B) and FACS (FIG. 60C) analyses.

The cp6830 protein was also identified in the 2D-PAGE experiment (Cpn0540) and showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp6830 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 61

The following C. pneumoniae protein (PID 4376854) was expressed <SEQ ID 121; cp6854>:

1   MSIAIAREQY AAILDMHPKP SIAMFSSEQA RTSWEKRQAH
    PYLYRLLEII
51  WGVVKFLLGL IFFIPLGLFW VLQKICQNFI LLGAGGWIFR
    PICRDSNLLR
101 QAYAARLFSA SFQDHVSSVR RVCLQYDEVF IDGLELRLPN
    AKPDRWMLIS
151 NGNSDCLEYR TVLQGEKDWI FRIAEESQSN ILIFNYPGVM
    KSQGHITRNN
201 VVKSYQACVR YLRDEPAGPQ ARQIVAYGYS LGASVQAEAL
    SKEIADGSDS
251 VRWFVVKDRG ARSTGAVAKQ FIGSLGVWLA NLTEWNINSE
    KRSKDLHCPE
301 LFIYGKDSQG NLIGDGLFKK ETCFAAPFLD PKNLEECSGK
    KIPVAQTGLR
351 EDHILSDDVI KEVAGHIQRH FDN*

The cp6854 nucleotide sequence <SEQ ID 122> is:

1    ATGTCAATAG CTATTGCAAG GGAACAATAC GCAGCTATAT
     TGGATATGCA
51   TCCTAAACCT TCGATCGCCA TGTTTTCTTC GGAGCAGGCG
     AGAACTTCTT
101  GGGAGAAACG ACAGGCTCAT CCTTACCTTT ATCGTCTTCT
     TGAGATCATA
151  TGGGGTGTTG TGAAATTTCT TCTCGGCTTA ATCTTCTTTA
     TTCCCTTGGG
201  TCTTTTCTGG GTCCTTCAGA AGATATGTCA GAATTTTATT
     CTTCTTGGTG
251  CAGGAGGGTG GATTTTTAGA CCCATATGCA GGGACTCTAA
     TTTATTGCGA
301  CAAGCTTACG CCGCGCGTCT TTTCTCCGCT TCATTCCAAG
     ATCATGTCTC
351  CTCTGTGCGA AGGGTTTGCT TACAGTATGA CGAGGTCTTT
     ATTGACGGAT
401  TGGAGTTACG TCTTCCCAAT GCTAAGCCAG ATCGATGGAT
     GTTAATCTCC
451  AATGGAAACT CCGATTGCTT AGAGTATAGG ACAGTGCTGC
     AAGGGGAAAA
501  GGACTGGATA TTCCGTATTG CTGAAGAGTC TCAATCCAAC
     ATTTTAATCT
551  TCAATTACCC AGGAGTCATG AAGAGCCAAG GGAATATAAC
     AAGAAACAAT
601  GTAGTCAAAT CTTATCAAGC ATGCGTACGC TATCTTAGAG
     ATGAACCCGC
651  AGGACCTCAG GCGCGTCAAA TCGTTGCTTA TGGCTATTCT
     TTAGGAGCTA
701  GTGTTCAAGC CGAAGCATTA AGTAAAGAGA TCGCAGACGG
     AAGTGATAGC
751  GTCCGTTGGT TTGTCGTTAA AGATCGAGGA GCTCGCTCTA
     CAGGAGCCGT
801  TGCTAAACAG TTTATTGGAA GTCTAGGAGT TTGGCTGGCG
     AATCTTACCC
851  ATTGGAATAT TAATTCTGAA AAGAGAAGCA AGGACTTGCA
     TTGCCCAGAA
901  CTCTTTATTT ATGGCAAGGA TTCCCAAGGT AATCTTATCG
     GGGATGGATT
951  GTTCAAAAAA GAGACGTGCT TCGCAGCACC ATTTTTAGAT
     CCTAAAAACT
1001 TGGAAGAGTG TTCAGGGAAG AAAATCCCTG TAGCTCAGAC
     CGGTCTAAGA
1051 CACGATCATA TCCTTTCCGA TGATGTGATT AAAGAAGTTG
     CAGGTCATAT
1101 TCAAAGACAT TTCGATAATT A

The PSORT algorithm predicts an inner membrane location (0.461).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 61A. The recombinant protein was used to immunise mice, whose sera were used in Western blot (FIG. 61B) and FACS (FIG. 61C) analyses. A his-tagged protein was also expressed.

These experiments show that cp6854 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 62

The following C. pneumoniae protein (PID 4377101) was expressed <SEQ ID 123; cp7101>:

1   MYSCYSKGIS HNYLIHPMSR LDIFVFDSLI ANQDQNLLEE
    IFCSEDTVLF
51  KAYRTTALQS PLAAKNLNIA RKVANYILAD NGEIDTVKLV
    EAIHHLSQCT
101 YPLGPHRHNE AQDREHLLKM LKALKENPKL KESIKTLFVP
    SYSTIQNLIR
151 HTLALNPQTI LSTIHVRQAA LTALFTYLRQ DVGSCFATAP
    AILIHQEYPE
201 RFLKDLNDLI SSGKLSRIVN QREIAVPINL SGCIGELFKP
    LRILDLYPDP
251 LVKLSSSPGL KKAFSAANLI ETLGDSAEQI QQLLSHQYLM
    QKLQNVHETL
301 TANDIIKSTL LHYYQLQEST VRAIFFKEGL FSKEQVAFST
    QHPRFLSSIQ
351 RVYHYLHAYE EAKSAFIHDT QNPLLKAWEY TLATLADASQ
    PTISNHIRLA
401 LGWKSEDPHS LVSLVTHFVE EEVENIRIDV QQCEQTYEEA
    RSQLEYIEGR
451 MRNPLNNQDS QILTMDHMRF RQELNKALYE WSDAQEKAKK
    FLHLPEFLLS
501 FYTKQIPLYF RSSYDAFIQE FAHLYANAPA GFRILFTHGR
    THPNTWSPIY
551 SINEFIRFLS EFFTSTESEL LSKHAVINLE KETSRLVENI
    TAMLHTDVFQ
601 EALLTRIIEA YQLPVPPSIL NHLDQLSQTP WVYVSGGTVD
    TLLLDYFESS
651 EPLTLTEKHP ENPHELAAFY ADALKDLPTG IKSYLEEGSH
    SLLSSSPTHV
701 FSIIAGSPLF REAWDNDWYS YTWLRDVWVK QHQDFLQDTI
    LPQLSIYAFI
751 ENFCNKYALQ HVVHDFHDFC SDHSLTLPEL YDKGSRFLSS
    LFTKDKTVAL
801 IYIRRLLYLM VREVPYVSEQ QLPEVLDNVS SYLGISSRIT
    YEKFRSLIEE
851 TIPKMTLISS ADLRHIYKSL LMQSYQKIYT EEDTYLRLTT
    AMRHHNLAYP
901 APLLFADSNW PSIYFGFILN PGTTEIDLWK FNYAGLQGQP
    LDNIQELFAT
951 SRPWTLYANP IDYGMPPPPG YRSRLPKEFF *

The cp7101 nucleotide sequence <SEQ ID 124> is:

1    ATGTATTCGT GTTACAGCAA AGGAATATCC GATAACTATC
     TTCTACATCC
51   TATGTCACGT TTGGATATTT TTGTTTTCGA TTCTCTGATC
     GCAAACCAGG
101  ATCAAAATCT TCTTGAGGAA ATTTTCTGTT CTGAAGACAC
     AGTTTTATTT
151  AAGCCTACC GTACTACGGCC TCTACAATCC CCTCTAGCTG
     CTAAGAACCT
201  AAATATCGCC CGTAAAGTCG CAAATTATAT CTTAGCTGAC
     AATGGGGAAA
251  TCGATACAGT AAAGCTTGTC GAAGCCATTC ACCATCTCTC
     ACAATGTACC
301  TATCCTTTAG GGCCTCATCG CCATAATGAA GCTCAAGATC
     GTGAACACCT
351  CCTTAAAATG CTAAAAGCTC TAAAGGAAAA TCCTAAATTA
     AAAGAAAGCA
401  TCAAAACTCT CTTTGTCCCT TCATACTCTA CAATCCAAAA
     CCTAATTCGC
451  CATACACTAG CATTGAATCC ACAGACAATT CTCTCTACGA
     TTCATGTGCG
501  TCAAGCAGCA CTCACAGCGC TCTTCACCTA CCTTCGGCAA
     GATGTAGGTT
551  CCTGTTTTGC TACGGCTCCT GCCATTCTCA TTCACCAAGA
     ATATCCAGAA
601  CGATTCCTTA AAGATCTCAA TGATCTCATT AGCATGGGCA
     AACTCTCTAG
651  AATCGTAAAC CAAAGGGAAA TTGCGGTTCC TATAAACCTT
     TCGGGATGCA
701  TTGGAGAGCT ATTCAAGCCT TTAAGGATTC TAGATCTTTA
     TCCTGATCCT
751  CTGGTTAAGC TCTCCTCATC TCCAGGACTC AAAAAAGCCT
     TTTCTGCTGC
801  CAATCTTATT GAAACTCTTG GGGATTCTGA AGCACAAATC
     CAACAGTTGC
851  TCTCGCATCA ATATTTGATG CAAAAACTAC AAAATGTCCA
     TGAGACCTTA
901  ACTGCTAACG ACATTATCAA ATCGACACTT CTGCACTACT
     ATCAGCTCCA
951  AGAAAGTACT GTACGAGCTA TTTTCTTCAA AGAAGGGTTG
     TTCAGCAAAG
1001 AACAAGTGGC ATTCTCGACG CAACACCCCA GAGAGCTCTC
     AGAAATACAA
1051 CGGGTATACC ACTACTTACA TGCCTATGAA GAAGCAAAAT
     CTGCTTTTAT
1101 CCATGACACT CAAAATCCCT TACTGAAAGC CTGGGAGTAT
     ACTTTAGCGA
1151 CTCTTGCGGA TGCTAGCCAA CCTACCATCT CAAACCATAT
     CCGCCTTGCC
1201 TTAGGATGGA AAAGTGAAGA CCCTCACAGT CTTGTATCTC
     TAGTTACACA
1251 CTTTGTTGAA GAGGAAGTAG AAAACATCCG AATTTTAGTC
     CAACAATGTG
1301 AACAGACCTA TCACGAAGCA CGCTCCCAAC TAGAATATAT
     TGAAGGGCGG
1351 ATGCGCAACC CACTAAATAA TCAAGACAGT CAGATTTTGA
     CGATGGATCA
1401 CATGCGCTTC CGTCAAGAAC TCAATAAAGC TCTTTATGAG
     TGGGATAGTG
1451 CTCAAGAAAA GGCAAAGAAA TTTCTACATC TTCCTGAATT
     CTTACTTTCT
1501 TTCTATACAA AGCAAATTCC CTTATACTTT CGTAGTTCTT
     ACGATGCCTT
1551 CATTGAAGAA TTTGCTCATC TCTATGCTAA TGCTCCCGCT
     GGCTTCCGTA
1601 TTCTTTTCAC GCATGGACGC ACCCATCCGA ACACATGGTC
     CCCCATCTAT
1651 TCGATTAATG AATTTATACG TTTTCTTTCT GAATTCTTCA
     CCTCCACAGA
1701 GTCAGAACTT CTGGGGAAAC ATGCCGTGAT CAATTTAGAG
     AAAGAAACAT
1751 CTCGGCTCGT CCACAACATC ACTGCCATGC TACACACGGA
     TGTTTTCCAA
1801 GAAGCTCTCC TTACAAGAAT TTTAGAAGCC TATCAGCTTC
     CTGTGCCTCC
1851 CTCCATCTTA AACCACTTAG ATCAGCTGTC ACAAACTCCC
     TGGGTTTATG
1901 TTTCTGGAGG AACAGTGGAC ACTCTTCTTT TGGATTATTT
     TGAAAGCTCA
1951 GAACCTCTGA CACTTACAGA AAAGCATCCT GAAAATCCTC
     ATGAGCTTGC
2001 AGCTTTCTAC GCAGACGCCC TTAAAGATCT CCCTACAGGA
     ATTAAAAGTT
2051 ATCTAGAAGA AGGATCCCAC TCTCTACTTA GCTCATCACC
     CACCCACGTT
2101 TTCTCTATAA TCGCAGGATC TCCTTTATTT CGGGAAGCTT
     GGGATAATGA
2151 TTGGTACAGC TATACCTGGC TTCGTGATGT CTGGGTGAAA
     CAACACCAAG
2201 ATTTCCTTCA AGATACTATA TTACCTCAGC TAAGTATCTA
     TGCTTTCATA
2251 GAGAATTTTT GTAACAAATA TGCTTTGCAA CATGTAGTTC
     ATGACTTTCA
2301 TGATTTCTGC TCCGACCACT CCTTGACTCT TCCGGAGCTC
     TATGACAAAG
2351 GATCGCGTTT TCTAAGCTCC TTATTCACCA AAGATAAGAC
     CGTAGCTCTT
2401 ATCTATATAC GCCGTCCTCT CTACCTTATG GTCCGTGAAG
     TCCCTTATGT
2451 TTCAGAACAA CAGCTTCCAG AAGTCTTAGA TAACGTCTCT
     TCATATCTCG
2501 GGATTTCCTA TCGTATTACC TATGAGAAAT TCCGCTCCCT
     GATAGAGGAA
2551 ACCATCCCTA AAATGACCTT ACTCTCCTCA GCAGACCTGA
     GGCATATCTA
2601 TAAAGGTCTC CTCATGCAAA GTTATCAAAA GATCTACACC
     GAAGAAGATA
2651 CGTACCTCCG CCTCACCACG GCAATGAGGC ATCATAATCT
     TGCCTATCCC
2701 GCTCCTTTGC TCTTTGCAGA CAGTAACTGG CCTTCTATTT
     ATTTTGGATT
2751 CATCCTAAAT CCAGGAACCA CAGAGATCGA TCTTTGGAAA
     TTTAACTATG
2801 CAGGGCTGCA AGGACAGCCT CTTGACAATA TCCAGGAGCT
     GTTCGCAACG
2851 TCAAGACCCT GGACCCTCTA TGCAAATCCT ATAGATTATG
     GCATGCCACC
2901 GCCTCCAGGC TACCGCAGCC GCCTCCCTAA AGAATTTTTC
     TAG

The PSORT algorithm predicts a cytoplasmic location (0.206).

The protein was expressed in E. coli and purified as a GST-fusion (FIG. 62A) or his-tagged product. The proteins were used to immunise mice, whose sera were used in Western blot (FIG. 62B) and FACS (FIG. 62C) analyses.

This protein also showed good cross-reactivity with human sera, including sera from patients with pneumonitis.

These experiments show that cp7101 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 63

The following C. pneumoniae protein (PID 4377107) was expressed <SEQ ID 125; cp7107>:

1   MSIVRNSALP LPCLSRSETF KKVRSHMKFM KVLTPWIYRK
    DLWVTAFLLT
51  AIPGSFAHTL VDIAGERRHA AQATGVSGDG KIVIGMKVPD
    DPFAITVGFQ
101 YIDGHLQPLE AVRPQCSVYP NGITPDGTVI VGTNYAIGMG
    SVAVKWVNGK
151 VSELPMLPDT LDSVASAVSA DGRVIGGNRN INLGASVAVK
    WEDDVITQLP
201 SLPDAMNACV NGISSDGSII VGTMVDVSWR NTAVQWIGDQ
    LSVIGTLGGT
251 TSVASAISTD GTVIVGGSEN ADSQTHAYAY KNGVMSDIGT
    LGGFYSLAHA
301 VSSDGSVIVG VSTNSERRYH AFQYADGQMV DLGTLGGPES
    YAQGVSGDGK
351 VIVGRAQVPS GDWHAFLCPF QAPSPAPVHG GSTVVTSQNP
    RGMVDINATY
401 SSLKNSQQQL QRLLIQHSAK VESVSSGAPS FTSVKGAISK
    QSPAVQNDVQ
451 KGTFLSYRSQ VHGNVQNQQL LTGAFMDWKL ASAPKCGFKV
    ALHYGSQDAL
501 VERAALPYTE QGLGSSVLSG FGGQVQGRYD FNLGETVVLQ
    PFMGIQVLHL
551 SREGYSEKNV RFPVSYDSVA YSAATSFMGA HVFASLSPKM
    STAATLGVER
601 DLNSHIDEFK GSVSAMGNFV LENSTVSVLR RFASLAMYYD
    VRQQQLVTLS
651 VVMNQQPLTG TLSLVSQSSY NLSF*

The cp7107 nucleotide sequence <SEQ ID 126> is:

1    ATGAGTATAG TCAGAAATTC TGCATTGCCA CTTCCGTGTT
     TAAGCAGATC
51   CGAAACCTTT AAAAAAGTTA GGTCGCATAT GAAATTTATG
     AGAGTCCTTA
101  CTCCATGGAT TTATCGAAAA GATCTTTGGG TAACAGCATT
     CTTACTGACA
151  GCAATTCCAG GATCTTTTGC ACATACTCTT GTTGATATAG
     CAGGAGAACC
201  TCGGCATGCT GCTCAAGCAA CAGGAGTTTC TGGAGATGGT
     AAAATTGTTA
251  TAGGAATGAA AGTTCCGGAT GATCCTTTTG CTATAACTGT
     AGGATTTCAA
301  TATATTGATG GGCATTTGCA ACCCTTAGAG GCAGTACGTC
     CTCAATGCTC
351  TGTATACCCT AATGGTATAA CCCCGGACGG AACGGTTATT
     GTGGGTACAA
401  ACTATGCCAT CGGGATGGGT AGTGTTGCTG TGAAATGGGT
     AAATGGCAAG
451  GTTTCTGAAC TTCCCATGCT CCCTGACACC CTCGATTCTG
     TAGCATCGGC
501  AGTTTCTGCA GATGGAAGAG TGATTGGAGG GAATAGAAAT
     ATAAATCTTG
551  GCGCTTCTGT TGCTGTGAAA TGGGAGGACG ACGTGATTAC
     ACAACTTCCT
601  TCTCTTCCTG ATGCTATGAA TGCTTGTGTT AACGGAATTT
     CTTCAGATGG
651  TTCTATAATT GTAGGAACCA TGGTAGACGT GTCATGGAGA
     AATACCGCAG
701  TACAATGGAT CGGGGATCAG CTCTCTGTTA TTGGGACTTT
     AGGAGGAACT
751  ACTTCTGTTG CTAGTGCAAT CTCAACAGAT GGCACTGTGA
     TTGTAGGAGG
801  TTCTGAAAAT GCAGATTCTC AGACTCATGC CTATGCTTAT
     AAAAACGGTG
851  TTATGAGCGA TATAGGGACC CTCGGAGGTT TTTATTCTTT
     AGCACATGCA
901  GTATCTTCAG ATGGTTCTGT GATTGTAGGA GTATCCACGA
     ACTCTGAGCA
951  TAGATATCAT GCATTCCAAT ATGCTGATGG ACAGATGGTA
     GATTTAGGAA
1001 CTTTAGGAGG GCCTGAATCT TATGCTCAAG GTGTGTCTGG
     AGATGGAAAG
1051 GTAATTGTGG GTAGAGCACA AGTACCATCT GGAGATTGGC
     ATGCGTTCCT
1101 ATGTCCTTTC CAAGCTCCGA GCCCTGCTCC TGTCCATGGG
     GGAAGCACTG
1151 TCGTAACTAG CCAGAATCCA CGTGGAATGG TAGATATCAA
     TGCTACGTAC
1201 TCCTCTTTGA AAAATAGCCA ACAACAACTA CAAAGATTGC
     TTATCCAGCA
1251 TAGTGCAAAA GTTGAAAGTG TATCCTCAGG AGCACCATCT
     TTTACAAGTG
1301 TGAAAGGTGC GATCTCAAAA CAGAGCCCTG CAGTGCAAAA
     TGATGTACAG
1351 AAAGGGACGT TTTTAAGTTA CCGTTCCCAA GTTCATGGAA
     ACGTGCAGAA
1401 TCAGCAATTG CTCACAGGAG CTTTTATGGA CTGGAAACTC
     GCTTCAGCTC
1451 CTAAATGCGG CTTTAAAGTA GCTCTCCACT ATGGCTCTCA
     AGATGCTCTC
1501 GTAGAACGTG CAGCTCTTCC TTACACAGAA CAAGGCTTAG
     GAAGCAGTGT
1551 CTTGTCAGGT TTTGGAGGAC AAGTTCAAGG ACGCTATGAC
     TTTAATTTAG
1601 GAGAAACTGT TGTTCTGCAA CCCTTTATGG GCATTCAAGT
     TCTCCACCTA
1651 AGTAGAGAAG GGTATTCTGA GAAGAATGTT CGATTTCCTG
     TAAGCTATGA
1701 TTCTGTAGCC TACTCAGCAG CTACTAGCTT TATGGGTGCG
     CATGTATTTG
1751 CCTCCCTAAG CCCTAAAATG AGTACAGCAG CAACTTTAGG
     TGTGGAGAGA
1801 GATCTGAATT CACATATAGA TGAATTTAAG GGATCCGTCT
     CTGCTATGGG
1851 AAACTTTGTC TTGGAAAATT CTACAGTGAG TGTTTTAAGA
     CCTTTTGCTT
1901 CTCTTGCTAT GTACTATGAC GTAAGACAAC AGCAACTCGT
     GACGTTGTCA
1951 GTAGTTATGA ATCAACAACC CTTAACAGGC ACACTAAGCT
     TAGTAAGCCA
2001 AAGTAGCTAT AATCTTAGCT TCTAA

The PSORT algorithm predicts an inner membrane location (0.100).

The protein was expressed in E. coli and purified as a GST-fusion (FIG. 63A) or his-tagged product. The proteins were used to immunise mice, whose sera were used in Western blot (FIG. 63B) and FACS (FIG. 63C) analyses.

These experiments show that cp7107 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 64

The following C. pneumoniae protein (PID 4376467) was expressed <SEQ ID 127; cp6467>:

1   MLRFFAVFIS TLWLITSGCS PSQSSKGIFV VNMKEMPRSL
    DPGKTRLIAD
51  QTLMRHLYEG LVEEHSQNGE IKPALAESYT ISEDGTRYTF
    KIKNILWSNG
101 DPLTAQDFVS SWKEILKEDA SSVYLYAFLP IKNARAIFDD
    TESPENLGVR
151 ALDKRHLEIQ LETPCAHFLH FLTLPIFFPV HETLRNYSTS
    FEEMPITCGA
201 FRPVSLEKGL RLHLEKNPMY HNKSRVKLHK IIVQFISNAN
    TAAILPKHKK
251 LDWQGPPWGE PIPPEISASL HQDDQLFSLP GASTTWLLFN
    IQKKPWNNAK
301 LRKALSLAID KDMLTKVVYQ GLAEPTDHIL HPRLYPGTYP
    ERKRQNERIL
351 EAQQLFEEAL DELQMTREDL EKETLTFSTF SFSYGRICQM
    LREQWKKVLK
401 FTIPIVGQEF FTIQKNFLEG NYSLTVNQWT AAFIDPMSYL
    MIFANPGGIS
451 PYHLQDSHFQ TLLIKITQEH KKHLRNQLII EALDYLEECH
    ILEPLCHPNL
501 RIALNKNIKN FNLFVRRTSD FRFIEKL*

A predicted signal peptide is highlighted.

The cp6467 nucleotide sequence <SEQ ID 128> is:

1    ATGCTCCGTT TCTTCGCTGT ATTTATATCA ACTCTTTGGC
     TCATTACCTC
51   AGGATGTTCC CCATCCCAAT CCTCTAAAGG AATTTTTGTG
     GTAAATATGA
101  AGGAAATGCC ACGCTCCTTG GATCCTGGAA AAACTCGTCT
     CATTGCAGAC
151  CAAACTCTAA TGCGTCATCT ATATGAAGGA CTCGTCGAAG
     AACATTCCCA
201  AAATGGAGAG ATTAAACCAG CCCTTGCAGA AAGCTACACC
     ATCTCCGAAG
251  ACGGGACTCG GTACACATTT AAAATCAAAA ACATCCTTTG
     GAGTAACGGA
301  GACCCTCTGA CAGCTCAAGA CTTTGTCTCC TCTTGGAAGG
     AAATCCTAAA
351  GGAAGATGCG TCCTCCGTAT ATCTCTATGC GTTTTTACCT
     ATCAAAAATG
401  CTCGGGCAAT CTTTGATGAT ACTGAGTCTC CAGAAAATCT
     AGGAGTCCGA
451  GCTTTAGATA AGCGTCATCT CGAAATTCAG TTAGAAACTC
     CCTGCGCGCA
501  TTTCCTACAT TTCTTGACTC TTCCTATTTT TTTCCCTGTT
     CATGAAACTC
551  TGCGAAACTA TAGCACCTCT TTTGAAGAGA TGCCCATTAC
     CTGCGGTGCT
601  TTCCGCCCTG TGTCTCTAGA AAAAGGCCTG AGACTCCATC
     TAGAGAAAAA
651  CCCTATGTAC CATAATAAAA GCCGTGTGAA ACTACATAAA
     ATTATTGTAC
701  AGTTTATCTC AAACGCTAAC ACTGCAGCCA TTCTATTCAA
     ACATAAGAAA
751  TTAGATTGGC AAGGACCTCC TTGGGGAGAA CCTATCCCTC
     CAGAAATCTC
801  AGCTTCTCTA CATCAAGATG ACCAGCTCTT TTCTCTTCCG
     CGCGCTTCGA
851  CTACATGGTT ACTCTTTAAT ATACAAAAAA AACCTTGGAA
     CAATGCTAAA
901  TTACGCAAGG CATTGAGCCT TGCAATAGAC AAAGATATGT
     TAACCAAAGT
951  GGTATACCAA GGTCTTGCAG AACCTACAGA TCATATCCTA
     CATCCAAGAC
1001 TTTATCCAGG GACCTATCCC GAACGGAAAA GACAAAACGA
     AAGAATTCTT
1051 GAGGCTCAAC AACTCTTTGA AGAAGCTCTA GACGAACTTC
     AAATGACACG
1101 CGAAGATCTA GAAAAGGAAA CTTTGACTTT CTCAACCTTT
     TCTTTTTCTT
1151 ACGGAAGGAT TTGCCAAATG CTAAGAGAAC AATGGAAGAA
     AGTCTTAAAA
1201 TTTACTATCC CTATAGTAGG CCAAGAGTTT TTCACAATAC
     AAAAAAACTT
1251 CCTAGAGGGG AACTATTCCC TAACCGTGAA CCAATGGACC
     GCAGCATTTA
1301 TTGATCCGAT GTCTTATCTC ATGATCTTTG CCAATCCTGG
     AGGAATTTCC
1351 CCCTATCACC TCCAAGATTC ACACTTTCAA ACTCTTCTCA
     TAAAGATCAC
1401 TCAAGAACAT AAAAAACACC TACGAAATCA GCTTATTATT
     GAAGCCCTTG
1451 ACTATTTAGA ACACTGTCAC ATTCTCGAAC CACTATGTCA
     TCCAAATCTT
1501 CGAATTGCTT TGAACAAAAA CATTAAAAAC TTTAATCTTT
     TTGTTCGACG
1551 AACTTCAGAC TTTCGTTTTA TAGAAAAACT ATAG

The PSORT algorithm predicts an outer membrane lipoprotein (0.790).

The protein was expressed in E. coli and purified as a his-tag product and a GST-fusion protein, as shown in FIG. 64A. The recombinant his-tag protein was used to immunise mice, whose sera were used in a Western blot (FIG. 64B). The recombinant GST-fusion protein was also used to immunise mice, whose sera were used in a Western blot (FIG. 64C) and for FACS analysis (FIG. 64D).

These experiments show that cp6467 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 65

The following C. pneumoniae protein (PID 4376679) was expressed <SEQ ID 129; cp6679>:

1   MRKMLVLLAS LGLLSPTLSS CTHLGSSGSY HPKLYTSGSK
    TKGVIAMLPV
51  FHRPGKSLEP LPWNLQGEFT EEISKRFYAS EKVFLIKHNA
    SPQTVSQFYA
101 PIANRLPETI IEQFLPAEFI VATELLEQKT GKEAGVDSVT
    ASVRVRVFDI
151 RHHKIALIYQ EIIECSQPLT TLVNDYHRYG WNSKHFDSTP
    MGLMHSRLFR
201 EVVARVEGYV CANYS*

A predicted signal peptide is highlighted.

The cp6679 nucleotide sequence <SEQ ID 130> is:

1   ATGCGAAAAA TGTTGGTATT ATTGGCATCT TTAGGACTTC
    TATCCCCAAC
51  CCTATCCAGC TCCACTCACT TAGGCTCTTC AGGAAGTTAT
    CATCCTAAGC
101 TATACACTTC AGGGAGCAAA ACTAAAGGTG TGATTGCGAT
    GCTTCCTGTA
151 TTTCATCGCC CAGGAAAGAG TCTTGAACCT TTACCTTGGA
    ACCTCCAAGG
201 AGAATTTACT GAAGAGATCA GCAAAAGGTT TTATGCTTCG
    GAAAAGGTCT
251 TCCTGATCAA GCACAATGCT TCACCTCAGA CAGTCTCTCA
    GTTCTATCCT
301 CCGATTGCGA ATCGTCTACC CGAAACAATT ATTGAGCAAT
    TTCTTCCTGC
351 AGAATTCATT GTTGCTACAG AACTGTTAGA ACAAAAGACA
    GGGAAAGAAG
401 CAGGTGTCGA TTCTGTAACA GCGTCTGTAC GTGTTCGCGT
    TTTTGATATC
451 CGTCATCATA AAATAGCTCT CATTTATCAA GAGATTATCG
    AATGCAGCCA
501 GCCTTTAACT ACCCTAGTCA ATGATTATCA TCGCTATGGC
    TGGAACTCAA
551 AACATTTTGA TTCAACGCCC ATGGGCTTAA TGCATAGCCG
    TCTTTTCCGC
601 GAAGTTGTTG CCAGAGTTGA GGGCTATGTT TGTGCTAACT
    ACTCGTAG

The PSORT algorithm predicts an inner membrane location (0.149).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 65A) and as a GST-fusion product (FIG. 65B). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 65C) and for FACS analysis.

These experiments show that cp6679 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 66

The following C. pneumoniae protein (PID 4376890) was expressed <SEQ ID 131; cp6890>:

1   MKQLLFCVCV FAMSCSAYAS RRRQDRSVMK ETFRNNYGII
    VSGQEWVKRG
51  SDGTITKVLK NGATLHEVYS GGLLHGEITL TFPHTTALDV
    VQIYDQGRLV
101 SRKTFFVNGL PSQEELFNED GTFVLTRWPD NNDSDTITKP
    YFIETTYQGH
151 VIEGSYISFN GKYSSSIHNG EGVRSVFSSN NILLSEETFN
    EGVMVKYTTF
201 YRNRDPESIT HYQNGQRHGL RLTYLQGGIP NTIEEWRYGF
    QDGTTIVFKN
251 GCKTSEIAYV KGVKEGLELR YNEQEIVAEE VSWRNDFLHG
    ERKIYAGGIQ
301 KHEWYYRGRS VSKAKFERLN AAG*

A predicted signal peptide is highlighted.

The cp6890 nucleotide sequence <SEQ ID 132> is:

1   ATGAAACAAT TACTTTTCTG TGTTTGCGTA TTTGCTATGT
    CATGTTCTGC
51  TTACGCATCC CCACGACGAC AAGATCCTTC TGTTATGAAG
    GAAACATTCC
101 GAAATAATTA TGGCATTATT GTTTCCGGTC AAGAATGGGT
    AAAGCGTGGT
151 TCTGACGGCA CCATCACCAA AGTACTCAAA AATGGAGCTA
    CCCTGCATGA
201 AGTTTATTCT GGAGGCCTCC TTCATGGGGA AATTACCTTA
    ACGTTTCCCC
251 ATACCACAGC ATTGGACGTT GTTCAAATCT ATGATCAAGG
    TAGACTCGTT
301 TCTCGCAAAA CCTTTTTTGT GAACGGTCTT CCATCTCAAG
    AAGAGCTGTT
351 CAATGAAGAT GGCACGTTTG TCCTCACACG ATGGCCGGAC
    AACAACGACA
401 GTGATACCAT CACAAAGCCT TACTTCATAG AAACGACATA
    TCAAGGGCAT
451 GTCATAGAAG GAAGTTATAC TTCCTTTAAT GGGAAATACT
    CCTCATCCAT
501 CCACAATGGA GAGGGAGTTC GTTCTGTGTT CTCCTCCAAT
    AACATCCTTC
551 TTTCTGAAGA GACCTTCAAT GAAGGTGTCA TGGTGAAATA
    TACCACATTC
601 TATCCGAATC GCGATCCCGA ATCGATTACT CATTATCAAA
    ATGGACAGCC
651 TCACGGCTTA CGGCTAACAT ATCTACAAGG TGGCATCCCC
    AATACGATAG
701 AGGAGTGGCG TTATGGCTTT CAAGACGGAA CGACCATCGT
    ATTTAAAAAT
751 GGTTGTAAGA CATCTGAGAT CGCTTATGTT AAGGGAGTGA
    AAGAAGGTTT
801 AGAACTGCGC TACAATGAAC AGGAAATTGT AGCTGAAGAA
    GTTTCTTGGC
851 GTAATGATTT TCTGCATGGA GAACGTAAGA TCTATGCTGG
    AGGAATCCAA
901 AAGCATGAAT GGTATTACCG CGGGAGATCT GTATCTAAAG
    CCAAATTCGA
951 GCGGCTAAAT GCTGCAGGAT AG

The PSORT algorithm predicts an outer membrane location (0.940).

The protein was expressed in E. coli and purified as a GST-fusion product, as shown in FIG. 66A. The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 66B) and for FACS analysis. A his-tagged protein was also expressed.

These experiments show that cp6890 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 67

The following C. pneumoniae protein (PID 6172323) was expressed <SEQ ID 133; cp0018>:

1   MKTSVSMLLA LLCSGASSIV LHAATTPLNP EDGFIGEGNT
    NTFSPKSTTD
51  AGGTTYSLTG EVLYIDPGKG GSITGTCFVE TAGDLTFLGN
    GNTLKFLSVD
101 AGANIAVAHV QGSKNLSFTD FLSLVITESP KSAVTTGKGS
    LVSLGAVQLQ
151 DINTLVLTSN ASVEDGGVIK GNSCLIQGIK NSAIFGQNTS
    SKKGGAISTT
201 QGLTIENNLG TLKFNENKAV TSGGALDLGA ASTETANHEL
    IFSQNKTSGN
251 AANGGAINCS GDLTFTDNTS LLLQENSTMQ DGGALCSTGT
    ISITGSDSIN
301 VIGNTSGQKG GAISAASLKI LGGQGGALFS NNVVTHATPL
    GGAIFINTGG
351 SLQLFTQGGD IVFEGNQVTT TAPNATTKRN VIHLESTAKW
    TGLAASQGNA
401 IYFYDPITTN DTGASDNLRI NEVSANQKLS GSIVFSGERL
    STAEAIAENL
451 TSRINQPVTL VEGSIVLKQG VTLITQGFSQ EPESTLLLDL
    GTSL*

A predicted signal peptide is highlighted.

The cp0018 nucleotide sequence <SEQ ID 134> is:

1    ATGAAGACTT CAGTTTCTAT GTTGTTGGCC CTGCTTTGCT
     CGGGGGCTAG
51   CTCTATTGTA CTCCATGCCG CAACCACTCC ACTAAATCCT
     GAAGATGGGT
101  TTATTGGGGA GGGCAATACA AATACTTTTT CTCCGAAATC
     TACAACGGAT
151  GCTGCAGGAA CTACCTACTC TCTCACAGGA GAGGTTCTGT
     ATATAGATCC
201  GGGGAAAGGT GGTTCAATTA CAGGAACTTG CTTTGTAGAA
     ACTGCTGGCG
251  ATCTTACATT TTTAGGTAAT GGAAATACCC TAAAGTTCCT
     GTCGGTAGAT
301  GCAGGTGCTA ATATCGCGGT TGCTCATGTA CAAGGAAGTA
     AGAATTTAAG
351  CTTCACAGAT TTCCTTTCTC TGGTGATCAC AGAATCTCCA
     AAATCCGCTG
401  TTACTACAGG AAAAGGTAGC CTAGTCAGTT TAGGTGCAGT
     CCAACTGCAA
451  GATATAAACA CTCTAGTTCT TACAAGCAAT GCCTCTGTCG
     AAGATGGTGG
501  CGTGATTAAA GGAAACTCCT GCTTGATTCA GGGAATCAAA
     AATAGTGCGA
551  TTTTTGGACA AAATACATCT TCGAAAAAAG GAGGGGCGAT
     CTCCACGACT
601  CAAGGACTTA CCATAGAGAA TAACTTAGGG ACGCTAAAGT
     TCAATGAAAA
651  CAAAGCAGTG ACCTCAGGAG GCGCCTTAGA TTTAGGAGCC
     GCGTCTACAT
701  TCACTGCGAA CCATGAGTTG ATATTTTCAC AAAATAAGAC
     TTCTGGGAAT
751  GCTGCAAATG GCGGAGCCAT AAATTGCTCA GGGGACCTTA
     CATTTACTGA
801  TAACACTTCT TTGTTACTTC AAGAAAATAG CACAATGCAG
     GATGGTGGAG
851  CTTTGTGTAG CACAGGAACC ATAAGCATTA CCGGTAGTGA
     TTCTATCAAT
901  GTGATAGGAA ATACTTCAGG ACAAAAAGGA GGAGCGATTT
     CTGCAGCTTC
951  TCTCAAGATT TTGGGAGGGC AGGGAGGCGC TCTCTTTTCT
     AATAACGTAG
1001 TGACTCATGC CACCCCTCTA GGAGGTGCCA TTTTTATCAA
     CACAGGAGGA
1051 TCCTTGCAGC TCTTCACTCA AGGAGGGGAT ATCGTATTCG
     AGGGGAATCA
1101 GGTCACTACA ACAGCTCCAA ATGCTACCAC TAAGAGAAAT
     GTAATTCACC
1151 TCGAGAGCAC CGCGAAGTGG ACGGGACTTG CTGCAAGTCA
     AGGTAACGCT
1201 ATCTATTTCT ATGATCCCAT TACCACCAAC GATACGGGAG
     CAAGCGATAA
1251 CTTACGTATC AATGAGGTCA GTGCAAATCA AAAGCTCTCG
     GGATCTATAG
1301 TATTTTCTGG AGAGAGATTG TCGACAGCAG AAGCTATAGC
     TGAAAATCTT
1351 ACTTCGAGGA TCAACCAGCC TGTCACTTTA GTAGAGGGGA
     GCTTAGTACT
1401 TAAACAGGGA GTGACCTTGA TCACACAAGG ATTCTCGCAG
     GAGCCAGAAT
1451 CCACGCTTCT TTTGGATCTG GGGACCTCAT TATAA

The PSORT algorithm predicts outer membrane (0.935).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 67A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 67B) and for FACS analysis.

These experiments show that cp0018 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 68

The following C. pneumoniae protein (PID 4376262) was expressed <SEQ ID 135; cp6262>:

1   MRKLRILAIV LIALSIILIA GGVVLLTVAI PGLSSVISSP
    AGMGACALGC
51  VMLALGIDVL LKKREVPIVL ASVTTTPGTG SPRSGISISG
    ADSTIRSLPT
101 YLLDEGHPQS MRKLRILAIV LIVFSIILIA SGVVLLTVAI
    PGLSSVISSP
151 AGMGACALGC VMLALGIDVL LKKREVPIVL ASVTTTPGTG
    SPRSGISISG
201 ADSTIRSLPT YPLDEGHPQS MRKLRILAIV LIVFSIILIA
    SGVVLLTVAI
251 PGLSSIISSP AEMGACALGC VMLALGIDVL LKKREVPIVV
    PAPIPEEVVI
301 DDIDEESIRL QQEAEAALAR LPEEMSAFEG YIKVVESHLE
    NMKSLPYDGH
351 GLEEKTKHQI RVVRSSLKAM VPEFLDIRRI FEEEEFFFLS
    ARKRLIDLAT
401 TLVERKILTE QLERNNLRKA FSYLYQDSIF KKIIDNFEKL
    AWKFMILSKS
451 ICRFTIIFEN HEHGVAKSLL HKNAVLLEKV IYRSLQKSYR
    DIGMSSAKMK
501 ILHGNPFFSL EDNKKTIMKE HAEMLESLSS YRKVFLALSD
    ENVVDTPSDP
551 KKWDLSGIPC RDALSEISRD EQWQKKAHLK HQESLYTQAR
    DRLTDQSSKE
601 NQKELEKAEQ EYISSWERVK KFEIERVQER IRAIQKLYPN
    ILEREEETIG
651 QETVTPTVQG TTASSDLTDI LGRIEVSSRE DNQNQESCVK
    VLRSHEVEMS
701 WEVKQEYGPK KKEFQDQMGS LERFFTEHIE ELEVLQKDYS
    KHLSYFKKVN
751 NKKEVQYAKF RLKVLESDLE GILAQTESAE SLLTQEELPI
    LATRGALEKA
801 VFKGSLCCAL ASKAKPYFEE DPRFQDSDTQ LRALTLRLQE
    AKASLEEEIK
851 RFSNLENDIA EERRILKESK QTFERAGLGV LREIAVESTY
    DLRSLTNTWE
901 GTPESEKVYF SMYLNYYNEE KRRAKTRLVE MTQRYRDFKM
    ALEAMQFNEE
951 ALLQEELSIQ APSE*

A predicted signal peptide is highlighted.

The cp6262 nucleotide sequence <SEQ ID 136> is:

1    ATGAGGAAAC TTCGTATTCT TGCGATCGTT CTCATAGCTT
     TGAGCATTAT
51   TTTGATTGCA GGTGGTGTGG TATTGCTTAC TGTAGCGATC
     CCTGGATTAA
101  GTTCAGTCAT TTCTTCCCCG GCAGGGATGG GTGCCTGTGC
     TTTGGGATGT
151  GTGATGCTTG CTTTAGGGAT CGATGTTCTT CTGAAGAAAC
     GAGAAGTCCC
201  TATAGTTCTC GCATCTGTAA CTACGACACC AGGAACTGGC
     AGCCCTAGAA
251  GTGGTATTTC TATTTCAGGA GCTGATAGCA CCATACGTTC
     TCTTCCTACG
301  TATCTCTTGG ACGAGGGACA TCCACAATCC ATGAGGAAAC
     TTCGTATTCT
351  TGCGATCGTT CTCATAGTTT TTAGCATTAT TTTGATTGCA
     AGTGGTGTGG
401  TATTGCTTAC TGTAGCGATC CCTGGATTAA GTTCAGTCAT
     TTCTTCCCCG
451  GCAGGGATGG GTGCCTGTGC TTTGGGATGT GTGATGCTTG
     CTTTAGGGAT
501  CGATGTTCTT CTGAAGAAAC GAGAAGTCCC TATAGTTCTC
     GCATCTGTAA
551  CTACGACACC AGGAACTGGC AGCCCTAGAA GTGGTATTTC
     TATTTCAGGA
601  GCTGATAGCA CCATACGTTC TCTTCCTACG TATCCCTTGG
     ACGAGGGACA
651  TCCACAATCC ATGAGGAAAC TTCGTATTCT TGCGATCGTT
     CTCATAGTTT
701  TTAGCATTAT TTTGATTGCA AGTGGTGTGG TATTGCTTAC
     TGTAGCGATC
751  CCTGGATTAA GCTCGATCAT TTCTTCCCCA GCGGAGATGG
     GTGCTTGTGC
801  TTTGGGATGT GTGATGCTTG CTTTGGGGAT CGACGTTCTT
     CTGAAGAAAC
851  GAGAAGTCCC TATAGTAGTT CCCGCACCTA TTCCTGAAGA
     AGTCGTCATA
901  GATGATATAG ATGAAGAGAG TATACGGCTG CAGCAGGAAG
     CTGAAGCCGC
951  TTTAGCAAGA CTTCCTGAGG AGATGAGTGC ATTTGAAGGT
     TACATAAAAG
1001 TTGTCGAGAG TCATTTGGAG AACATGAAAA GCCTGCCTTA
     TGATGGTCAT
1051 GGGCTAGAAG AGAAAACGAA ACATCAGATA AGAGTCGTCA
     GATCTTCTTT
1101 GAAGGCTATG GTTCCAGAAT TTTTAGATAT CAGAAGAATT
     TTTGAAGAAG
1151 AAGAGTTCTT TTTTCTCTCA GCTCGCAAAC GACTTATAGA
     TTTAGCTACT
1201 ACTTTAGTAG AGAGAAAAAT TTTAACAGAG CAACTTGAGC
     GCAATAATTT
1251 AAGGAAAGCG TTTTCTTATT TATATCAGGA CTCAATTTTT
     AAAAAAATTA
1301 TTGATAACTT CGAGAAGTTA GCATGGAAAT TTATGATTTT
     GAGTAAATCA
1351 ATTTGTCGAT TTACAATTAT TTTTGAAAAT CATGAACATG
     GTGTAGCAAA
1401 GAGCCTGTTA CACAAGAATG CAGTGTTACT GGAGAAGGTA
     ATCTATAGGA
1451 GTTTGCAAAA AAGCTATAGA GATATAGGCA TGTCATCTGC
     AAAGATGAAA
1501 ATCTTGCACG GCAACCCTTT TTTCTCTTTG GAAGATAATA
     AAAAGACGAT
1551 AATGAAAGAA CACGCAGAGA TGCTTGAAAG TCTCAGTAGC
     TATAGGAAGG
1601 TATTTTTAGC TCTATCTGAT GAGAACGTTG TAGATACACC
     TAGCGATCCA
1651 AAGAAATGGG ATTTGTCAGG AATCCCCTGT AGGGACGCGT
     TGTCTGAGAT
1701 TTCTCGTGAT GAACAGTGGC AGAAGAAAGC ACATCTAAAG
     CATCAAGAGT
1751 CCCTCTATAC GCAAGCTAGG GATCGTTTAA CAGACCAGAG
     CTCTAAAGAA
1801 AATCAGAAAG AGTTAGAGAA AGCTGAACAA GAGTACATAT
     CTTCTTGGGA
1851 ACGGGTTAAA AAATTTGAGA TTGAGAGAGT ACAGGAGAGG
     ATACGGGCAA
1901 TTCAAAAGCT TTATCCTAAT ATCCTCGAGA GAGAAGAAGA
     AACCACAGGT
1951 CAGGAGACTG TGACTCCAAC TGTTCAAGGG ACGACGGCTT
     CATCCGATTT
2001 AACAGATATT TTAGGAAGAA TAGAGGTCTC CAGTAGGGAG
     GATAATCAGA
2051 ATCAAGAGTC TTGTGTAAAA GTCTTAAGAA GTCATGAGGT
     AGAAATGAGC
2101 TGGGAAGTCA AACAAGAGTA TGGCCCTAAG AAAAAAGAAT
     TTCAGGATCA
2151 AATGGGTTCT TTAGAGAGGT TTTTTACAGA GCATATTGAA
     GAGTTAGAAG
2201 TATTACAGAA GGACTACTCT AAACACTTGT CTTATTTTAA
     AAAAGTAAAC
2251 AATAAGAAAG AGGTTCAATA TGCGAAGTTT AGGTTGAAGG
     TTTTAGAGTC
2301 AGATTTAGAA GGGATTCTAG CTCAGACTGA GAGTGCTGAG
     AGTCTGTTAA
2351 CTCAAGAAGA ACTTCCGATT CTTGCAACTC GGGGAGCCTT
     AGAGAAAGCT
2401 GTTTTCAAAG GGAGTCTATG TTGCGCGCTA GCAAGCAAAG
     CAAAACCCTA
2451 TTTTGAAGAG GATCCCAGAT TCCAAGATTC TGATACGCAA
     TTGCGAGCTC
2501 TGACTCTAAG GTTACAGGAG GCTAAGGCAA GCCTGGAAGA
     AGAGATAAAG
2551 AGATTTTCAA ATCTTGAGAA CGATATTGCA GAGGAAAGAC
     GCCTTCTTAA
2601 AGAGAGCAAG CAGACGTTCG AAAGAGCAGG TTTAGGGGTT
     CTCCGAGAAA
2651 TTGCAGTCGA GTCTACTTAT GATTTGCGTT CCTTAACAAA
     TACATGGGAA
2701 GGGACCCCAG AGAGTGAGAA GGTCTATTTT AGCATGTATC
     TTAATTATTA
2751 CAACGAAGAG AAACGTAGGG CTAAAACAAG ATTGGTTGAA
     ATGACACAGA
2801 GGTATAGAGA TTTTAAAATG GCCTTGGAAG CTATGCAGTT
     TAATGAAGAA
2851 GCCCTTTTGC AAGAGGAACT CTCTATTCAA GCTCCCAGTG
     AATAA

The PSORT algorithm predicts inner membrane (0.660).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 68A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 68B) and for FACS analysis.

These experiments show that cp6262 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 69

The following C. pneumoniae protein (PID 4376269) was expressed <SEQ ID 137; cp6269>:

1   MYQENLRLLE RLLYNSVQKS YADRLFSYEK TKMVHDTPLI
    PWEEDKEKCA
51  EAEKAFLEQQ KILLDYGKSI FWLNENDEIN LNDPWSWGLN
    TVRTRKVFQE
101 VDDSERWNHK VLIQKLEDDY EKLLEESSKE STEANKKLLS
    DLVDRLEDAK
151 TKFFLKKQEE VETRVKDLRA RYGGTVDPKQ DTEAKKKVEL
    EASLETFLDS
201 IESELVQCLE DQDIYWKEQD VKDLARTQEL EEQDIEAKRE
    EAAEDLRSLN
251 ERLKKSKTML DRAKWHIENA EDSITWWTSQ IEMKDMKARL
    KILKEDITSV
301 LPEIDEIETC LSLEELPLLT TRELLTKSYL KFKICSETLL
    KMTSVFENNI
351 YVQEYEVQLQ NLGFKLQGIS QRFGKKQDDF ANLEEQVALQ
    KKRLRELTQN
401 FEIQGFNFMK EDFKAAAKDL YIRSTAEQKM NFDVPCMELF
    RRYHEEVNKP
451 LLELMYNCAD SYRDAKKKLC SLRLDEKELL QKEIKKEEFY
    QKKQQRHADR
501 SRHTTYQKLR IAEELALELK KKI*

The cp6269 nucleotide sequence <SEQ ID 138> is:

1    ATGTACCAGG AGAATCTAAG ATTGTTGGAA AGGCTTCTTT
     ATAATAGTGT
51   TCAAAAGAGC TATGCGGATC GGCTGTTTTC CTATGAAAAG
     ACAAAGATGG
101  TGCACGATAC TCCGCTGATT CCTTGGGAAG AGGATAAGGA
     AAAATGTGCT
151  GAAGCTGAGA AAGCTTTCTT AGAGCAACAG AAGATTCTCC
     TAGATTATGG
201  AAAATCTATC TTTTGGCTGA ATGAGAACGA TGAGATCAAT
     TTAAACGATC
251  CTTGGAGTTG GGGTCTTAAT ACGGTGAGGA CTAGGAAAGT
     ATTCCAAGAG
301  GTTGACGACA GTGAACGTTG GAATCATAAG GTACTCATTC
     AAAAACTCGA
351  GGACGATTAT GAGAAACTTC TAGAGGAAAG TTCAAAAGAG
     TCTACTGAAG
401  CAAATAAGAA GCTTTTATCT GACTTAGTAG ATCGTCTTGA
     AGATGCTAAG
451  ACAAAATTTT TCCTGAAGAA ACAGGAGGAG GTGGAGACTC
     GCGTTAAGGA
501  TCTTAGACCT CGATATGGAG GCACAGTAGA TCCTAAGCAG
     GATACGGAAG
551  CTAAGAAGAA AGTCGAATTG GAGGCTAGCT TAGAAACCTT
     TTTAGATTCC
601  ATCGAATCAG AGCTAGTACA GTGTTTAGAA GATCAAGATA
     TATATTGGAA
651  AGAACAGGAT GTCAAAGATC TAGCACGTAC GCAAGAGCTC
     GAGGAACAAG
701  ATATTGAAGC GAAGAGGGAA GAAGCTGCCG AAGACCTAAG
     AAGTCTTAAT
751  GAGCGTTTAA AGAAGTCAAA AACTATGTTA GATAGGGCTA
     AATGGCATAT
801  TGAAAATGCT GAGGACAGTA TTACCTGGTG GACTAGTCAG
     ATAGAAATGA
851  AGGATATGAA AGCAAGACTG AAGATCTTAA AAGAAGATAT
     AACAAGTGTT
901  CTACCTGAAA TAGATGAGAT TGAAACGTGT TTAAGCTTAG
     AGGAGCTTCC
951  TTTGCTTACG ACCAGGGAAC TCTTAACTAA GTCCTACCTA
     AAGTTTAAGA
1001 TTTGTTCGGA AACACTATTA AAAATGACTT CTGTGTTTGA
     GAACAATATC
1051 TATGTTCAGG AGTACGAGGT TCAGCTGCAA AATCTAGGGT
     TTAAGTTACA
1101 AGGTATATCT CAGAGATTCG GAAAGAAACA AGACGATTTT
     GCGAATCTAG
1151 AGGAACAGGT TGCTTTGCAA AAGAAACGAC TCAGAGAGCT
     CACTCAGAAT
1201 TTTGAAATAC AAGGATTCAA TTTCATGAAA GAAGATTTTA
     AGGCAGCCGC
1251 TAAAGATCTT TATATAAGAA GTACAGCTGA ACAAAAGATG
     AACTTTGATG
1301 TGCCTTGCAT GGAGCTCTTC CGTAGGTATC ATGAGGAGGT
     CAACAAGCCG
1351 CTTCTTGAGT TGATGTACAA TTGTGCAGAC AGTTATAGAG
     ATGCTAAGAA
1401 AAAGCTTTGC TCTCTACGTC TTGATGAAAA AGAGTTATTA
     CAAAAAGAAA
1451 TCAAGAAAGA GGAATTTTAT CAAAAGAAAC AACAAAGGCA
     TGCAGATAGA
1501 TCACGTCATA CTACGTATCA AAAGCTACGA ATTGCTGAAG
     AGCTTGCTCT
1551 TGAGCTGAAG AAGAAAATCT AA

The PSORT algorithm predicts cytoplasmic location (0.412).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 69A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 69B) and for FACS analysis.

These experiments show that cp6269 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 70

The following C. pneumoniae protein (PID 4376270) was expressed <SEQ ID 139; cp6270>:

1   MKIPLRFLLI SLVPTLSMSN LLGAATTEEL SASNSFDGTT
    STTSFSSKTS
51  SATDGTNYVF KDSVVIENVP KTGETQSTSC FKNDAAAGDL
    NFLGGGFSFT
101 FSNIDATTAS GAAIGSEAAN KTVTLSGFSA LSFLKSPAST
    VTNGLGAINV
151 KGNLSLLDND KVLIQDNFST GDGGAINCAG SLKIANNKSL
    SFIGNSSSTR
201 GGAIHTKNLT LSSGGETLFQ GNTAPTAAGK GGAIAIADSG
    TLSISGDSGD
251 IIFEGNTIGA TGTVSHSAID LGTSADITAL RAAQGHTIYF
    YDPITVTGST
301 SVADALNINS PDTGDNKEYT GTIVFSGEKL TEAEAKDEKN
    RTSKLLQNVA
351 FKNGTVVLKG DVVLSANGFS QDANSKLIMD LGTSLVANTE
    SIELTNLEIN
401 IDSLRNGKKI KLSAATAGKD IRIDRPVVLA ISDESFYQNG
    FLNEDHSYDG
451 ILELDAGKDI VISADSRSIT AVQSPYGYQG KWTINWSTDD
    KKATVSWAKQ
501 SFNPTAEQEA PLVPNLLWGS FIDVRSFQNF IELGTEGAPY
    EKRFWVAGIS
551 NVLHRSGREN QRKFRHVSGG AVVGASTRMP GGDTLSLGFA
    QLFARDKDYF
601 MNTNFAKTYA GSLRLQHDAS LYSVVSILLG EGGLREILLP
    YVSKTLPCSF
651 YGQLSYGHTD HRMKTESLPP PPPTLSTDHT SWGGYVWAGE
    LGTRVAVENT
701 SGRGFFQEYT PFVKVQAVYA RQDSFVELGA ISRDFSDSHL
    YNLAIPLGIK
751 LEKRFAEQYY HVVAMYSPDV CRSNPKCTTT LLSNQGSWKT
    KGSNLARQAG
801 IVQASGFRSL GAAAELFGNF GFEWRGSSRS YNVDAGSKIK F*

A predicted signal peptide is highlighted.

The cp6270 nucleotide sequence <SEQ ID 140> is:

1    ATGAAGATTC CACTCCGCTT TTTATTGATA TCATTAGTAC
     CTACGCTTTC
51   TATGTCGAAT TTATTAGGAG CTGCTACTAC CGAAGAGTTA
     TCGGCTAGCA
101  ATAGCTTCGA TGGAACTACA TCAACAACAA GCTTTTCTAG
     TAAAACATCA
151  TCGGCTACAG ATGGCACCAA TTATGTTTTT AAAGATTCTG
     TAGTTATAGA
201  AAATGTACCC AAAACAGGGG AAACTCAGTC TACTAGTTGT
     TTTAAAAATG
251  ACGCTGCAGC TGGAGATCTA AATTTCTTAG GAGGGGGATT
     TTCTTTCACA
301  TTTAGCAATA TCGATGCAAC CACGGCTTCT GGAGCTGCTA
     TTGGAAGTGA
351  AGCAGCTAAT AAGACAGTCA CGTTATCAGG ATTTTCGGCA
     CTTTCTTTTC
401  TTAAATCCCC AGCAAGTACA GTGACTAATG GATTGGGAGC
     TATCAATGTT
451  AAAGGGAATT TAAGCCTATT GGATAATGAT AAGGTATTGA
     TTCAGGACAA
501  TTTCTCAACA GGAGATGGCG GAGCAATTAA TTGTGCAGGC
     TCCTTGAAGA
551  TCGCAAACAA TAAGTCCCTT TCTTTTATTG GAAATAGTTC
     TTCAACACGT
601  GGCGGAGCGA TTCATACCAA AAACCTCACA CTATCTTCTG
     GTGGGGAAAC
651  TCTATTTCAG GGGAATACAG CGCCTACGGC TGCTGGTAAA
     GGAGGTGCTA
701  TCGCGATTGC AGACTCTGGC ACCCTATCCA TTTCTGGAGA
     CAGTGGCGAC
751  ATTATCTTTG AAGGCAATAC GATAGGAGCT ACAGGAACCG
     TCTCTCATAG
801  TGCTATTGAT TTAGGAACTA GCGCTAAGAT AACTGCGTTA
     CGTGCTGCGC
851  AAGGACATAC GATATACTTT TATGATCCGA TTACTGTAAC
     AGGATCGACA
901  TCTGTTGCTG ATGCTCTCAA TATTAATAGC CCTGATACTG
     GAGATAACAA
951  AGAGTATACG GGAACCATAG TCTTTTCTGG AGAGAAGCTC
     ACGGAGGCAG
1001 AAGCTAAAGA TGAGAAGAAC CGCACTTCTA AATTACTTCA
     AAATGTTGCT
1051 TTTAAAAATG GGACTGTAGT TTTAAAAGGT GATGTCGTTT
     TAAGTGCGAA
1101 CGGTTTCTCT CAGGATGCAA ACTCTAAGTT GATTATGGAT
     TTAGGGACGT
1151 CGTTGGTTGC AAACACCGAA AGTATCGAGT TAACGAATTT
     GGAAATTAAT
1201 ATAGACTCTC TCAGGAACGG GAAAAAGATA AAACTCAGTG
     CTGCCACAGC
1251 TCAGAAAGAT ATTCGTATAG ATCGTCCTGT TGTACTGGCA
     ATTAGCGATG
1301 AGAGTTTTTA TCAAAATGGC TTTTTGAATG AGGACCATTC
     CTATGATGGG
1351 ATTCTTGAGT TAGATGCTGG GAAAGACATC GTGATTTCTG
     CAGATTCTCG
1401 CAGTATAGAT GCTGTACAAT CTCCGTATGG CTATCAGGGA
     AAGTGGACGA
1451 TCAATTGGTC TACTGATGAT AAGAAAGCTA CGGTTTCTTG
     GGCGAAGCAG
1501 AGTTTTAATC CCACTGCTGA GCAGGAGGCT CCGTTAGTTC
     CTAATCTTCT
1551 TTGGGGTTCT TTTATAGATG TTCGTTCCTT CCAGAATTTT
     ATAGAGCTAG
1601 GTACTGAAGG TGCTCCTTAC GAAAAGAGAT TTTGGGTTGC
     AGGCATTTCC
1651 AATGTTTTGC ATAGGAGCGG TCGTGAAAAT CAAAGGAAAT
     TCCGTCATGT
1701 GAGTGGAGGT GCTGTAGTAG GTGCTAGCAC GAGGATGCCG
     GGTGGTGATA
1751 CCTTGTCTCT GGGTTTTGCT CAGCTCTTTG CGCGTGACAA
     AGACTACTTT
1801 ATGAATACCA ATTTCGCAAA GACCTACGCA GGATCTTTAC
     GTTTGCAGCA
1851 CGATGCTTCC CTATACTCTG TGGTGAGTAT CCTTTTAGGA
     GAGGGAGGAC
1901 TCCGCGAGAT CCTGTTGCCT TATGTTTCCA AGACTCTGCC
     GTGCTCTTTC
1951 TATGGGCAGC TTAGCTACGG CCATACGGAT CATCGCATGA
     AGACCGAGTC
2001 TCTACCCCCC CCCCCCCCGA CGCTCTCGAC GGATCATACT
     TCTTGGGGAG
2051 GATATGTCTG GGCTGGAGAG CTGGGAACTC GAGTTGCTGT
     TGAAAATACC
2101 AGCGGCAGAG GATTTTTCCA AGAGTACACT CCATTTGTAA
     AAGTCCAAGC
2151 TGTTTACGCT CGCCAAGATA GCTTTGTAGA ACTAGGAGCT
     ATCAGTCGTG
2201 ATTTTAGTGA TTCGCATCTT TATAACCTTG CGATTCCTCT
     TGGAATCAAG
2251 TTAGAGAAAC GGTTTGCAGA GCAATATTAT CATGTTGTAG
     CGATGTATTC
2301 TCCAGATGTT TGTCGTAGTA ACCCCAAATG TACGACTACC
     CTACTTTCCA
2351 ACCAAGGGAG TTGGAAGACC AAAGGTTCGA ACTTAGCAAG
     ACAGGCTGGT
2401 ATTGTTCAGG CCTCAGGTTT TCGATCTTTG GGAGCTGCAG
     CAGAGCTTTT
2451 CGGGAACTTT GGCTTTGAAT GGCGGGGATC TTCTCGTAGC
     TATAATGTAG
2501 ATGCGGGTAG CAAAATCAAA TTTTAG

The PSORT algorithm predicts outer membrane (0.92).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 70A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot and for FACS analysis (FIG. 70B).

The cp6270 protein was also identified in the 2D-PAGE experiment (Cpn0013).

These experiments show that cp6270 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 71

The following C. pneumoniae protein (PID 4376402) was expressed <SEQ ID 141; cp6402>:

1   MNVADLLSHL ETLLSSKIFQ DYGPNGLQVG DPQTPVKKIA
    VAVTADLETI
51  KQAVAAEANV LIVHHGIFWK GMPYPITGMI HKRIQLLIEH
    NIQLIAYHLP
101 LDAHPTLGNN WRVALDLNWH DLKPFGSSLP YLGVQGSFSP
    IDIDSFIDLL
151 SQYYQAPLKG SALGGPSRVS SAALISGGAY RELSSAATSQ
    VDCFITGNFD
201 EPAWSTALES NINFLAFGHT ATEKVGPKSL AEHLKSEFPI
    STTFIDTANP
251 F*

The cp6402 nucleotide sequence <SEQ ID 142> is:

1   ATGAATGTTG CGGATCTCCT TTCTCATCTT GAGACTCTTC
    TCTCATCAAA
51  AATATTTCAG GATTATGGAC CCAACGGACT TCAAGTTGGA
    GATCCCCAAA
101 CTCCGGTAAA GAAAATCGCT GTTGCAGTTA CCGCAGATCT
    AGAAACCATA
151 AAACAAGCTG TTGCGGCCGA AGCAAACGTT CTCATTGTAC
    ACCACGGAAT
201 TTTTTGGAAA GGTATGCCCT ATCCTATTAC CGGCATGATC
    CATAAGCGCA
251 TCCAATTACT AATAGAACAC AATATCCAAC TCATTGCCTA
    CCACCTTCCT
301 TTGGATGCTC ACCCTACCTT AGGAAATAAC TGGAGAGTTG
    CCCTGGATCT
351 AAATTGGCAT GACTTGAAGC CCTTTGGTTC TTCCCTCCCT
    TATTTAGGAG
401 TGCAAGGCTC TTTCTCTCCT ATCGATATAG ATTCTTTCAT
    TGACCTGTTA
451 TCTCAATATT ACCAAGCTCC CCTAAAAGGA TCTGCCTTGG
    GCGGCCCCTC
501 TAGAGTCTCC TCAGCAGCTC TGATCTCAGG AGGAGCTTAT
    AGAGAACTCT
551 CTTCGGCAGC CACGTCCCAA GTCGATTGCT TCATCACAGG
    AAATTTTGAT
601 GAACCTGCAT GGTCGACAGC TCTAGAAAGC AATATCAACT
    TCCTAGCATT
651 TGGACATACA GCCACAGAAA AAGTAGGTCC AAAATCTCTT
    GCAGAGCATC
701 TAAAAAGCGA ATTTCCTATT TCCACAACCT TTATAGATAC
    GGCCAACCCC
751 TTCTAA

The PSORT algorithm predicts cytoplasmic (0.158).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 71A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 71B) and for FACS analysis.

These experiments show that cp6402 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 72

The following C. pneumoniae protein (PID 4376520) was expressed <SEQ ID 143; cp6520>:

1   MKHYLSFSPS ADFFSKQGAI ETQVLFGERV LVKGSTCYAY
    SQLFHNELLW
51  KPYPGHSFRS TLVPCTPEFH IHPNVSVVSV DAFLDPWGIP
    LPFGTLLHVN
101 SQNTVIFPKD ILNHMNTIWG SGTPQCDPRH LRRLNYNFFA
    ELLIKDADLL
151 LNFPYVWGGR SVHESLEKPG VDCSGFINIL YQAQGYNVPR
    NAADQYADCH
201 WISSFENLPS GGLIFLYPKE EKRISHVMLK QDSSTLIHAS
    GGGKKVEYFI
251 LEQDGKFLDS TYLFFRNNQR GRAFFGIPRK RKAFL*

The cp6520 nucleotide sequence <SEQ ID 144> is:

1   ATGAAACACT ACCTATCATT TTCTCCTTCT GCTGATTTTT
    TCTCTAAACA
51  GGGTGCTATT GAAACTCAAG TCCTTTTTGG AGAGCGCGTC
    TTAGTCAAAG
101 GGAGCACCTG CTATGCATAT TCCCAATTAT TCCACAATGA
    GCTGTTATGG
151 AAGCCCTATC CAGGTCATAG CTTTCGTTCT ACCCTAGTCC
    CCTGCACTCC
201 TGAATTTCAT ATCCATCCAA ATGTTTCTGT GGTTTCTGTG
    GATGCATTTT
251 TAGATCCTTG GGGGATCCCT CTTCCTTTTG GAACTTTACT
    CCATGTGAAT
301 TCTCAAAATA CCGTTATTTT CCCTAAGGAT ATTCTCAATC
    ATATGAACAC
351 CATCTGGGGC TCCGGCACAC CTCAATGCGA TCCTAGACAT
    CTACGTCGTC
401 TAAATTATAA CTTCTTTGCT GAACTTTTAA TTAAAGACGC
    AGACCTTTTA
451 CTGAACTTTC CCTATGTATG GGGAGGACGG TCTGTACACG
    AAAGTCTGGA
501 AAAGCCGGGT GTTGATTGTT CGGGATTTAT CAATATCCTT
    TACCAGGCAC
551 AGGGATACAA CGTCCCTAGA AACGCTGCAG ATCAATATGC
    GGATTGTCAT
601 TGGATCTCTA GCTTTGAGAA CCTTCCTTCT GGTGGGTTAA
    TATTTCTTTA
651 CCCTAAAGAA GAAAAGCGTA TTTCTCATGT TATGTTGAAA
    CAGGATAGTT
701 CCACCCTCAT TCATGCTTCT GGTGGAGGGA AAAAAGTGGA
    GTATTTCATT
751 TTAGAACAAG ATGGGAAGTT TTTAGATTCG ACTTATCTAT
    TTTTTAGAAA
801 TAATCAGAGG GGACGGGCAT TTTTTGGGAT CCCTAGAAAA
    AGAAAAGCCT
851 TTCTGTAA

The PSORT algorithm predicts cytoplasmic (0.265).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 72A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 72B) and for FACS analysis.

These experiments show that cp6520 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 73

The following C. pneumoniae protein (PID 4376567) was expressed <SEQ ID 145; cp6567>:

1   MTSPIPFQSS GDASFLAEQP QQLPSTSESQ LVTQLLTMMK
    HTQALSETVL
51  QQQRDRLPTA SILLQVGGAP TGGAGAPFQP GPADDHHHPI
    PPPVVPAQIE
101 TEITTIRSEL QLMRSTLQQS TKGARTGVLV VTAILMTISL
    LAIIIIILAV
151 LGFTGVLPQV ALLMQGETNL IWAMVSGSII CFIALIGTLG
    LILTNKNTPL
201 PAS*

The cp6567 nucleotide sequence <SEQ ID 146> is:

1   ATGACCTCAC CGATCCCCTT TCAGTCTAGT GGCGATGCCT
    CTTTCCTTGC
51  CGAGCAGCCA CAGCAACTCC CGTCTACTTC TGAATCTCAG
    CTAGTAACTC
101 AATTGCTAAC CATGATGAAG CATACTCAAG CATTATCCGA
    AACGGTTCTT
151 CAACAACAAC GCGATCGATT ACCAACCGCA TCTATTATCC
    TTCAAGTAGG
201 AGGAGCTCCT ACAGGAGGAG CGGGTGCGCC TTTTCAACCA
    GGACCGGCAG
251 ATGATCATCA TCATCCCATA CCGCCGCCTG TTGTACCAGC
    TCAAATAGAA
301 ACAGAAATCA CCACTATAAG ATCCGAGTTA CAGCTCATGC
    GATCTACTCT
351 ACAACAAAGC ACAAAAGGAG CTCGTACAGG AGTTCTAGTG
    GTTACTGCAA
401 TCTTAATGAC GATCTCCTTA TTGGCTATTA TTATCATAAT
    ACTAGCTGTG
451 CTTGGATTTA CGGGCGTCTT GCCTCAAGTA GCTTTATTGA
    TGCAGGGTGA
501 AACAAATCTG ATTTGGGCTA TGGTGAGCGG TTCTATTATT
    TGCTTTATTG
551 CGCTAATTGG AACTCTAGG ATTAATTTTAA CAAATAAGAA
    CACGCCTCTA
601 CCGGCTTCTT AA

The PSORT algorithm predicts inner membrane (0.694).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 73A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 73B) and for FACS analysis.

These experiments show that cp6567 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 74

The following C. pneumoniae protein (PID 4376576) was expressed <SEQ ID 147; cp6576>:

1   MLIMRNKVIL QISILALIQT PLTLFSTEKV KEGHVVVDSI
    TIITEGENAS
51  NKHPLPKLKT RSGALFSQLD FDEDLRILAK EYDSVEPKVE
    FSEGKTNIAL
101 HLIAKPSIRN IHISGNQVVP EHKILKTLQI YRNDLFEREK
    FLKGLDDLRT
151 YYLKRGYFAS SVDYSLEHNQ EKGHIDVLIK INEGPCGKIK
    QLTFSGISRS
201 EKSDIQEFIQ TKQHSTTTSW FTGAGLYHPD IVEQDSLAIT
    NYLHNNGYAD
251 AIVNSHYDLD DKGNILLYMD IDRESRYTLG HVHIQGFEVL
    PKRLIEKQSQ
301 VGPNDLYCPD KIWDGAHKIK QTYAKYGYIN TNVDVLFIPH
    ATRPIYDVTY
351 EVSFGSPYKV GLIKITGNTH TKSDVILHET SLFPGDTFNR
    LKLEDTEQRL
401 RNTGYFQSVS VYTVRSQLDP MGNADQYRDI FVEVKETTTG
    NLGLFLGFSS
451 LDNLFGGIEL SESNFDLFGA RNIFSKGFRC LRGGGEHLFL
    KANFGDKVTD
501 YILKWTKPHF LNTPWILGIE LDKSINRALS KDYAVQTYGG
    NVSTTYILNE
551 HLKYGLFYRG SQTSLHEKRK FLLGPNIDSN KGFVSAAGVN
    LNYDSVDSPR
601 TPTTGIRGGV TFEVSGLGGT YHFIKLSLNS SIYRKLTRKG
    ILKIKGEAQF
651 IKPYSNTTAE GVPVSERFFL GGETTVRGYK SFIIGPKYSA
    TEPQGGLSSL
701 LISEEFQYPL IRQPNISAFV FLDSGFVGLQ EYKISLKDLR
    SSAGFGLRFD
751 VMNNVRVMLG FGWPFRPTET LNGEKIDVSQ RFFFALGGMF *

A predicted signal peptide is highlighted.

The cp6576 nucleotide sequence <SEQ ID 148> is:

1    ATGCTCATCA TGCGAAATAA AGTTATCTTG CAAATATCTA
     TTCTAGCGTT
51   AATCCAAACC CCTTTAACTT TATTTTCTAC TGAAAAAGTT
     AAAGAAGGCC
101  ATGTGGTGGT AGACTCTATC ACAATCATAA CGGAAGGAGA
     AAATGCTTCA
151  AATAAACATC CCTTACCCAA ATTAAAGACC AGAAGTGGGG
     CTCTTTTTTC
201  TCAATTAGAT TTTGATGAAG ACTTGAGAAT TCTAGCTAAA
     GAATACGACT
251  CTGTTGAGCC TAAAGTAGAA TTTTCTGAAG GGAAAACTAA
     CATAGCCCTT
301  CACCTAATAG CTAAACCCTC AATTCGAAAT ATTCATATCT
     CAGGAAATCA
351  AGTCGTTCCT GAACATAAAA TTCTTAAAAC CCTACAAATT
     TACCGTAATG
401  ATCTCTTTGA ACGAGAAAAA TTTCTTAAGG GTCTTGATGA
     TCTAAGAACG
451  TATTATCTCA AGCGAGGATA TTTCGCATCC AGTGTAGACT
     ACAGTCTGGA
501  ACACAATCAA GAAAAAGGTC ACATCGATGT TTTAATTAAA
     ATCAATGAAG
551  GTCCTTGCGG GAAAATTAAA CAGCTTACGT TCTCAGGAAT
     CTCTCGATCA
601  GAAAAATCAG ATATCCAAGA ATTTATTCAA ACCAAGCAGC
     ACTCTACAAC
651  TACAAGTTGG TTTACTGGAG CTGGACTCTA TCACCCAGAT
     ATTGTTGAAC
701  AAGATAGCTT GGCAATTACG AATTACCTAC ATAATAACGG
     GTACGCTGAT
751  GCTATAGTCA ACTCTCACTA TGACCTTGAC GACAAAGGGA
     ATATTCTTCT
801  TTACATGGAT ATTGATCGAG GGTCGCGATA TACCTTAGGA
     CACGTCCATA
851  TCCAAGGGTT TGAGGTTTTG CCAAAACGCC TTATAGAAAA
     GCAATCCCAA
901  GTCGGCCCCA ATGATCTTTA TTGCCCCGAT AAAATATGGG
     ATGGGGCTCA
951  TAAGATCAAA CAAACTTATG CAAAGTATGG CTACATCAAT
     ACCAATGTAG
1001 ACGTTCTCTT CATCCCTCAC GCAACCCGCC CTATTTATGA
     TGTAACTTAT
1051 GAGGTAAGTG AAGGGTCTCC TTATAAAGTT GGGTTAATTA
     AAATTACTGG
1101 GAATACCCAT ACAAAATCTG ACGTTATTTT ACACGAAACC
     AGTCTCTTCC
1151 CAGGAGATAC ATTCAATCGC TTAAAGCTAG AAGATACTGA
     GCAACGTTTA
1201 AGAAATACAG GCTACTTCCA AAGCGTTAGT GTCTATACAG
     TTCGTTCTCA
1251 ACTTGATCCT ATGGGCAATG CGGATCAATA CCGAGATATT
     TTTGTAGAAG
1301 TCAAAGAAAC AACAACAGGA AACTTAGGCT TATTCTTAGG
     ATTTAGTTCT
1351 CTTGACAATC TTTTTGGAGG AATTGAACTA TCTGAAAGTA
     ATTTTGATCT
1401 ATTTGGAGCT AGAAATATAT TTTCTAAAGG TTTTCGTTGT
     CTAAGAGGCG
1451 GTGGAGAACA TCTATTCTTA AAAGCCAACT TCGGGGACAA
     AGTCACAGAC
1501 TATACTTTGA AGTGGACCAA ACCTCATTTT CTAAACACTC
     CTTGGATTTT
1551 AGGAATTGAA TTAGATAAAT CAATTAACAG AGCATTATCT
     AAAGATTATG
1601 CTGTCCAAAC CTATGGCGGG AACGTCAGCA CAACGTATAT
     CTTGAACGAA
1651 CACCTGAAAT ACGGTCTATT TTATCGAGGA AGTCAAACGA
     GTTTACATGA
1701 AAAACGTAAG TTCCTCCTAG GGCCAAATAT AGACAGCAAT
     AAAGGATTTG
1751 TCTCTGCTGC AGGTGTCAAC TTGAATTACG ATTCTGTAGA
     TAGTCCTAGA
1801 ACTCCAACTA CAGGGATTCG CGGGGGGGTG ACTTTTGAGG
     TTTCTGGTTT
1851 GGGAGGAACT TATCATTTTA CAAAACTCTC TTTAAACAGC
     TCTATCTATA
1901 GAAAACTTAC GCGTAAAGGT ATTTTGAAAA TCAAAGGGGA
     AGCTCAATTT
1951 ATTAAACCCT ATAGCAATAC TACAGCTGAA GGAGTTCCTG
     TCAGTGAGCG
2001 CTTCTTCCTA GGTGGAGAGA CTACAGTTCG GGGATATAAA
     TCCTTTATTA
2051 TCGGTCCAAA ATACTCTGCT ACAGAACCTC AGGGAGGACT
     CTCTTCGCTC
2101 CTTATTTCAG AAGAGTTTCA ATACCCTCTC ATCAGACAAC
     CTAATATTAG
2151 TGCCTTTGTA TTCTTAGACT CAGGTTTTGT CGGTTTACAA
     GAGTATAAGA
2201 TTTCGTTAAA AGATCTACGT AGTAGTGCTG GATTTGGTCT
     GCGCTTCGAT
2251 GTAATGAATA ATGTTCCTGT TATGTTAGGA TTTGGTTGGC
     CCTTCCGTCC
2301 AACCGAGACT TTGAATGGAG AAAAAATTGA TGTATCTCAG
     CGATTCTTCT
2351 TTGCTTTAGG GGGCATGTTC TAA

The PSORT algorithm predicts outer membrane (0.7658).

The protein was expressed in E. coli and purified as GST-fusion (FIG. 74A), his-tag and his-tag/GST-fusion products. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 74B) and for FACS analysis (FIG. 74C).

The cp6576 protein was also identified in the 2D-PAGE experiment (Cpn0300).

These experiments show that cp6576 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 75

The following C. pneumoniae protein (PID 4376607) was expressed <SEQ ID 149; cp6607>:

1   MNKRQKDKLK ICVIISTLIL VGIFARAPRG DTFKTFLKSE
    EAIIYSNQCN
51  EDMRKILCDA IEHADEEIFL RIYNLSEPKI QQSLTRQAQA
    KNKVTIYYQK
101 FKIPQILKQA SNVTLVEQPP AGRKLMHQKA LSIDKKDAWL
    GSANYTLLSL
151 RLDNNLILGM HSSELCDLII TNTSGDFSIK DQTGKYFVLP
    QDRKIAIQAV
201 LEKIQTAQKT IQVAMFALTH SEIIQALHQA KQRGIHVDII
    IDRSHSKLTF
251 KQLRQLNINK DFVSINTAPC TLHHKFAVID NKTLLAGSIN
    WSKGRFSLND
301 ESLIILENLT KQQNQCLRMI WKDLAKHSEH PTVDDEEKEI
    IEKSLPVEFQ
351 EAA*

A predicted signal peptide is highlighted.

The cp6607 nucleotide sequence <SEQ ID 150> is:

1    ATGAATAAAA GACAAAAAGA TAAATTAAAA ATCTGTGTTA
     TTATTAGCAC
51   GTTGATTTTA GTAGGAATTT TTGCAAGAGC TCCTCGTGGT
     GACACTTTTA
101  AGACTTTTTT AAAGTCTGAA GAAGCTATCA TCTACTCAAA
     TCAATGCAAT
151  GAGGACATGC GTAAAATTCT ATGCGATGCT ATAGAACACG
     CTGATGAAGA
201  GATCTTCCTA CGTATTTATA ACCTCTCAGA ACCCAAGATC
     CAACAGAGTT
251  TAACTCGACA AGCTCAAGCA AAAAACAAAG TTACGATCTA
     CTATCAAAAA
301  TTTAAAATTC CCCAAATCTT AAAGCAAGCC AGCAATGTAA
     CTTTAGTCGA
351  GCAACCTCCA GCAGGGCGTA AACTGATGCA TCAAAAAGCT
     CTTTCCATAG
401  ATAAGAAAGA TCGTTGGCTA GGATCTGCGA ACTACACCAA
     TCTTTCTCTA
451  CGTTTAGATA ATAATCTCAT TCTAGGAATG CATAGCTCGG
     AGCTCTGTGA
501  TCTCATTATC ACAAATACCT CTGGAGACTT TTCTATAAAG
     GATCAAACAG
551  GAAAGTATTT TCTTCTTCCT CAAGATCGTA AAATTGCAAT
     ACAAGCTGTA
601  CTCGAAAAAA TCCAGACAGC TCAGAAAACC ATCCAAGTTG
     CTATGTTTGC
651  TCTGACCCAC TCGGAGATTA TTCAAGCCTT ACATCAAGCA
     AAACAACGAG
701  GAATCCATGT AGATATTATC ATTGATAGAA GTCATAGCAA
     ACTTACTTTT
751  AAGCAATTAC GACAATTAAA TATCAATAAA GACTTTGTTT
     CTATAAATAC
801  CGCACCCTGT ACTCTTCACC ATAAGTTTGC AGTTATAGAT
     AATAAAACTC
851  TACTTGCAGG ATCTATAAAT TGGTCTAAAG GAAGATTCTC
     CTTAAATGAT
901  GAAAGCTTGA TCATACTGGA AAACCTGACC AAACAACAAA
     ATCAGAAACT
951  TCGAATGATT TGGAAAGATC TAGCTAAGCA TTCAGAACAT
     CCTACAGTAG
1001 ACGATGAAGA AAAAGAAATT ATAGAAAAAA GTCTTCCAGT
     AGAAGAGCAA
1051 GAAGCAGCGT GA

The PSORT algorithm predicts periplasmic (0.934).

The protein was expressed in E. coli and purified as a his-tagged product (FIG. 75A) and also as a GST-fusion. The GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 75B) and for FACS analysis.

These experiments show that cp6607 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 76

The following C. pneumoniae protein (PID 4376624) was expressed <SEQ ID 151; cp6624>:

1   MDAKMGYIFK VMRWIFCFVA CGITFGCTNS GFQNANSRPC
    ILSMNRMIHD
51  CVERVVGNRL ATAVLIKGSL DPHAYEMVKG DKDKIAGSAV
    IFCNGLGLEH
101 TLSLRKHLEN NPNSVKLGER LIARGAFVPL EEDGICDPHI
    WMDLSIWKEA
151 VIEITEVLIE KFPEWSAEFK ANSEELVCEM SILDSWAKQC
    LSTIPENLRY
201 LVSGHNAFSY FTRRYLATPE EVASGAWRSR CISPEGLSPE
    AQISVRDIMA
251 VVDYINEHDV SVVFPEDTLN QDALKKIVSS LKKSHLVRLA
    QKPLYSDNVD
301 DNYFSTFKHN VCLITEELGG VALECQR*

The cp6624 nucleotide sequence <SEQ ID 152> is:

1   ATGGATGCGA AAATGGGATA TATATTTAAA GTGATGCGTT
    GGATTTTCTG
51  TTTCGTGGCA TGTGGTATAA CTTTTGGATG TACCAATTCT
    GGGTTTCAGA
101 ATGCAAATTC AGCTCCTTGT ATACTATCCA TGAATCGCAT
    GATTCATGAT
151 TGTGTTGAAA GAGTCGTGGG GAATAGGCTT GCTACCGCTG
    TTTTGATCAA
201 TGGATCCTTA GACCCTCATG CGTATGAGAT GGTTAAAGGG
    GATAAGGACA
251 AGATTGCTGG AAGTGCCGTA ATTTTTTGTA ACGGCCTGGG
    TCTTGAGCAT
301 ACATTAAGTT TGCGGAAGCA TTTAGAAAAT AATCCCAATA
    GTGTCAAGTT
351 AGGGGAGCGG TTGATAGCGC GTGGGGCCTT TGTTCCTCTA
    GAAGAAGACG
401 GTATTTGCGA TCCTCATATC TGGATGGATC TTTCTATTTG
    GAAGGAAGCT
451 GTCATAGAAA TTACAGAAGT TCTCATTGAA AAGTTCCCTG
    AATGGTCTGC
501 TGAATTTAAA GCAAATAGTG AGGAACTTGT TTGTGAAATG
    TCTATTTTAG
551 ATTCTTGGGC GAAACAATGC TTGAGCACAA TTCCTGAAAA
    TTTACGGTAT
601 CTTGTCTCAG GTCATAATGC GTTCAGTTAC TTTACACGTC
    GCTATTTAGC
651 TACTCCTGAA GAAGTGGCTT CCGGAGCATG GAGGTCTCGT
    TGTATTTCTC
701 CTGAGGGTCT ATCTCCAGAA GCTCAAATCA GTGTTCGTGA
    TATTATGGCG
751 GTTGTAGATT ATATTAATGA GCATGATGTC AGTGTGGTTT
    TCCCTGAGGA
801 TACTCTGAAC CAAGATGCGT TGAAAAAAAT TGTTTCTTCT
    CTGAAGAAAA
851 GTCATTTAGT TCGTCTAGCT CAAAAACCAT TGTATAGTGA
    TAATGTGGAC
901 GACAATTATT TTAGCACCTT TAAACATAAT GTCTGCCTTA
    TCACAGAAGA
951 ATTAGGAGGG GTGGCTCTTG AATGTCAAAG ATGA

The PSORT algorithm predicts inner membrane (0.168).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 76A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 76B) and for FACS analysis.

The cp6624 protein was also identified in the 2D-PAGE experiment.

These experiments show that cp6624 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 77

The following C. pneumoniae protein (PID 4376728) was expressed <SEQ ID 153; cp6728>:

1   MKSSVSWLFF SSIPLFSSLS IVAAEVTLDS SNNSYDGSNG
    TTFTVESTTD
51  AAAGTTYSLL SDVSFQNAGA LGIPLASGCF LEAGGDLTFQ
    GNQHALKFAF
101 INAGSSAGTV ASTSAADKNL LENDFSRLSI ISCPSLLLSP
    TGQCALKSVG
151 NLSLTGNSQI IFTQNFSSDN GGVINTKNFL LSGTSQFASF
    SRNQAFTGKQ
201 GGVVYATGTI TIENSPGIVS FSQNLAKGSG GALYSTDNCS
    ITDNFQVIFD
251 GNSAWEAAQA QGGAICCTTT DKTVTLTGNK NLSFTNNTAL
    TYGGAISGLK
301 VSISAGGPTL FQSNISGSSA GQGGGGAINI ASAGELALSA
    TSGDITFNNN
351 QVTNGSTSTR NAINIIDTAK VTSIRAATGQ SIYFYDPITN
    PGTAASTDTL
401 NLNLADANSE IEYGGAIVFS GEKLSPTEKA IAANVTSTIR
    QPAVLARGDL
451 VLRDGVTVTF KDLTQSPGSR ILMDGGTTLS AKEANLSLNG
    LAVNLSSLDG
501 TNKAALKTEA ADKNISLSGT IALIDTEGSF YENHNLKSAS
    TYPLLELTTA
551 GANGTITLGA LSTLTLQEPE THYGYQGNWQ LSWANATSSK
    IGSINWTRTG
601 YIPSPERKSN LPLNSLWGNF IDIRSINQLI ETKSSGEPFE
    RELWLSGIAN
651 FFYRDSMPTR HGFRHISGGY ALGITATTPA EDQLTFAFCQ
    LFARDRNHIT
701 GKNHGDTYGA SLYFHHTEGL FDIANFLWGK ATRAPWVLSE
    ISQIIPLSFD
751 AKFSYLHTDN HMKTYYTDNS IIKGSWRNDA FCADLGASLP
    FVISVPYLLK
801 EVEPFVKVQY IYAHQQDFYE RHAEGRAFNK SELINVEIPI
    GVTFERDSKS
851 EKGTYDLTLM YILDAYRRNP KCQTSLIASD ANWMAYGTNL
    ARQGFSVRAA
901 NHFQVNPHME LFGQFAFEVR SSSRNYNTNL GSKFCF*

The cp6728 nucleotide sequence <SEQ ID 154> is:

1    ATGAAGTCCT CTGTCTCTTG GTTGTTCTTT TCTTCAATCC
     CGCTCTTTTC
51   ATCGCTCTCT ATAGTCGCGG CAGAGGTGAC CTTAGATAGC
     AGCAATAATA
101  GCTATGATGG ATCTAACGGA ACTACCTTCA CGGTCTTTTC
     CACTACGGAC
151  GCTGCTGCAG GAACTACCTA TTCCTTACTT TCCGACGTAT
     CCTTTCAAAA
201  TGCAGGGGCT TTAGGAATTC CCTTAGCCTC AGGATGCTTC
     CTAGAAGCGG
251  GCGGCGATCT TACTTTCCAA GGAAATCAAC ATGCACTGAA
     GTTTGCATTT
301  ATCAATGCGG GCTCTAGCGC TGGAACTGTA GCCAGTACCT
     CAGCAGCAGA
351  TAAGAATCTT CTCTTTAATG ATTTTTCTAG ACTCTCTATT
     ATCTCTTGTC
401  CCTCTCTTCT TCTCTCTCCT ACTGGACAAT GTGCTTTAAA
     ATCTGTGGGG
451  AATCTATCTC TAACTGGCAA TTCCCAAATT ATATTTACTC
     AGAACTTCTC
501  GTCAGATAAC GGCGGTGTTA TCAATACGAA AAACTTCTTA
     TTATCAGGGA
551  CATCTCAGTT TGCGAGCTTT TCGAGAAACC AAGCCTTCAC
     AGGGAAGCAA
601  GGCGGTGTAG TTTACGCTAC AGGAACTATA ACTATCGAGA
     ACAGCCCTGG
651  GATAGTTTCC TTCTCTCAAA ACCTAGCGAA AGGATCTGGC
     GGTGCTCTGT
701  ACAGCACTGA CAACTGTTCG ATTACAGATA ACTTTCAAGT
     GATCTTTGAC
751  GGCAATAGTG CTTGGGAAGC CGCTCAAGCT CAGGGCGGGG
     CTATTTGTTG
801  CACTACGACA GATAAAACAG TGACTCTTAC TGGGAACAAA
     AACCTCTCTT
851  TCACAAATAA TACAGCATTG ACATATGGCG GAGCCATCTC
     TGGACTCAAG
901  GTCAGTATTT CCGCTGGAGG TCCTACTCTA TTTCAAAGTA
     ATATCTCAGG
951  AAGTAGCGCC GGTCAGGGAG GAGGAGGAGC GATCAATATA
     GCATCTGCTG
1001 GGGAACTCGC TCTCTCTGCT ACTTCTGGAG ATATTACCTT
     CAATAACAAC
1051 CAAGTCACCA ACGGAAGCAC AAGTACAAGA AACGCAATAA
     ATATCATTGA
1101 TACCGCTAAA GTCACATCGA TACGAGCTGC TACGGGGCAA
     TCTATCTATT
1151 TCTATGATCC CATCACAAAT CCAGGAACCG CAGCTTCTAC
     CGACACATTG
1201 ACCTTAAACT TAGCAGATGC GAACAGTGAG ATGGAGTATG
     GGGGTGCGAT
1251 TGTCTTTTCT GGAGAAAAGC TTTCCCCTAC AGAAAAAGCA
     ATCGCTGCAA
1301 ACGTCACCTC TACTATCCGA CACCCTGCAG TATTAGCGCG
     GGGAGATCTT
1351 GTACTTCGTG ATGGAGTCAC CGTACCTTTC AAGGATCTGA
     CTCAAAGTCC
1401 AGGATCCCGC ATCTTACTGG ATGGGGGGAC TACACTTAGT
     GCTAAAGAGG
1451 CACATCTTTC GCTTACTGGC TTAGCAGTAA ATCTCTCCTC
     TTTAGATGGA
1501 ACCAACAAGG CAGCTTTAAA AACAGAAGCT GCAGATAAAA
     ATATCAGCCT
1551 ATCGGGAACG ATTGCGCTTA TTGACACGGA AGGGTCATTC
     TATGAGAATC
1601 ATAACTTAAA AAGTGCTAGT ACCTATCCTC TTCTTGAACT
     TACCACCGCA
1651 GGAGCCAACG GAACGATTAC TCTGGGAGCT CTTTCTACCC
     TGACTCTTCA
1701 AGAACCTGAA ACCCACTACG GGTATCAAGG AAACTGGCAG
     TTGTCTTGGG
1751 CAAATGCAAC ATCCTCAAAA ATAGGAAGCA TCAACTGGAC
     CCGTACAGGA
1801 TACATTCCTA GTCCTGAGAG AAAAAGTAAT CTCCCTCTAA
     ATAGCTTATG
1851 GGGAAACTTT ATAGATATAC GCTCGATCAA TCAGCTTATA
     GAACCCAAGT
1901 CCAGTGGGGA GCCTTTTGAG CGTGAGCTAT GGCTTTCAGG
     AATTGCGAAT
1951 TTCTTCTATA GAGATTCTAT GCCCACCCGC CATGGTTTCC
     GCCATATCAG
2001 CGGGGGTTAT GCACTAGGGA TCACAGCAAC AACTCCTGCC
     GAGGATCAGC
2051 TTACTTTTGC CTTCTGCCAG CTCTTTGCTA GAGATCGCAA
     TCATATTACA
2101 GGTAAGAACC ACGGAGATAC TTACGGTGCC TCTTTGTATT
     TCCACCATAC
2151 AGAAGGGCTC TTCGACATCG CCAATTTCCT CTGGGGAAAA
     GCAACCCGAG
2201 CTCCCTGGGT GCTCTCTGAG ATCTCCCAGA TCATTCCTTT
     ATCGTTCGAT
2251 GCTAAATTCA GTTATCTCCA TACAGACAAC CACATGAAGA
     CATATTATAC
2301 CGATAACTCT ATCATCAAGG GTTCTTGGAG AAACGATGCC
     TTCTGTGCAG
2351 ATCTTGGAGC TAGCCTGCCT TTTGTTATTT CCGTTCCGTA
     TCTTCTGAAA
2401 GAAGTCGAAC CTTTTGTCAA AGTACAGTAT ATCTATGCGC
     ATCAGCAAGA
2451 CTTCTACGAG CTGCATGCTG AAGGACGCGC TTTCAATAAA
     AGCGAGCTTA
2501 TCAACGTAGA GATTCCTATA GGCGTCACCT TCGAAAGAGA
     CTCAAAATCA
2551 GAAAAGGGAA CTTACGATCT TACTCTTATG TATATACTCG
     ATGCTTACCG
2601 ACGCAATCCT AAATGTCAAA CTTCCCTAAT AGCTAGCGAT
     GCTAACTGGA
2651 TGGCCTATGG TACCAACCTC GCACGACAAG GTTTTTCTGT
     TCGTGCTGCG
2701 AACCATTTCC AAGTGAACCC CCACATGGAA ATCTTCGGTC
     AATTCGCTTT
2751 TGAAGTACGA AGTTCTTCAC GAAATTATAA TACAAACCTA
     GGCTCTAAGT
2801 TTTGTTTCTA G

The PSORT algorithm predicts inner membrane (0.187).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 77A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 77B) and for FACS analysis.

The cp6728 protein was also identified in the 2D-PAGE experiment.

These experiments show that cp6728 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 78

The following C. pneumoniae protein (PID 4376847) was expressed <SEQ ID 155; cp6847>:

1   MFVMKKLVRL CVVLISLLPN VLFSSDLLRE EGIKKMMDKL
    IEYHVDAQEV
51  STDILSRSLS SYIQSFDPHK SYLSNQEVAV FLQSPETKKR
    LLKVYKAGNF
101 AIYRNINQLI HESIIRARQW RNEWVKNPKE LVLEASSYQI
    SKQPMQWSKS
151 LDEVKQRQRA LLLSYLSLHL AGASSSRYEG KEEQLAALCL
    RQIENHENVY
201 LGINDHGVAM DRDEEAYQFH IRVVKALAHS LDAHTAYFSK
    DEALAMRIQL
251 EKGMCGIGVV LKEDIDGVVV REIIPGGPAA KSGDLQLGDI
    IYRVDGKDIE
301 HLSFRGVLDC LRGGHGSTVV LDIHRGESDH TIALRREKIL
    LEDRRVDVSY
351 EPYGDGVIGK VTLHSFYEGE NQVSSEQDLR RAIQGLKEKN
    LLGLVLDIRE
401 NTGGFLSQAI KVSGLFMTNG VVVVSRYADG TMKCYRTVSP
    KKFYDGPLAI
451 LVSKSSASAA EIVAQTLQDY GVALVVGDEQ TYGKGTIQHQ
    TITGDASQDD
501 CFKVTVGKYY SPSGKSTQLQ GVKSDILIPS LYAEDRLGER
    FLEHPLPADC
551 CDNVLHDPLT DLDTQTRPWF QKYYLPNLQK QETLWREMLP
    QLTKNSEQRL
601 SENSNFQAFL SQIKSSEKTD LSYGSNDLQL EESINILKDM
    ILLQQCRK*

A predicted signal peptide is highlighted.

The cp6847 nucleotide sequence <SEQ ID 156> is:

1    ATGTTCGTAA TGAAAAAACT TGTCCGTCTA TGCGTAGTTC
     TTCTTTCTTT
51   ACTTCCGAAT GTATTATTTT CTTCGGATCT TTTACGAGAA
     GAGGGCATCA
101  AAAAGATGAT GGACAAGCTG ATCGAGTATC ATGTCGATGC
     TCAAGAGGTT
151  TCTACGGATA TACTCTCGCG TTCTTTATCT AGTTACATTC
     AATCTTTTGA
201  TCCTCATAAA TCTTATCTTT CAAACCAAGA GGTTGCAGTT
     TTTCTACAGT
251  CTCCGGAAAC AAAGAAACGT CTCTTAAAGA ATTATAAGGC
     AGGCAACTTT
301  GCTATTTATC GCAACATCAA TCAATTAATT CATGAGAGTA
     TTCTTCGTGC
351  CAGGCAGTGG AGAAACGAAT GGGTTAAGAA TCCAAAAGAG
     CTTGTATTGG
401  AGGCATCCTC ATATCAGATA TCGAAGCAAC CTATGCAATG
     GAGCAAATCT
451  TTAGACGAAG TGAAGCAGAG ACAACGCGCT CTACTCCTTT
     CCTATCTTTC
501  TTTACATCTT GCTGGAGCTT CTTCCTCTCG TTATGAGGGT
     AAAGAAGAGC
551  AGCTTGCTGC TCTGTGTCTA CGTCAAATCG AGAACCATGA
     GAATGTATAT
601  TTAGGTATCA ACGATCATGG TGTTGCTATG GATCGGGATG
     AAGAAGCCTA
651  CCAATTCCAT ATCCGTGTTG TTAAAGCTTT AGCTCATAGC
     TTAGATGCAC
701  ATACGGCGTA TTTCAGTAAG GACGAAGCGT TGGCGATGCG
     AATCCAACTA
751  GAAAAAGGCA TCTGTGGAAT TGGTGTTGTT CTGAAGGAAG
     ATATTGATGG
801  AGTTGTTGTT AGAGAAATCA TTCCTGGGGG ACCTGCGGCT
     AAATCTGGGG
851  ATCTTCAGCT TGGAGATATC ATCTATCGGG TGGATGGCAA
     GGATATCGAG
901  CATCTTTCTT TCCGCGGTGT TTTAGATTGT TTACGTGGAG
     GTCATGGCTC
951  TACTGTAGTC TTAGATATCC ATCGTGGGGA GAGCGATCAT
     ACGATCGCCT
1001 TGAGAAGGGA GAAAATCCTT TTAGAAGACC GTCGTGTGGA
     TGTTTCCTAT
1051 GAGCCTTATG GAGATGGTGT GATTGGGAAA GTTACGTTAC
     ATTCTTTTTA
1101 TGAAGGAGAA AATCAGGTTT CTAGTGAACA AGATCTACGT
     CGAGCGATTC
1151 AGGGATTAAA GGAGAAGAAC CTTCTTGGAT TAGTTTTAGA
     TATCCGAGAA
1201 AATACGGGTG GATTTTTATC TCAAGCGATC AAAGTTTCTG
     GTTTATTTAT
1251 GACCAATGGC GTTGTGGTTG TATCTCGCTA TGCTGATGGT
     ACCATGAAGT
1301 GCTACCGCAC AGTATCTCCT AAAAAATTCT ATGATGGTCC
     TTTGGCTATT
1351 TTAGTATCTA AAAGTTCCGC ATCAGCAGCG GAGATTGTAG
     CACAAACTCT
1401 CCAAGATTAT GGAGTTGCTT TAGTTGTTGG AGATGAGCAG
     ACCTATGGGA
1451 AGGGAACGAT TCAGCATCAA ACAATTACTG GAGATGCCTC
     TCAGGACGAT
1501 TGTTTTAAGG TTACTGTAGG GAAATATTAT TCCCCTTCTG
     GGAAATCGAC
1551 TCAACTTCAG GGAGTAAAAT CCGATATTTT AATTCCTTCT
     CTCTATGCTG
1601 AAGATCGTCT AGGAGAGCGT TTTCTAGAGC ATCCCTTACC
     TGCAGATTGC
1651 TGTGATAATG TACTTCACGA TCCTCTCACG GAGTTGGATA
     CTCAAACACG
1701 TCCTTGGTTT CAAAAATACT ATCTTCCTAA TCTACAAAAG
     CAAGAGACTC
1751 TTTGGAGAGA GATGCTACCT CAGCTTACGA AAAACAGTGA
     GCAAAGGCTT
1801 TCTGAGAATT CGAATTTTCA GGCATTTTTG TCGCAGATAA
     AATCATCTGA
1851 AAAAACGGAC CTATCCTATG GTTCCAATGA TTTACAATTG
     GAAGAGTCGA
1901 TAAACATTTT GAAGGACATG ATTTTATTAC AACAGTGTAG
     AAAATAA

The PSORT algorithm predicts periplasmic (0.932).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 78A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 78B) and for FACS analysis.

These experiments show that cp6847 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 79

The following C. pneumoniae protein (PID 4376969) was expressed <SEQ ID 157; cp6969>:

1   MRLFSLGTIY LFFSLALSSC CGYSILNSPY HLSSLGKSLL
    QERIFIAPIK
51  EDPHGQLCSA LTYELSKRSF AISGRSSCAG YTLKVELLNG
    IDKNIGFTYA
101 PNKLGDKTHR HFIVSNEGRL SLSAKVQLIN NDTQEVLIDQ
    CVARESVDFD
151 FEPDLTGANA HEFALGQFEM HSEAIKSARR ILSIRLAETI
    AQQVYYDLF*

A predicted signal peptide is highlighted.

The cp6969 nucleotide sequence <SEQ ID 158> is:

1   ATGAGATTGT TTTCTTTAGG CACGATTTAT CTTTTTTTTT
    CTCTAGCACT
51  TTCGTCATGC TGTGGTTACT CTATTTTAAA CAGCCCGTAT
    CACTTATCGT
101 CTTTAGGTAA GTCTTTATTA CAGGAAAGAA TTTTCATTGC
    TCCCATAAAA
151 GAAGATCCTC ATGGTCAGCT CTGCTCAGCT CTAACTTATG
    AGCTTAGTAA
201 GCGTTCTTTT GCTATCTCTG GAAGGAGTTC TTGCGCAGGC
    TATACTCTTA
251 AAGTAGAGCT TCTGAATGGT ATTGACAAGA ATATAGGTTT
    TACGTATGCC
301 CCAAATAAAC TCGGAGATAA GACTCACAGG CATTTTATAG
    TCTCTAATGA
351 AGGCAGACTA TCACTATCTG CAAAAGTACA GCTTATCAAT
    AATGACACTC
401 AAGAAGTCCT TATAGACCAA TGTGTTGCTC GAGAGTCTGT
    AGACTTTGAC
451 TTTGAGCCTG ACTTAGGAAC AGCAAACGCT CATGAATTTG
    CTTTAGGCCA
501 ATTTGAAATG CATAGTGAAG CCATAAAAAG TGCTCGCCGT
    ATACTATCTA
551 TACGCCTAGC CGAGACGATT GCTCAACAGG TATACTATGA
    CCTTTTTTGA

The PSORT algorithm predicts inner membrane (0.126).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 79A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 79B) and for FACS analysis.

These experiments show that cp6969 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 80

The following C. pneumoniae protein (PID 4377109) was expressed <SEQ ID 159; cp7109>:

1   MKKTCCQNYR SIGVVFSVVL FVLTTQTLFA GHFIDIGTSG
    LYSWARGVSG
51  DGRVVVGYEG GNAFKYVDGE KFLLEGLVPR SEALVFKASY
    DGSVIIGISD
101 QDPSCRAVKW VNGALVDLGI FSEGMQSFAE GVSSDGKTIV
    GCLYSDDTET
151 NFAVKWDETG MVVLPNLPED RHSCAWDASE DGSVIVGDAM
    GSEEIAKAVY
201 WKDGEQHLLS NIPGAKRSSA HAVSKDGSFI VGEFISEENE
    VHAFVYHNGV
251 IKDIGTLGGD YSVATGVSRD GKVIVGHSTR TDGEYRAFKY
    VDGRMIDLGT
301 LGGSASFAFG VSDDGKTIVG KFETELGECH AFIYLDD*

A predicted signal peptide is highlighted.

The cp7109 nucleotide sequence <SEQ ID 160> is:

1    ATGAAAAAGA CATGTTGCCA AAATTACAGA TCGATAGGCG
     TTGTGTTCTC
51   TGTGGTACTT TTCGTTCTTA CAACACAGAC GCTGTTTGCA
     GGACATTTTA
101  TTGATATTGG AACTTCTGGA TTATATTCTT GGGCTCGAGG
     TGTATCTGGA
151  GATGGCCGCG TTGTCGTAGG TTATGAAGGT GGCAATGCAT
     TTAAATATGT
201  TGATGGTGAG AAATTTCTGT TAGAAGGTTT GGTCCCGAGA
     TCCGAGGCCT
251  TGGTATTTAA AGCTTCTTAT GATGGCTCTG TAATTATAGG
     AATCTCGGAT
301  CAAGATCCGT CTTGCCGCGC TGTGAAGTGG GTAAACGGTG
     CACTTGTTGA
351  TCTTGGAATA TTTTCTGAGG GAATCCAATC TTTTGCAGAG
     GGTGTTTCCA
401  GTGATGGAAA GACGATTGTA GGGTGCCTAT ATAGTGATGA
     TACAGAGACA
451  AACTTTGCTG TGAAGTGGGA TGAAACAGGA ATGGTTGTTC
     TCCCTAACTT
501  ACCAGAAGAT CGACATTCTT GCGCTTGGGA TGCCTCTGAA
     GATGGCTCTG
551  TGATTGTAGG GGACGCCATG GGTAGCGAGG AAATTGCCAA
     GGCAGTGTAC
601  TGGAAGGACG GTGAACAACA TCTGCTTTCT AATATCCCAG
     GAGCTAAAAG
651  ATCGTCAGCA CATGCAGTTT CTAAAGATGG ATCTTTTATC
     GTAGGCGAGT
701  TCATCAGTGA AGAAAATGAA GTTCATGCCT TTGTTTATCA
     CAACGGTGTT
751  ATCAAAGATA TCGGGACTTT AGGAGGAGAT TACTCTGTAG
     CAACTGGAGT
801  TTCTAGGGAT GGTAAGGTCA TCGTGGGTCA TTCTACAAGA
     ACAGATGGTG
851  AATACCGTGC ATTTAAATAT GTGGATGGAA GAATGATAGA
     TTTGGGGACT
901  TTAGGAGGTT CAGCATCTTT TGCTTTTGGT GTTTCTGACG
     ATGGCAAAAC
951  AATCGTAGGA AAATTTGAAA CAGAGCTAGG AGAATGTCAT
     GCCTTTATCT
1001 ACCTTGATGA TTAG

The PSORT algorithm predicts outer membrane (0.887).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 80A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 80B) and for FACS analysis.

These experiments show that cp7109 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 81

The following C. pneumoniae protein (PID 4377110) was expressed <SEQ ID 161; cp7110>:

1   MAAIKQILRS MLSQSSLWMV LFSLYSLSGY CYVITDKPED
    DFHSSSAVKW
51  DHWGKTTLSR LSNKKASAKA VSGTGATTVG FIKDTWSRTY
    AVRWNYWGTK
101 ELPTSSWVKK SKATGISSDG SIIAGIVENE LSQSFAVTWK
    NNEMYLLPST
151 WAVQSKAYGI SSDGSVIVGS AKDAWSRTFA VKWTGHEAQV
    LPVGWAVKSV
201 ANSVSANGSI IVGSVQDASG ILYAVKWEGN TITHLGTLGG
    YSAIAKAVSN
251 NGKVIVGRSE TYYGEVHAFC HKNGVMSDLG TLGGSYSAAK
    GVSATGKVIV
301 GMSTTANGKL HAFKYVGGRM IDKGEYSWKE ACANAVSIDG
    EIIVGVQSE*

A predicted signal peptide is highlighted.

The cp7110 nucleotide sequence <SEQ ID 162> is:

1    ATGGCAGCTA TAAAACAAAT TTTACGTTCT ATGCTATCTC
     AGAGTAGCTT
51   ATGGATGGTC CTATTTTCAT TATATTCTCT ATCTGGTTAT
     TGCTATGTAA
101  TTACAGACAA ACCAGAAGAT GACTTCCATT CTTCATCCGC
     AGTAAAATGG
151  GATCATTGGG GAAAGACAAC TCTCTCAAGA TTATCAAATA
     AAAAAGCCTC
201  TGCAAAAGCT GTTTCAGGAA CTGGTGCTAC AACTGTCGGC
     TTTATAAAAG
251  ACACTTGGTC TCGAACATAC GCAGTAAGAT GGAATTATTG
     GGGGACCAAA
301  GAACTCCCTA CCAGCTCATG GGTAAAAAAA TCAAAAGCAA
     CAGGAATCTC
351  CTCTGATGGG TCTATAATCG CGGGGATTGT CGAGAATGAG
     CTTTCTCAAA
401  GTTTCGCAGT CACATGGAAA AACAATGAAA TGTATTTGCT
     CCCTTCCACA
451  TGGGCAGTGC AATCTAAAGC GTATGGAATT TCTTCTGATG
     GCTCTGTTAT
501  TGTAGGGAGT GCTAAGGATG CTTGGTCGCG AACTTTCGCT
     GTGAAGTGGA
551  CGGGACACGA GGCTCAGGTG TTACCAGTAG GCTGGGCTGT
     CAAATCTGTA
601  GCGAATTCTG TATCTGCCAA TGGATCTATA ATTGTAGGGT
     CTGTACAAGA
651  CGCCTCTGGA ATTCTTTATG CTGTAAAGTG GGAAGGGAAC
     ACTATTACAC
701  ATCTAGGAAC TTTAGGAGGC TATTCTGCCA TTGCAAAAGC
     TGTATCCAAT
751  AATGGCAAGG TCATTGTAGG GAGATCCGAA AGATATTATG
     GAGAGGTCCA
801  TGCTTTCTGT CATAAGAATG GCGTCATGTC AGACCTCGGC
     ACCCTCGGAG
851  GATCTTATTC TGCAGCTAAG GGAGTCTCTG CAACTGGAAA
     AGTTATTGTC
901  GGTATGTCCA CAACAGCAAA TGGGAAATTG CATGCCTTTA
     AATATGTCGG
951  TGGAAGAATG ATCGACTTAG GAGAGTATAG CTGGAAAGAA
     GCCTGTGCAA
1001 ACGCTGTTTC TATTCATGGA GAAATTATTG TTGGAGTCCA
     ATCAGAATAA

The PSORT algorithm predicts outer membrane (0.827).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 81A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 81B) and for FACS analysis.

These experiments show that cp7110 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

FIG. 191 shows a schematic representation of the structural relationships between of cp7105, cp7106, cp7107, cp7108, cp7109 and cp7110, each of which is identified herein. These six proteins may be grouped in a new family of related outer membrane-associated proteins. These proteins have a repeat structure in common (cf. the pmp family).

Example 82

The following C. pneumoniae protein (PID 4377127) was expressed <SEQ ID 163; cp7127>:

1   MVFFRNSLLH LVALSGMLCC SSGVALTIAE KMASLEHSGR
    GADDYEGMAS
51  FNANMREYSL QLSKLYEEAR KLRASGTEDE ALWKDLIRRI
    GEVRGYLREI
101 EELWAAEIRE KGGNLEDYAL WNHPETTIYN LVTDYGTEDS
    IYLIPQEIGA
151 IKIATLSKFV VPKESFEDCL TQILSRLGIG VRQVNSWIKE
    LYMMRKEGCS
201 VAGVFSSRKD LEALPETAYI GFVLNSNVDA HTNQHVLKKF
    INPETTHVDV
251 IAGRVWIFGS AGEVGELLKI YNFVQSESIR QEYRVIPLTK
    IDPGEMISIL
301 NAAFREDLTK DVSEESLGLR VVPLQYQGRS LFLSGTAALV
    QQALTLIREL
351 EEGIENPTDK TVFWYNVKHS DPQELAALLS QVHDVFSGEN
    KASVGAADGC
401 GSQLNASIQI DTTVSSSAKD GSVKYGNFIA DSKTGTLIMV
    VEKEVLPRIQ
451 MLLKKLDVPK KMVRIEVLLF ERKLAHEQKS GLNLLRLGEE
    VCKKGCSPSV
501 SWAGGTGILE FLFKGSTGSS IVPGYDLAYQ FLMAQEDVRI
    NASPSVVTMN
551 QTPARIAVVD EMSIAVSSDK DKAQYNRAQY GIMIKMLPVI
    NVGEEDGKSY
601 ITLETDITFD TTGKNHDDRP DVTRRNITNK VRIADGETVI
    IGGLRCKQMS
651 DSHDGIPFLG DIPGIGKLFG MSSTSDSLTE MFVFITPKIL
    ENPVEQQERK
701 EEALLSSRPG EREEYYQALA ASEAAARAAH KKLEMFPASG
    VSLSQVERQE
751 YDGC*

A predicted signal peptide is highlighted.

The cp7127 nucleotide sequence <SEQ ID 164> is:

1    ATGGTTTTTT TCCGTAATTC TTTACTGCAT TTAGTTGCCC
     TATCCGGAAT
51   GCTCTGTTGT TCTTCTGGAG TGGCTTTAAC CATAGCCGAG
     AAGATGGCTT
101  CTTTAGAGCA CTCGGGGAGA GGAGCAGACG ATTATGAGGG
     GATGGCTTCG
151  TTTAATGCCA ATATGAGGGA GTATAGCCTT CAGCTGAGCA
     AGTTGTATGA
201  GGAAGCACGA AAGCTACGCG CTTCTGGAAC TGAGGATGAA
     GCTCTGTGGA
251  AGGACTTAAT TCGACGGATT GGTGAGGTGC GAGGCTATCT
     TCGAGAGATC
301  GAGGAGCTTT GGGCTGCAGA AATTCGTGAG AAAGGGGGCA
     ATCTCGAGGA
351  CTACGCCCTC TGGAATCACC CAGAGACTAC GATTTACAAT
     CTTGTTACCG
401  ATTACGGAAC CGAAGACTCT ATTTATTTGA TTCCTCAAGA
     AATCGGAGCG
451  ATTAAAATCG CAACCTTATC GAAATTTGTA GTTCCTAAAG
     AGTCTTTCGA
501  AGACTGTCTC ACTCAGATCC TATCTCGCTT AGGTATTGGC
     GTGCGTCAGG
551  TCAATTCTTG GATTAAGGAA CTTTATATGA TGCGTAAGGA
     GGGCTGCAGT
601  GTTGCTGGAG TTTTTTCCTC CAGAAAAGAT TTAGAGGCGC
     TCCCAGAAAC
651  AGCCTATATT GGTTTTGTAT TGAATTCGAA CGTAGATGCG
     CATACCAATC
701  AACATGTCTT AAAAAAGTTC ATTAACCCTG AAACAACGCA
     TGTAGATGTG
751  ATTGCAGGAC GTGTGTGGAT TTTTGGTTCT GCGGGGGAAG
     TCGGCGAGCT
801  TCTGAAGATT TATAATTTTG TGCAGTCGGA GAGCATACGT
     CAAGAGTATC
851  GGGTGATTCC CTTAACTAAG ATCGATCCAG GGGAGATGAT
     TTCCATTCTC
901  AACGCAGCAT TTCGTGAGGA TCTGACTAAA GATGTTAGTG
     AAGAATCTTT
951  AGGCCTTCGT GTAGTTCCTT TACAGTATCA AGGGCGTTCG
     TTGTTTTTAA
1001 GTGGAACCGC GGCGTTAGTG CAGCAAGCGC TGACTCTCAT
     TCGAGAGCTT
1051 GAAGAAGGGA TTGAGAACCC TACGGATAAA ACAGTATTTT
     GGTATAACGT
1101 CAAGCACTCC GATCCCCAAG AGTTGGCGGC ATTGCTTTCC
     CAAGTCCATG
1151 ATGTCTTCTC TGGCGAGAAT AAGGCGAGTG TCGGAGCTGC
     AGATGGATGT
1201 GGGTCGCAAT TAAATGCCTC GATCCAAATT GATACTACAG
     TAAGTTCTTC
1251 TGCGAAAGAT GGCTCAGTGA AGTACGGAAA CTTCATCGCG
     GATTCTAAGA
1301 CAGGAACTCT GATTATGGTG GTTGAGAAAG AAGTTCTTCC
     ACGTATTCAG
1351 ATGCTACTTA AGAAACTAGA TGTCCCTAAA AAGATGGTCC
     GTATCGAGGT
1401 GCTGTTATTT GAAAGAAAAT TGGCACATGA GCAGAAATCT
     GGGTTAAATC
1451 TTCTACGTCT TGGTGAGGAA GTTTGTAAAA AAGGGTGCAG
     TCCTTCTGTG
1501 TCTTGGGCCG GGGGTACTGG CATACTAGAA TTTTTATTTA
     AAGGAAGTAC
1551 GGGATCTTCG ATAGTTCCTG GTTATGATCT CGCCTATCAA
     TTTTTAATGG
1601 CTCAAGAGGA CGTTCGGATT AATGCGAGTC CTTCTGTAGT
     TACTATGAAC
1651 CAAACCCCAG CACGGATTGC TGTTGTTGAT GAAATGTCAA
     TAGCGGTGTC
1701 TTCAGATAAA GATAAAGCGC AATACAATCG TGCGCAGTAC
     GGTATCATGA
1751 TAAAAATGCT CCCCGTAATT AATGTGGGAG AGGAAGACGG
     AAAAAGTTAC
1801 ATTACTTTAG AGACAGACAT CACCTTTGAT ACTACGGGAA
     AAAATCATGA
1851 TGATCGTCCT GATGTTACAA GGCGTAATAT TACTAATAAG
     GTGCGCATTG
1901 CTGACGGAGA GACTGTGATT ATTGGAGGTT TGCGTTGCAA
     ACAGATGTCA
1951 GATTCTCATG ATGGCATTCC TTTCCTTGGA GACATTCCTG
     GTATAGGGAA
2001 GTTATTTGGA ATGAGTTCCA CATCAGACAG TCTCACGGAG
     ATGTTTGTAT
2051 TTATCACTCC GAAGATCCTA GAAAATCCTG TAGAGCAACA
     AGAACGTAAA
2101 GAAGAAGCTT TACTCTCTTC GCGCCCTGGA GAGAGAGAAG
     AATACTATCA
2151 GGCTTTAGCA GGTAGTGAGG CTGCAGCACG AGCAGCTCAT
     AAAAAATTAG
2201 AGATGTTCCC GGCATCAGGA GTATCTTTAT CTCAGGTAGA
     GAGGCAAGAA
2251 TACGATGGCT GCTAG

The PSORT algorithm predicts periplasmic (0.920).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 82A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 82B) and for FACS analysis.

These experiments show that cp7127 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 83

The following C. pneumoniae protein (PID 4377133) was expressed <SEQ ID 165; cp7133>:

1   MQPFIFTLLC LTSLVSLVAF DAANARKRCA CAQTIERGEN
    FFSIKRSACA
51  EIEYQEKSRH ASAIERISKD KGKVTPKQIA KVATKKKQRY
    RLLQVPFSRP
101 PNNSRYNLYA LLSEPPECYS DTASWYAIFI RLLRRAYVDT
    GNVPPGSEYA
151 IANALISNKQ EILERGAQLG PDVIETLTLP EEQAEIFYKM
    LKGSSNSQSL
201 LNFLHYEEKS LGHCKLNLIF MDPLLLEAVL DHPDAYRETS
    LLRDGIWEAV
251 KRQEHAIQEH GQAAALELFK TRTDFRLELR DKMQLLLSRY
    DLLPLLNKKM
301 FDYTLGSAGD YLFLVDPDTK AISRCRCPSK SIKL

A predicted signal peptide is highlighted.

The cp7133 nucleotide sequence <SEQ ID 166> is:

1    ATGCAACCTT TTATCTTTAC TTTACTGTGC TTGACATCTT
     TGGTTTCTTT
51   AGTCGCCTTT GATGCTGCGA ATGCTCGTAA ACGTTGTGCC
     TGTGCTCAAA
101  CTATAGAACG TGGAGAGAAC TTCTTTTCCA TAAAACGCTC
     TGCTTGTGCT
151  GAAATCGAAT ATCAAGAAAA ATCTCGCCAC GCCTCAGCAA
     TTGAAAGAAT
201  CTCAAAAGAT AAAGGCAAAG TCACTCCAAA GCAGATTGCG
     AAAGTAGCTA
251  CTAAGAAAAA GCAAAGATAC CGTTTATTGC AGGTTCCTTT
     TTCAAGGCCT
301  CCGAATAACT CAAGGTATAA CCTCTATGCT TTGCTTAGTG
     AACCTCCCGA
351  ATGCTATAGC GATACAGCAT CATGGTATGC TATTTTTATT
     CGGTTACTTC
401  GACGTGCTTA TGTAGACACG GGAAATGTAC CTCCTGGATC
     TGAGTATGCC
451  ATCGCTAATG CTTTGATAAG TAACAAACAA GAGATTTTAG
     AGAGGGGAGC
501  GCAGCTTGGA CCCGATGTTA TTGAAACTCT AACATTGCCT
     GAGGAACAAG
551  CCGAGATTTT TTATAAAATG CTCAAAGGGT CGTCAAACTC
     TCAGTCGCTA
601  CTGAATTTTC TGCATTATGA AGAGAAAAGC TTAGGCCACT
     GTAAGCTAAA
651  TCTGATCTTC ATGGATCCCC TACTGTTAGA AGCTGTTCTA
     GATCATCCCG
701  ATGCTTATAG GGAAACGTCG CTCCTGCGCG ATGGCATTTG
     GGAAGCGGTG
751  AAGCGTCAAG AACATGCCAT CCAAGAACAT GGCCAGGCAG
     CTGCTTTGGA
801  GCTTTTTAAA ACACGCACCG ACTTCCGCCT GGAGCTGCGA
     GATAAGATGC
851  AGTTACTTCT AAGTCGATAC GATTTGCTCC CCTTATTAAA
     TAAAAAAATG
901  TTCGACTACA CCTTAGGAAG TGCCGGAGAT TACTTATTTT
     TGGTAGACCC
951  AGATACTAAG GCAATTTCTC GATGTCGCTG CCCTTCAAAG
     AGTATTAAAT
1001 TATAA

The PSORT algorithm predicts outer membrane (0.92).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 83A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 83B) and for FACS analysis.

These experiments show that cp7133 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 84

The following C. pneumoniae protein (PID 4377222) was expressed <SEQ ID 167; cp7222>:

1   MNRRDMVITA VVVNAILLVA LFVTSKRIGV KDYDEGFRNF
    ASSKVTQAVV
51  SEEKVIEKPV VAEVPSRPIA KETLAAQFIE SKPVIVTTPP
    VPVVSETPEV
101 PTVAVPPQPV RETVKEEQAP YATVVVKKGD FLERIARANH
    TTVAKLMQIN
151 DLTITQLKIG QVIKVPTSQD VSNEKTPQTQ TANPENYYIV
    QEGDSPWTIA
201 LRNHIRLDDL LKMNDLDEYK ARRLKPGDQL RIR*

A predicted signal peptide is highlighted.

The cp7222 nucleotide sequence <SEQ ID 168> is:

1   ATGAATCGTA GAGACATGGT AATAACAGCT GTCGTAGTGA
    ATGCTATATT
51  GCTTGTGGCT CTTTTCGTCA CATCAAAGCG TATTGGCGTC
    AAGGACTATG
101 ACGAGGGATT CCGTAATTTT GCTTCTAGCA AGGTTACACA
    AGCAGTAGTT
151 TCAGAAGAAA AAGTCATAGA AAAGCCTGTA GTCGCAGAAG
    TGCCTAGCCG
201 TCCTATCGCT AAAGAGACTC TAGCTGCACA GTTTATTGAA
    AGTAAGCCGG
251 TTATTGTAAC CACACCACCC GTGCCTGTTG TTAGCGAAAC
    CCCAGAAGTG
301 CCTACTGTGG CAGTTCCGCC TCAGCCTGTT CGTGAGACAG
    TAAAAGAGGA
351 ACAAGCTCCT TATGCTACTG TTGTAGTGAA AAAAGGAGAT
    TTTCTCGAAC
401 GCATTGCGAG AGCAAATCAT ACTACCGTTG CAAAATTGAT
    GCAGATCAAT
451 GATCTTACCA CCACCCAACT TAAAATTGGT CAGGTCATCA
    AAGTCCCTAC
501 GTCTCAAGAT GTCAGCAACG AAAAAACTCC TCAAACACAG
    ACCGCAAACC
551 CTGAAAATTA TTATATCGTC CAAGAAGGGG ATAGCCCGTG
    GACAATAGCA
601 TTGCGTAACC ATATTCGATT GGATGATTTG CTAAAAATGA
    ATGATCTCGA
651 TGAATATAAA GCCCGGCGCC TTAAGCCTGG AGATCAGTTG
    CGCATACGTT
701 GA

The PSORT algorithm predicts periplasmic (0.935).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 84A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 84B) and for FACS analysis.

These experiments show that cp7222 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 85

The following C. pneumoniae protein (PID 4377225) was expressed <SEQ ID 169; cp7225>:

1   MKGTPQYHFI GIGGIGMSAL AHILLDRGYE VSGSDLYESY
    TIESLKAKGA
51  RCFSGHDSSH VPHDAVVVYS SSIAPDNVEY LTAIQRSSRL
    LHRAELLSQL
101 MEGYESILVS GSHGKTGTSS LIRAIFQEAQ KDPSYAIGGL
    AANCLNGYSG
151 SSKIFVAEAD ESDGSLKHYT PRAVVITNID NEHLNNYAGN
    LDNLVQVIQD
201 FSRKVTDLNK VFYNGDCPIL KGNVQGISYG YSPECQLHIV
    SYNQKAWQSH
251 FSFTFLGQEY QDIELNLPGQ HNAANAAAAC GVALTFGIDI
    NIIRKALKKF
301 SGVHRRLERK NISESFLFLE DYAHHPVEVA HTLRSVRDAV
    GLRRVIAIFQ
351 PHRFSRLEEC IQTFPKAFQE ADEVILTDVY SAGESPRESI
    ILSDKAEQIR
401 KSSYVHCCYV PHGDIVDYLR NYIRIHDVCV SLGAGNIYTI
    GEALKDFNPK
451 KLSIGLVCGG KSCEHDISLL SAQHVSKYIS PEFYDVSYFI
    INRQGLWRTG
501 KDFPHLIEET QGDSPLSSEI ASALAKVDCL FPVLHGPFGE
    DGTIQGFFEI
551 LGKPYAGPSL SLAATAMDKL LTKRIASAVG VPVVPYQPLN
    LCFWKRNPEL
601 CIQNLIETFS FPMIVKTAHL GSSIGIFLVR DKEELQEKIS
    EAFLYDTDVF
651 VEESRLGSRE IEVSCIGHSS SWYCMAGPNE RCGASGFIDY
    QEKYGPDGID
701 CAKISFDLQL SQESLDCVRE LAERVYRAMQ GKGSARIDFF
    LDEEGNYWLS
751 EVNPIPGMTA ASPFLQAFVH AGWTQEQIVD HFIIDALHKF
    DKQQTIEQAF
801 TKEQDLVKR*

The cp7225 nucleotide sequence <SEQ ID 170> is:

1    ATGAAGGGAA CTCCTCAGTA TCATTTTATC GGTATCGGTG
     GTATAGGAAT
51   GAGCGCTTTA GCTCATATTT TCGTTGATCG TGGCTATGAG
     GTCTCTGGAA
101  GCGACTTATA TGAAAGCTAT ACGATCGAAA GCCTGAAAGC
     TAAAGGTGCG
151  AGGTGTTTCT CAGGCCATGA TTCCTCCCAT GTTCCTCATG
     ATGCCGTCGT
201  TGTTTATAGC TCAAGTATAG CCCCTGATAA TGTAGAGTAT
     CTTACCGCTA
251  TTCAAAGATC ATCACGTCTT CTTCATAGAG CAGAGCTCTT
     GAGTCAGCTT
301  ATGGAGGGTT ATGAAAGCAT TCTGGTTTCA GGAAGCCATG
     GGAAGACAGG
351  GACCTCATCT CTAATTCGAG CGATTTTCCA GGAAGCTCAG
     AAAGATCCCT
401  CCTATGCTAT TGGAGGACTC GCTGCAAACT GCCTGAATGG
     GTATTCTGGA
451  TCATCGAAAA TCTTCGTTGC CGAAGCCGAT GAAAGTGATG
     GGTCTTTAAA
501  GCACTACACT CCCCGTGCAG TAGTCATTAC AAATATAGAT
     AATGAACATT
551  TGAATAATTA CGCTGGGAAT CTTGATAACC TGGTTCAGGT
     AATCCAGGAC
601  TTCTCTAGAA AAGTAACAGA TCTCAATAAG GTATTCTATA
     ACGGGGATTG
651  TCCTATTTTG AAAGGAAATG TCCAAGGGAT TTCTTATGGA
     TATTCACCAG
701  AATGTCAATT GCATATCGTT TCCTATAATC AAAAGGCATG
     GCAATCTCAC
751  TTTTCCTTTA CCTTTTTAGG CCAGGAGTAT CAAGACATTG
     AGCTCAATCT
801  CCCTGGACAA CATAACGCTG CAAATGCAGC AGCAGCCTGT
     GGAGTTGCTC
851  TTACCTTTGG CATAGACATA AACATCATTC GAAAAGCTCT
     CAAAAAATTC
901  TCGGGAGTTC ATCGACGTCT AGAAAGAAAA AATATATCCG
     AAAGCTTTCT
951  TTTCTTAGAA GATTATGCTC ATCATCCTGT AGAGGTTGCA
     CATACCCTGC
1001 GCTCTGTGCG TGATGCTGTG GGTTTGCGAA GAGTCATCGC
     AATTTTTCAA
1051 CCACATCGAT TCTCTCGTTT AGAAGAGTGC TTACAAACCT
     TCCCCAAAGC
1101 TTTCCAAGAA GCTGATGAAG TCATACTTAC AGATGTCTAT
     AGTGCCGGAG
1151 AAAGTCCTAG AGAGTCTATC ATTCTTTCCG ACCTTGCGGA
     ACAGATTCGT
1201 AAGTCTTCTT ATGTCCATTG TTGTTATGTT CCCCATGGAG
     ACATCGTAGA
1251 TTATCTACGA AACTACATTC GCATTCATGA TGTCTGTGTT
     TCTCTAGGAG
1301 CTGGAAATAT CTATACTATT GGAGAGGCTT TAAAAGACTT
     TAACCCTAAA
1351 AAATTATCCA TAGGACTCGT CTGTGGAGGG AAATCTTGCG
     AACACGATAT
1401 TTCTCTACTT TCTGCTCAAC ATGTCTCTAA ATATATTTCT
     CCTGAATTCT
1451 ATGATGTGAG TTACTTCATC ATAAATCGTC AGGGCTTATG
     GAGAACAGGA
1501 AAGGATTTTC CTCATCTTAT TGAAGAGACT CAAGGGGATT
     CGCCACTTTC
1551 TTCTGAAATC GCTTCAGCTT TAGCAAAAGT CGACTGTTTG
     TTTCCCGTGC
1601 TCCATGGCCC ATTTGGAGAG GATGGTACGA TCCAGGGATT
     TTTTGAAATC
1651 TTAGGAAAAC CTTATGCCGG ACCCTCACTA TCTTTAGCAG
     CAACTGCAAT
1701 GGATAAGCTG TTAACAAAAC GAATTGCATC AGCAGTGGGT
     GTTCCTGTAG
1751 TCCCTTACCA ACCTTTAAAT CTCTGTTTCT GGAAACGCAA
     TCCAGAACTA
1801 TGTATTCAGA ATCTTATAGA GACATTTTCT TTCCCTATGA
     TTGTAAAAAC
1851 TGCACATTTG GGATCTAGTA TTGGGATATT TTTAGTCCGT
     GATAAAGAGG
1901 AATTACAAGA AAAGATCTCA GAAGCATTTC TATATGACAC
     GGATGTGTTT
1951 GTGGAGGAAA GTCGCTTAGG GTCTCGTGAA ATCGAAGTGT
     CCTGTATCGG
2001 CCATTCTTCT AGCTGGTATT GTATGGCAGG GCCTAATGAA
     CGCTGTGGTG
2051 CTAGTGGGTT TATTGATTAT CAAGAGAAAT ATGGATTTGA
     TGGCATAGAT
2101 TGCGCAAAGA TCTCTTTTGA TTTACAGCTC TCACAAGAAT
     CTTTAGATTG
2151 TGTTAGAGAA CTTGCAGAGC GTGTCTACCG AGCAATGCAA
     GGAAAAGGTT
2201 CAGCTCGAAT AGATTTTTTC TTGGATGAAG AGGGGAATTA
     TTGGTTGTCA
2251 GAGGTCAATC CTATTCCAGG AATGACAGCA GCTAGCCCAT
     TTTTACAAGC
2301 TTTTGTTCAC GCAGGATGGA CGCAAGAACA AATTGTAGAT
     CACTTTATTA
2351 TAGATGCTCT ACATAAGTTT GATAAGCAGC AGACTATCGA
     ACAGGCATTC
2401 ACTAAAGAAC AAGATTTAGT TAAAAGATAA

The PSORT algorithm predicts inner membrane (0.16).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 85A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 85B) and for FACS analysis.

These experiments show that cp7225 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 86

The following C. pneumoniae protein (PID 4377248) was expressed <SEQ ID 171; cp7248>:

1   MKFWLQGCAF VGCLLLTLPC CAARRRASGE NLQQTRPIAA
    ANLQWESYAE
51  ALEHSKQDHK PICKFFTGSD WCMWCIKMQD QILQSSEFKH
    FAGVHLHMVE
101 VDFPQKNHQP EEQRQKNQEL KAQYKVTGFP ELVFIDAEGK
    QLARMGFEPG
151 GGAAYVSKVK SALKLR*

A predicted signal peptide is highlighted.

The cp7248 nucleotide sequence <SEQ ID 172> is:

1   ATGAAATTTT GGTTGCAAGG ATGTGCTTTT GTCGGTTGTC
    TGCTATTGAC
51  TTTACCTTGT TGTGCTGCAC GAAGACGTGC TTCTGGAGAA
    AATTTGCAAC
101 AAACTCGTCC TATAGCAGCT GCAAATCTAC AATGGGAGAG
    CTATGCAGAA
151 GCTCTTGAAC ATTCTAAACA AGATCACAAA CCTATTTGTC
    TTTTCTTTAC
201 AGGATCAGAC TGGTGTATGT GGTGCATAAA AATGCAAGAC
    CAGATTTTGC
251 AAAGCTCTGA GTTTAAGCAT TTTGCGGGTG TGCATCTGCA
    TATGGTTGAA
301 GTTGATTTCC CCCAAAAGAA TCATCAACCT GAAGAGCAGC
    GCCAAAAAAA
351 TCAAGAACTG AAAGCTCAAT ATAAAGTTAC AGGATTCCCC
    GAACTGGTCT
401 TCATAGATGC AGAAGGAAAA CAGCTTGCTC GCATGGGATT
    TGAGCCTGGT
451 GGTGGAGCTG CTTACGTAAG CAAGGTGAAG TCTGCTCTTA
    AACTACGTTA
501 A

The PSORT algorithm predicts periplasmic (0.932).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 86A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 86B) and for FACS analysis.

The cp7248 protein was also identified in the 2D-PAGE experiment.

These experiments show that cp7248 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 87

The following C. pneumoniae protein (PID 4377249) was expressed <SEQ ID 173; cp7249>:

1   MIPSPTPINF RDDTILETDP KPSLIMFSSK KTEIASERRK
    AHPTLFKVLG
51  TIWNIVKFII SIILFLPLAL LWVLKKTCQF FILPSSIISQ
    SMSKTAVAIR
101 RMTFLSHIKQ LLSLKEISAA DRVVIQYDDL VVDSLAIKIP
    HALPHRWILY
151 SQGNSGLMEN LFDRGDSSLH QLAKATGSNL LVFNYPGIMS
    SKGEAKRENL
201 VKSYQACVRY LRDEEIGPKA NQIIAFGYSL GTSVQAAALD
    REVIDGSDGT
251 SWIVVKDRGP RSLADVANQI CKPIASAIIK LVGWNIDSVK
    PSERLRCPEI
301 FIYNSNHDQE LISDGLFERE NCVATPFLEL PEVKTSGTKI
    PIPERDLLHL
351 NPLSPNVVDR LAAVISNYLD SENRKSQQPD *

The cp7249 nucleotide sequence <SEQ ID 174> is:

1    ATGATCCCAT CCCCTACCCC AATAAACTTT CGTGATGATA
     CGATTCTAGA
51   GACGGATCCA AAGCCGTCTT TAATCATGTT CTCTTCAAAA
     AAAACAGAGA
101  TAGCTTCTGA AAGACGGAAG GCCCATCCCA CCTTATTTAA
     AGTTCTAGGA
151  ACGATTTGGA ATATTGTGAA GTTTATTATC TCAATCATTC
     TGTTCCTTCC
201  CTTAGCGTTA TTGTGGGTAC TCAAGAAAAC CTGTCAGTTT
     TTCATTCTCC
251  CATCTTCTAT CATATCTCAG AGCATGTCAA AAACAGCTGT
     GGCAATTCGG
301  CGAATGACCT TTCTGTCCCA TATTAAACAA CTCCTAAGCC
     TTAAGGAAAT
351  CTCAGCTGCC GATCGTGTGG TTATACAATA TGACCATTTG
     GTGGTTGATA
401  GCTTAGCTAT AAAGATACCT CATGCTCTTC CCCACAGGTG
     GATTCTTTAT
451  TCTCAAGGAA ACTCTGGATT GATGGAAAAC CTGTTCCATC
     GGGGCGATTC
501  CTCTCTACAC CAGCTAGCCA AAGCAACCGG CTCGAATCTT
     CTTGTGTTCA
551  ACTATCCTGG AATTATGTCC AGCAAAGGAG AAGCGAAACG
     AGAAAATCTG
601  GTTAAATCGT ATCAGGCATG CGTACGCTAC CTACGAGATG
     AAGAGACAGG
651  TCCTAAAGCC AATCAAATCA TAGCTTTCGG ATACTCTTTG
     GGAACTAGTG
701  TCCAAGCTGC TGCTCTAGAT CGTGAGGTCA CTGATGGCAG
     TGATGGAACT
751  TCATGGATTG TTGTAAAAGA TCGGGGCCCT CGCTCTCTAG
     CAGATGTCGC
801  GAGTCAAATT TGTAAGCCCA TAGCTTCCGC GATTATAAAA
     CTCGTTGGTT
851  GGAACATAGA CTCTGTGAAA CCTAGCGAAA GATTGCGTTG
     TCCCGAAATT
901  TTCATTTACA ACTCTAATCA TGATCAAGAA CTCATTAGCG
     ACGGCCTCTT
951  CGAAAGAGAA AATTGCGTAG CAACACCTTT TCTAGAGCTT
     CCTGAAGTAA
1001 AAACCTCGGG GACTAAAATT CCTATACCCG AAAGGGATCT
     TCTCCATCTA
1051 AATCCTCTCA GTCCAAATGT AGTAGACAGA TTAGCAGCAG
     TGATCTCTAA
1101 TTATTTAGAT TCTGAAAACA GAAAGTCTCA GCAACCTGAT
     TAA

The PSORT algorithm predicts inner membrane (0.571).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 87A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 87B) and for FACS analysis.

These experiments show that cp7249 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 88

The following C. pneumoniae protein (PID 4377261) was expressed <SEQ ID 175; cp7261>:

1   MLPISILLFY VILGCLSAYI ADKKKRNVIG WFFAGAFFGF
    IGLVVLLLLP
51  SRRNALEKPQ NDPFDNSDLF DDLKKSLAGN DEIPSSGDLQ
    EIVIDTEKWF
101 YLNKDRENVG PISFEELVVL LKGKTYPEFE WVWKKGMKDW
    QRVKDVPSLQ
151 QALKEASK*

The cp7261 nucleotide sequence <SEQ ID 176> is:

1   ATGCTCCCTA TTTCGATTTT ATTATTTTAT GTGATTCTAG
    GTTGTCTATC
51  TGCCTACATA GCAGATAAGA AAAAACGAAA TGTTATTGGC
    TGGTTTTTTG
101 CAGGAGCATT TTTTGGATTT ATTGGTCTAG TTGTCCTTCT
    TCTTCTTCCT
151 TCTCGTCGAA ACGCTTTAGA AAAGCCACAA AACGATCCTT
    TTGATAACTC
201 CGATCTTTTT GATGATTTGA AAAAAAGTTT AGCAGGTAAT
    GACGAGATAC
251 CCTCATCGGG AGATCTTCAA GAAATCGTTA TCGATACAGA
    GAAGTGGTTT
301 TATTTAAATA AAGATAGAGA AAACGTAGGT CCGATATCTT
    TTGAGGAGTT
351 GGTCGTACTT TTAAAGGGAA AAACGTATCC AGAAGAAATT
    TGGGTATGGA
401 AAAAGGGAAT GAAAGATTGG CAACGAGTGA AGGATGTTCC
    ATCACTACAA
451 CAGGCTTTGA AAGAAGCATC AAAATAA

The PSORT algorithm predicts inner membrane (0.848).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 88A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 88B) and for FACS analysis.

These experiments show that cp7261 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 89

The following C. pneumoniae protein (PID 4377305) was expressed <SEQ ID 177; cp7305>:

1   MEVYSFHPAV RTSFQHRVMA ALDAWFFLGG HRLKVVSLDS
    CNSGWAYQEL
51  VSISTTEKVL KLLSYLLVPI VIIALLIRCL LHSNFRIDVE
    KERWLKIREL
101 GIDIESCKLP SSYVNQVSSF IWFEKDKSKR PRIDVDYHTL
    HSKDWVVFPI
151 VFQKIPKTSR FSYWFSQKET RKRDYVRNML DHVIGYLTSE
    GGEWLQYISK
201 ISYQSATSLD PERVLQYCLT DNQELQGEVQ RLLNEESATK
    SSGDKEVLLS
251 HVSDIICQCW WPKFLEVIQS RAFIFELVEE VSGKLNLDFL
    CLEKANTLDQ
301 ELRNSLLRAV VHHGSEGVDI KKVGAGLIIY TEAIQLQIPF
    SRS*

The cp7305 nucleotide sequence <SEQ ID 178> is:

1    ATGGAAGTTT ATAGTTTTCA CCCTGCGGTA AGGACTTCGT
     TTCAGCACCG
51   TGTAATGGCA GCACTAGATG CTTGGTTTTT TCTAGGAGGG
     CACCGTTTAA
101  AGGTAGTTTC TCTAGATAGT TGTAACTCAG GTTGGGCGTA
     TCAAGAACTT
151  GTGTCTATTT CAACGACAGA AAAAGTCTTG AAACTACTCT
     CTTACCTACT
201  CGTACCGATT GTCATAATAG CTCTGTTAAT TCGTTGTCTT
     TTACATAGCA
251  ATTTTAGGAT AGACGTAGAG AAGGAACGTT GGTTAAAAAT
     AAGGGAGTTA
301  GGAATTGATA TAGAAAGCTG CAAACTCCCC AGTTCTTATG
     TAAACCAGGT
351  TTCCTCGTTT ATTTGGTTTG AAAAAGATAA ATCCAAACGG
     CCACGTATTG
401  ATGTAGATTA TCATACGCTA CATAGCAAAG ACTGGGTAGT
     TTTCCCTATC
451  GTTTTTCAGA AAATTCCAAA GACCTCGCGT TTCAGTTATT
     GGTTCTCACA
501  AAAAGAAACA AGGAAGAGGG ATTATGTGAG AAATATGCTG
     GACCACGTCA
551  TTGGTTATCT AACGTCAGAA GGTGGGGAGT GGTTGCAGTA
     TATATCGAAA
601  ACCTCTTATC AAAGCGCTAC TTCCTTGGAT CCTGAAAGAG
     TTCTTCAATA
651  TTGCTTAACT GATAACCAGG AGCTCCAGGG AGAAGTGCAA
     CGTTTGCTTA
701  ATGAGGAGAG TGCGACCAAA AGCTCTGGGG ATAAGGAAGT
     TTTGTTAAGT
751  CATGTATCTG ACATTATTTG CCAGTGTTGG TGGCCAAAGT
     TTCTTGAAGT
801  TATACAATCT CCGGCCTTTA TTGAAGAATT AGTAGAAGAA
     GTGAGTGGTA
851  AACTTAATTT AGATTTTTTA TGCCTAGAAA AGGCTAATAC
     ATTAGATCAG
901  GAGTTGAGAA ACAGTCTTCT AAGAGCAGTC GTACACCACG
     GTTCTGAAGG
951  AGTTGATATT AAGAAAGTTG GTGCCGGCCT CATTATTTAT
     ACGGAAGCTA
1001 TTCAATTACA GATTCCCTTC TCAAGGAGTT AA

The PSORT algorithm predicts inner membrane (0.508).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 89A) and also as a double GST/his fusion. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 89B) and for FACS analysis.

These experiments show that cp7305 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 90

The following C. pneumoniae protein (PID 4377347) was expressed <SEQ ID 179; cp7347>:

1   MKKGKLGAIV FGLLFTSSVA GFSKDLTKDN AYQDLNVIEH
    LISKLYAPLP
51  WKELLFGWDL SQQTQQARLQ LVLEEKPTTN YCQKVLSNYV
    RSLNDYHAGI
101 TFYRTESAYI PYVLKLSEDG HVFVVDVQTS QGDIYLGDEI
    LEVDGMGIRE
151 AIESLRFGRG SATDYSAAVR SLTSRSAAFG DAVPSGIAML
    KLRRPSGLIR
201 STPVRWRYTP EHIGDFSLVA PLIPEHKPQL PTQSCVLFRS
    GVNSQSSSSS
251 LFSSYMVPYF WEELRVQNKQ RFDSNHHIGS RNGFLPTFGP
    ILWEQDKGPY
301 RSYIFKAKDS QGNPHRIGFL RISSYVWTDL EGLEEDHKDS
    PWELFGEIID
351 HLEHETDALI IDQTHNPGGS VFYLYSLLSM LTDHPLDTPK
    HRMIFTQDFV
401 SSALHWQDLL EDVFTDWQAV AVLGETMEGY CMDMHAVASL
    QNFSQSVLSS
451 WVSGDINLSK PMPLLGFAQV RPHPKHQYTK PLFMLIDEDD
    FSCGDLAPAI
501 LKDNGRATLI GKPTAGAGGF VFQVTFPNRS GIKGLSLTGS
    LAVRKDGEFI
551 ENLGVAPHID LGFTSRDLQT SRFTDYVEAV KTIVLTSLSE
    NAKKSEEQTS
601 PQETPEVIRV SYPTTTSAS*

A predicted signal peptide is highlighted.

The cp7347 nucleotide sequence <SEQ ID 180> is:

1    ATGAAAAAAG GGAAATTAGG AGCCATAGTT TTTGGCCTTC
     TATTTACAAG
51   TAGTGTTGCT GGTTTTTCTA AGGATTTGAC TAAAGACAAC
     GCTTATCAAG
101  ATTTAAATGT CATAGAGCAT TTAATATCGT TAAAATATGC
     TCCTTTACCA
151  TGGAAGGAAC TATTATTTGG TTGGGATTTA TCTCAGCAAA
     CACAGCAAGC
201  TCGCTTGCAA CTGGTCTTAG AAGAAAAACC AACAACCAAC
     TACTGCCAGA
251  AGGTACTCTC TAACTACGTG AGATCATTAA ACGATTATCA
     TGCAGGGATT
301  ACGTTTTATC GTACTGAAAG TGCGTATATC CCTTACGTAT
     TGAAGTTAAG
351  TGAAGATGGT CATGTCTTTG TAGTCGACGT ACAGACTAGC
     CAAGGGGATA
401  TTTACTTAGG GGATGAAATC CTTGAAGTAG ATGGAATGGG
     GATTCGTGAG
451  GCTATCGAAA GCCTTCGCTT TGGACGAGGG AGTGCCACAG
     ACTATTCTGC
501  TGCAGTTCGT TCCTTGACAT CGCGTTCCGC CGCTTTTGGA
     GATGCGGTTC
551  CTTCAGGAAT TGCCATGTTG AAACTTCGCC GACCCAGTGG
     TTTGATCCGT
601  TCGACACCGG TCCGTTGGCG TTATACTCCA GAGCATATCG
     GAGATTTTTC
651  TTTAGTTGCT CCTTTGATTC CTGAACATAA ACCTCAATTA
     CCTACACAAA
701  GTTGTGTGCT ATTCCGTTCC GGGGTAAATT CACAGTCTTC
     TAGTAGCTCT
751  TTATTCAGTT CCTACATGGT GCCTTATTTC TGGGAAGAAT
     TGCGGGTTCA
801  AAATAAGCAG CGTTTTGACA GTAATCACCA TATAGGGAGC
     CGTAATGGAT
851  TTTTACCTAC GTTTGGTCCT ATTCTTTGGG AACAAGACAA
     GGGGCCCTAT
901  CGTTCCTATA TCTTTAAAGC AAAAGATTCT CAGGGCAATC
     CCCATCGCAT
951  AGGATTTTTA AGAATTTCTT CTTATGTTTG GACTGATTTA
     GAAGGACTTG
1001 AAGAGGATCA TAAGGATAGT CCTTGGGAGC TCTTTGGAGA
     GATCATCGAT
1051 CATTTGGAAA AAGAGACTGA TGCTTTGATT ATTGATCAGA
     CCCATAATCC
1101 TGGAGGCAGT GTTTTCTATC TCTATTCGTT ACTATCTATG
     TTAACAGATC
1151 ATCCTTTAGA TACTCCTAAA CATAGAATGA TTTTCACTCA
     GGATGAAGTC
1201 AGCTCGGCTT TGCACTGGCA AGATCTACTA GAAGATGTCT
     TCACAGATGA
1251 GCAGGCAGTT GCCGTGCTAG GGGAAACTAT GGAAGGATAT
     TGCATGGATA
1301 TGCATGCTGT AGCCTCTCTT CAAAACTTCT CTCAGAGTGT
     CCTTTCTTCC
1351 TGGGTTTCAG GTGATATTAA CCTTTCAAAA CCTATGCCTT
     TGCTAGGATT
1401 TGCACAGGTT CGACCTCATC CTAAACATCA ATATACTAAA
     CCTTTGTTTA
1451 TGTTGATAGA CGAGGATGAC TTCTCTTGTG GAGATTTAGC
     GCCTGCAATT
1501 TTGAAGGATA ATGGCCGCGC TACTCTCATT GGAAAGCCAA
     CAGCAGGAGC
1551 TGGAGGTTTT GTATTCCAAG TCACTTTCCC TAACCGTTCT
     GGAATTAAAG
1601 GTCTTTCTTT AACAGGATCT TTAGCTGTTA GGAAAGATGG
     TGAGTTTATT
1651 GAAAACTTAG GAGTGGCTCC TCATATTGAT TTAGGATTTA
     CCTCCAGGGA
1701 TTTGCAAACT TCCAGGTTTA CTGATTACGT TGAGGCAGTG
     AAAACTATAG
1751 TTTTAACTTC TTTGTCTGAG AACGCTAAGA AGAGTGAAGA
     GCAGACTTCT
1801 CCGCAAGAGA CGCCTGAAGT TATTCGAGTC TCTTATCCCA
     CAACGACTTC
1851 TGCTTCGTAA

The PSORT algorithm predicts periplasmic space (0.2497).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 90A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 90B) and for FACS analysis.

These experiments show that cp7347 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 91

The following C. pneumoniae protein (PID 4377353) was expressed <SEQ ID 181; cp7353>:

1   MNMPVPSAVP SANITLKEDS STVSTASGIL KTATGEVLVS
    CTALEGSSST
51  CALISLALGQ IILATQQELL LQSTNVHQLL FLPPEVVELE
    IQVVDLLVQL
101 EHAETITSEP QETQTQSRSE QTLPQQSSSK QSALSPRSLK
    PEISDSKQQQ
151 ALQTPKDSAV RKHSEAPSPE TQARASLSQA SSSSQRSLPP
    QESAPERTLL
201 EQQKASSFSP LSQFSAEKQK EALTTSKSHE LYKERDQDRQ
    QREQHDRKHD
251 QEEDAESKKK KKKRGLGVEA VAEEPGENLD IAALIFSDQM
    RPPAEETSKK
301 ETTFKKKLPS PMSVFSRFIP SKNPLSVGSS IHGPIQTPKV
    ENVFLRFMKL
351 MARILGQAEA EANELYMRVK QRTDDVDTLT VLISKINNEK
    KDIDWSENEE
401 MKALLNRAKE IGVTIDKEKY TWTEEEKRLL KENVQMRKEN
    MEKITQMERT
451 DMQRHLQEIS QCHQARSNVL KLLKELMDTF IYNLRP*

The cp7353 nucleotide sequence <SEQ ID 182> is:

1    ATGAATATGC CTGTTCCTTC TGCAGTTCCC TCTGCAAATA
     TAACTCTAAA
51   AGAAGACAGC TCAACAGTTT CCACAGCCTC TGGAATATTA
     AAGACTGCAA
101  CAGGTGAAGT CTTAGTCTCT TGTACAGCGC TAGAAGGAAG
     CTCTTCTACA
151  GATGCTTTAA TTAGCTTAGC TTTAGGACAA ATCATTCTTG
     CGACCCAACA
201  AGAACTGCTC TTACAAAGCA CAAATGTTCA TCAACTCCTC
     TTCCTCCCTC
251  CTGAAGTTGT AGAATTAGAA ATCCAAGTTG TTGACTTGCT
     AGTGCAATTG
301  GAACATGCAG AGACAATCAC AAGTGAACCA CAAGAAACAC
     AAACGCAAAG
351  TAGGAGTGAG CAGACCCTCC CTCAACAAAG CAGCAGTAAA
     CAATCTGCTC
401  TCTCCCCACG CTCCTTAAAA CCTGAAATTT CTGATTCTAA
     ACAACAGCAA
451  GCTCTTCAAA CACCAAAAGA CTCTGCTGTA AGAAAACACA
     GCGAAGCACC
501  GTCACCTGAG ACACAAGCTC GCGCTTCCTT ATCTCAGGCA
     AGCTCAAGTT
551  CTCAGAGATC CTTACCTCCG CAAGAAAGTG CGCCAGAAAG
     AACACTATTA
601  GAACAACAAA AAGCAAGCTC CTTCTCTCCT CTATCCCAGT
     TCTCTGCAGA
651  GAAACAAAAA GAGGCCCTGA CGACCTCAAA ATCTCATGAA
     CTCTATAAAG
701  AACGCGATCA AGATCGCCAA CAAAGAGAGC AGCACGACAG
     AAAGCACGAT
751  CAGGAAGAAG ACGCTGAATC TAAAAAGAAA AAGAAGAAAC
     GTGGTCTCGG
801  TGTAGAGGCA GTCGCTGAGG AACCCGGAGA AAATCTAGAT
     ATTGCCGCTT
851  TAATCTTCTC AGATCAAATG CGACCTCCTG CTGAAGAAAC
     TTCTAAAAAA
901  GAAACGACAT TCAAAAAGAA GCTACCTTCT CCAATGTCTG
     TGTTTAGCAG
951  ATTCATCCCT AGTAAGAATC CGTTATCTGT AGGCTCTTCA
     ATACACGGGC
1001 CTATACAAAC TCCAAAAGTA GAAAATGTGT TCTTAAGGTT
     CATGAAGCTC
1051 ATGGCAAGAA TCTTAGGCCA AGCCGAAGCC GAAGCTAATG
     AACTCTACAT
1101 GCGAGTCAAA CAACGTACCG ATGATGTAGA CACACTCACA
     GTCCTTATCT
1151 CTAAGATCAA TAATGAAAAG AAAGACATTG ATTGGAGTGA
     AAATGAAGAG
1201 ATGAAAGCTC TTTTAAATCG AGCTAAAGAG ATTGGAGTCA
     CTATAGACAA
1251 AGAAAAATAT ACTTGGACAG AAGAGGAAAA AAGACTTCTA
     AAAGAGAATG
1301 TCCAAATGCG CAAAGAGAAT ATGGAGAAAA TCACTCAAAT
     GGAAAGGACG
1351 GACATGCAAA GGCACCTCCA AGAGATTTCT CAATGTCATC
     AAGCGCGCTC
1401 TAATGTATTG AAGTTATTGA AAGAACTTAT GGACACCTTC
     ATTTACAACC
1451 TACGCCCCTA A

The PSORT algorithm predicts cytoplasm (0.1308).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 91A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 91B) and for FACS analysis.

These experiments show that cp7353 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 92

The following C. pneumoniae protein (PID 4377408) was expressed <SEQ ID 183; cp7408>:

1   MLKIQKKRMC VSVVITVGAI VGFFNSADAA RKKKKIPIQI
    LYSFTKVSSY
51  LKNEDASTIF CVDVDRGLLQ HRYLGSPGWQ ETRRRQLFKS
    LENQSYGNER
101 LGEETLAIDI FRNKECLESE IPEQMEAILA NSSALVLGIS
    SFGITGIPAT
151 LHSLLRQNLS FQKRSIASES FLLKIDSAPS DASVFYKGVL
    FRGETAIVDA
201 LSQLFAQLDL SRKKIIFLGE DREVVQAVGS ACIGAGANFL
    GLVYYPAQES
251 LFSYVHPYST ATELQEAQGL QVISDEVAQL TLNALPKMN*

The cp7408 nucleotide sequence <SEQ ID 184> is:

1   ATGTTGAAAA TCCAGAAAAA AAGAATGTGT GTCAGCGTAG
    TCATCACGGT
51  AGGCGCCATA GTGGGGTTTT TCAATTCTGC AGACGCAGCA
    CCAAAGAAAA
101 AGAAGATCCC TATACAGATT CTCTACTCCT TTACTAAAGT
    CTCTTCCTAT
151 TTAAAAAACG AAGACGCAAG TACTATATTT TGCGTCGATG
    TGGATCGTGG
201 ACTTCTCCAG CATCGGTATT TAGGTAGTCC AGGATGGCAG
    GAAACCAGAC
251 GTCGGCAGTT ATTTAAATCC TTAGAAAATC AATCATACGG
    CAACGAACGT
301 TTAGGAGAAG AAACTCTTGC TATTGATATT TTCAGGAACA
    AAGAGTGCTT
351 GGAGAGCGAG ATCCCAGAGC AGATGGAAGC TATCCTTGCA
    AATTCCTCGG
401 CCTTGGTCTT AGGCATCTCT TCTTTTGGGA TCACAGGAAT
    TCCTGCGACT
451 TTGCATAGTT TGCTTCGACA GAATCTATCT TTCCAAAAAC
    GCTCTATAGC
501 ATCGGAGAGC TTCCTTTTAA AGATCGATAG TGCCCCCTCA
    GATGCCTCTG
551 TTTTTTATAA AGGCGTGCTT TTCCGCGGAG AGACTGCGAT
    CGTGGATGCG
601 TTAAGCCAAT TATTTGCCCA GCTCGATCTT TCTCCTAAAA
    AAATTATCTT
651 TCTAGGAGAA GACCCTGAGG TCGTTCAAGC TGTTGGGTCT
    GCTTGTATAG
701 GTTGGGGCAT GAACTTTTTA GGCCTGGTAT ACTATCCTGC
    TCAAGAAAGC
751 CTTTTTTCTT ATGTTCATCC TTACTCTACA GCAACGGAGC
    TCCAAGAAGC
801 ACAGGGTTTA CAAGTAATTT CAGATGAAGT CGCACAGCTT
    ACTTTAAACG
851 CTCTTCCGAA AATGAATTAA

The PSORT algorithm predicts inner membrane (0.123).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 92A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 92B) and for FACS analysis.

These experiments show that cp7408 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 93

The following C. pneumoniae protein (PID 4376424) was expressed <SEQ ID 185; cp6424>:

1   MMHNIVVLSE EPGRSAFLGR TAFFPNAYPI AQGGVGIPST
    IGNLFTIWYC
51  FYFYPAATPQ SDHPDGCGFI LLERLKELGA GFFYCDLRES
    NTTGETLFFE
101 GSNKGVLKNH LFIRDE*

The cp6424 nucleotide sequence <SEQ ID 186> is:

1   ATGATGCACA ATATTGTTGT TCTTAGTGAG GAACCTGGAC
    GAAGCGCTTT
51  TCTTGGTAGG ACGGCATTTT TCCCTAATAA GTATCCAATA
    GCTCAGGGTG
101 GTGTTGGAAT ACCATCTACA ATAGGCAATC TCTTTACTAT
    ATGGTACTGT
151 TTCTATTTTT AGAGAGCTGC AACTCCACAA TCTGATCATC
    CTGACGGATG
201 TGGCTTTATT CTACTAGAAA GGCTTAAGGA GCTCGGTGCA
    GGGTTCTTTT
251 ATTGTGATCT TCGTGAGTCC AATACCACTG GCTTTACTCT
    TTTTTTTGAA
301 GGCTCCAATA AAGGTGTGTT AAAGAATCAC TTGTTTATTA
    GAGATGAGTA
351 A

The PSORT algorithm predicts cytoplasm (0.2502).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 93A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIG. 93B) and for FACS analyses (FIG. 93C; GST-fusion).

These experiments show that cp6424 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 94

The following C. pneumoniae protein (PID 4376449) was expressed <SEQ ID 187; cp6449>:

1   VASETYPSQI LHAQREVRDA YFNQADCHPA RANQILEAKK
    ICLLDVYHTN
51  HYSVFTFCVD NYPNLRFTFV SSKNNEMNGL SNPLDNVLVE
    AMVRRTHARN
101 LLAACKIRNI EVPRVVGLDL RSGILISKLE LKQPQFQSLT
    EDFVNHSTNQ
151 EEARVHQKHV LLISLILLCK QAVLESFQEK KRSS*

The cp6449 nucleotide sequence <SEQ ID 188> is:

1   GTGGCGTCTG AAACGTATCC TTCTCAGATA TTGCACGCTC
    AGAGGGAAGT
51  ACGTGATGCC TATTTTAATC AAGCGGATTG CCATCCTGCT
    CGGGCTAATC
101 AGATTCTCGA GGCTAAGAAA ATCTGTTTAT TAGATGTTTA
    TCATACTAAT
151 CATTATTCCG TATTTACTTT TTGTGTAGAT AATTATCCGA
    ATCTCCGCTT
201 TACATTTGTA TCTTCAAAAA ACAATGAGAT GAATGGCTTA
    TCTAATCCTC
251 TAGATAATGT TCTTGTAGAG GCTATGGTAC GTAGAACACA
    TGCAAGAAAC
301 CTACTTGCAG CGTGTAAAAT TCGAAATATT GAGGTTCCAA
    GGGTTGTTGG
351 GCTTGACCTA AGATCTGGGA TACTCATTTC GAAACTAGAA
    TTGAAGCAAC
401 CTCAGTTCCA AAGTTTAACA GAAGACTTCG TAAATCATTC
    CACAAATCAG
451 GAAGAAGCTC GCGTCCATCA AAAGCATGTG TTGCTAATTT
    CTTTAATTTT
501 ACTTTGCAAG CAGGCCGTTC TGGAATCATT CCAGGAAAAA
    AAGCGATCCT
551 CTTAA

The PSORT algorithm predicts inner membrane (0.2084).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 94A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIG. 94B) and for FACS analyses (FIG. 94C; GST-fusion).

These experiments show that cp6449 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 95

The following C. pneumoniae protein (PID 4376495) was expressed <SEQ ID 189; cp6495>:

MRELNAFELTQPEEYRNRWVLMPCLKCRFCRTQHAKVWSYRCVHEASLYEKNCELTLTYDDKHLPQYGSLVKLHLQLFLKR
LRKMISPHKIRYFECGAYGTKLQRPHYHLLLS

The cp6495 nucleotide sequence <SEQ ID 190> is:

TTGCGAGAATTAAATGCTTTTGAATTAACTCAACCTGAAGAGTATCGAAACCGTTGGGTTTTGATGCCTTGTCTTAAGTGT
CGTTTTTGTAGAACGCAACATGCAAAAGTCTGGTCTTATCGTTGTGTCCATGAAGCTTCTTTGTATGAGAAAAATTGTTTT
CTTACTTTGACTTATGATGATAAGCATTTACCTCAGTATGGTTCGTTGGTAAAGCTGCATTTACAGCTGTTTCTTAAGAGA
TTAAGAAAGATGATTTCTCCTCATAAAATTCGTTATTTTGAATGTGGTGCGTATGGAACCAAATTACAAAGACCTCATTAT
CATCTACTTTTATCATGA

The PSORT algorithm predicts cytoplasmic (0.280).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 95A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 95B) and for FACS analysis (FIG. 95C).

These experiments show that cp6495 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 96

The following C. pneumoniae protein (PID 4376506) was expressed <SEQ ID 191; cp6506>:

1   MRRFLFLILS SLRPVAFSAD NFTILEEKQS PLSRVSIIFA
    LPGVTPVSFD
51  GNCPIPWFSH SKKTLEGQRI YYSGDSFGKY FVVSALWPNK
    VSSAVVACNM
101 ILKHRVDLIL IIGSCYSRSQ DSRFGSVLVS KGYINYDADV
    RPFFERFEIP
151 DIKKSVFATS EVHREAILRG GEEFISTHKQ EIEELLKTHG
    YLKSTTKTEH
201 TLMEGLVATG ESFAMSRNYF LSLQKLYPEI HGFDSVSGAV
    SQVCYEYSIP
251 CLGVNILLPH PLESRENEDW KHLQSEASKI YMDTLLKSVL
    KELCSSH*

The cp6506 nucleotide sequence <SEQ ID 192> is:

1   ATGCGTCGTT TTCTGTTTCT TATTCTTAGC TCTCTTCCTT
    TGGTCGCATT
51  CTCTGCTGAT AGTTTCACTA TTCTAGAAGA AAAACAGAGT
    CCTTTAAGTC
101 GTGTAGGTAT TATTTTTGCT TTACCTGGGG TTACTCCCGT
    TTCTTTTGAT
151 GGTAATTGTC CTATTCCTTG GTTTTCTCAT AGTAAAAAGA
    CTCTAGAGGG
201 ACAGAGAATT TATTACTCTG GCGACTCCTT TGGGAAATAC
    TTTGTAGTTT
251 CTGCTCTTTG GCCTAATAAA GTTTCTTCAG CTGTTGTGGC
    TTGTAATATG
301 ATTCTTAAAC ATCGAGTGGA TCTTATTCTA ATTATAGGCT
    CGTGTTACTC
351 TAGGTCTCAA GATAGCCGTT TTGGCAGCGT CTTAGTTTCT
    AAAGGCTACA
401 TTAATTATGA TCCAGATGTG AGGCCTTTCT TTGAAAGATT
    TGAGATTCCA
451 GACATTAAAA AGAGTGTTTT TGCAACCAGT GAGGTTCATC
    GGGAGGCAAT
501 TCTTCGTGGA GGCGAAGAGT TTATTTCTAC CCATAAACAA
    GAAATCGAAG
551 AGCTTTTGAA GACTCATGGG TATTTGAAAT CAACAACCAA
    AACGGAGCAC
601 ACCTTAATGG AAGGTTTGGT TGCTACAGGC GAGTCTTTCG
    CGATGTCGCG
651 AAACTATTTT CTTTCCTTAC AAAAATTGTA TCCAGAGATT
    CATGGTTTTG
701 ATAGTGTCAG CGGCGCTGTT TCTCAGGTAT GCTATGAATA
    TAGCATTCCT
751 TGTTTAGGTG TGAATATCCT TCTCCCTCAT CCTTTAGAAT
    CACGGAGTAA
801 CGAGGATTGG AAGCATCTTC AAAGTGAGGC AAGTAAAATT
    TATATGGATA
851 CCTTGCTCAA GAGTGTATTA AAAGAACTCT GTTCTTCTCA
    TTAA

The PSORT algorithm predicts periplasmic space (0.571).

The protein was expressed in E. coli and purified as his-tag (FIG. 96A) and GST-fusion (FIG. 96B) products. The GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 96C) and for FACS analysis (FIG. 96D).

These experiments show that cp6506 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 97

The following C. pneumoniae protein (PID 4376882) was expressed <SEQ ID 193; cp6882>:

1   MSLLNLRSSQ DSASEDSTSQ SQIFDRIRNR ELVSTPEEKV
    RQRLLSFLMH
51  KLNYPKKLII IEKELKTLFP LLMRKGTLIP KRRPDILIIT
    PPTYTDAQGN
101 THNLGDPKPL LLIECKALAV NQNALKQLLS YNYSIGATCI
    AMAGKHSQVS
151 ALFNPKTQTL DFYPGLPEYS QLLNYFISLN L*

The cp6882 nucleotide sequence <SEQ ID 194> is:

1   ATGTCCTTAT TGAACCTTCC CTCAAGCCAG GATTCTGCAT
    CTGAGGACTC
51  CACATCGCAA TCTCAAATCT TCGATCCCAT TAGAAATCGG
    GAGTTAGTTT
101 CTACTCCCGA AGAAAAAGTC CGCCAAAGGT TGCTCTCCTT
    CCTAATGCAT
151 AAGCTGAACT ACCCTAAGAA ACTCATCATC ATAGAAAAAG
    AACTCAAAAC
201 TCTTTTTCCT CTGCTTATGC GTAAAGGAAC CCTAATCCCA
    AAACGCCGCC
251 CAGATATTCT CATCATCACT CCCCCCACAT ACACAGACGC
    ACAGGGAAAC
301 ACTCACAACC TAGGCGACCC AAAACCCCTG CTACTTATCG
    AATGTAAGGC
351 CTTAGCCGTA AACCAAAATG CACTCAAACA ACTCCTTAGC
    TATAACTACT
401 CTATCGGAGC CACCTGCATT GCTATGGCAG GGAAACACTC
    TCAAGTGTCA
451 GCTCTCTTCA ATCCAAAAAC ACAAACTCTT GATTTTTATC
    CTGGCCTCCC
501 AGAGTATTCC CAACTCCTAA ACTACTTTAT TTCTTTAAAC
    TTATAG

The PSORT algorithm predicts cytoplasm (0.362).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 97A). The protein was used to immunise mice, whose sera were used in a Western blot (FIG. 97B) and for FACS analysis (FIG. 97C).

These experiments show that cp6882 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 98

The following C. pneumoniae protein (PID 4376979) was expressed <SEQ ID 195; cp6979>:

1   MSVNPSGNSK NDLWITGAHD QHPDVKESGV TSANLGSHRV
    TASGGRQGLL
51  ARIKEAVTGF FSRMSFFRSG APRGSQQPSA PSADTVRSPL
    PGGDARATEG
101 AGRNLIKKGY QPGMKVTIPQ VPGGGAQRSS GSTTLKPTRP
    APPPPKTGGT
151 NAKRPATHGK GPAPQPPKTG GTNAKRAATH GKGPAPQPPK
    GILKQPGQSG
201 TSGKKRVSWS DED*

The cp6979 nucleotide sequence <SEQ ID 196> is:

1   ATGTCTGTTA ATCCATCAGG AAATTCCAAG AACGATCTCT
    GGATTACGGG
51  AGCTCATGAT CAGCATCCCG ATGTTAAAGA ATCCGGGGTT
    ACAAGTGCTA
101 ACCTAGGAAG TCATAGAGTG ACTGCCTCAG GAGGACGCCA
    AGGGTTATTA
151 GCACGAATCA AAGAAGCAGT AACCGGGTTT TTTAGTCGGA
    TGAGCTTCTT
201 CAGATCGGGA GCTCCAAGAG GTAGCCAACA ACCCTCTGCT
    CCATCTGCAG
251 ATACTGTACG TAGCCCGTTG CCGGGAGGGG ATGCTCGCGC
    TACCGAGGGA
301 GCTGGTAGGA ACTTAATTAA AAAAGGGTAC CAACCAGGGA
    TGAAAGTCAC
351 TATCCCACAG GTTCCTGGAG GAGGGGCCCA ACGTTCATCA
    GGTAGCACGA
401 CACTAAAGCC TACGCGTCCG GCACCCCCAC CTCCTAAAAC
    GGGTGGAACT
451 AATGCAAAAC GTCCGGCAAC GCACGGGAAG GGTCCAGCAC
    CCCAGCCTCC
501 TAAAACAGGT GGGACCAATG CTAAGCGCGC AGCAACGCAT
    GGGAAAGGTC
551 CAGCACCTCA ACCTCCTAAG GGCATTTTGA AACAGCCTGG
    GCAGTCTGGG
601 ACTTCAGGAA AGAAGCGTGT CAGCTGGTCT GACGAAGATT
    AA

The PSORT algorithm predicts cytoplasm (0.360).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 98A). The GST-fusion protein was used to immunise mice, whose sera were used in a Western blot (FIG. 98B) and for FACS analysis (FIG. 98C).

These experiments show that cp6979 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 99

The following C. pneumoniae protein (PID 4377028) was expressed <SEQ ID 197; cp7028>:

1   MLLGFLCDCP CASWQCAAVA NCYDSVFMSR PEHKPNIPYI
    TKATRRGLRM
51  KTLAYLASLK DARQLAYDFL KDPGSLARLA KALIAPKEAL
    QEGNLFFYGC
101 SNIEDILEEM RRPHRILLLG FSYCQKPKAC PEGRFNDACR
    YDPSHPTCAS
151 CSIGTMMRLN ARRYTTVIIP TFIDIAKHLH TLKKRYPGYQ
    ILFAVTACEL
201 SLKMFGDYAS VMNLKGVGIR LTGRICNTFK AFKLAERGVK
    PGVTILEEDG
251 FEVLARILTE YSSAPFPRDF CEIH*

The cp7028 nucleotide sequence <SEQ ID 198> is:

1   ATGCTTCTAG GGTTTTTGTG TGACTGCCCC TGTGCTTCGT
    GGCAGTGTGC
51  GGCCGTTGCT AATTGTTATG ATTCCGTATT TATGTCTAGA
    CCAGAGCACA
101 AACCTAATAT TCCTTATATT ACTAAAGCTA CAAGACGGGG
    TCTGCGTATG
151 AAGACGCTTG CTTATCTGGC CTCTTTAAAA GATGCTAGAC
    AGCTTGCCTA
201 TGATTTTCTG AAAGATCCTG GTTCTTTAGC TCGGTTAGCT
    AAGGCTTTGA
251 TAGCTCCTAA GGAGGCCTTA CAGGAGGGCA ACCTATTTTT
    TTATGGCTGT
301 AGTAATATTG AGGATATTTT AGAGGAGATG CGTCGTCCTC
    ATAGAATCCT
351 TTTGTTAGGA TTTTCTTATT GTCAAAAGCC TAAGGCATGT
    CCTGAAGGGC
401 GTTTCAATGA TGCTTGTCGG TATGATCCTT CACATCCTAC
    ATGTGCCTCA
451 TGTTCTATAG GGACCATGAT GCGGCTGAAT GCTCGTAGAT
    ACACTACTGT
501 GATCATCCCT ACATTTATAG ATAGCGCAAA ACATTTACAC
    ACTTTAAAAA
551 AGCGCTACCC TGGATATCAA ATTCTCTTTG CAGTTACTGC
    TTGTGAACTT
601 TCCTTAAAAA TGTTTGGAGA TTATGCCTCC GTAATGAACT
    TAAAGGGTGT
651 GGGCATCAGA CTCACAGGAC GTATTTGCAA TACATTTAAG
    GCATTTAAAT
701 TAGCTGAGCG AGGAGTCAAA CCAGGAGTCA CTATCCTAGA
    AGAAGATGGC
751 TTTGAGGTAT TAGCAAGGAT TCTTACAGAA TACAGTAGCG
    CTCCTTTCCC
801 TAGAGACTTT TGTGAGATCC ATTAG

The PSORT algorithm predicts cytoplasm (0.1453).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 99A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 99B) and for FACS analysis (FIG. 99C).

These experiments show that cp7028 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 100

The following C. pneumoniae protein (PID 4377355) was expressed <SEQ ID 199; cp7355>:

1  MKKVVTLSII FFATYCASEL SAVTVVAVPL SEAPGKIQVR
   PVVGLQFQEE
51 QGSVPYSFYY PYDYGYYYPE TYGYTKNTGQ ESRECYTRFE
   DGTIDYECD*

The cp7355 nucleotide sequence <SEQ ID 200> is:

1   ATGAAGAAAG TCGTAACACT ATCCATTATA TTTTTCGCAA
    CGTATTGTGC
51  ATCAGAGCTT AGTGCTGTAA CTGTAGTGGC TGTGCCTTTA
    TCAGAGGCTC
101 CAGGGAAGAT TCAAGTTCGT CCCGTCGTTG GTCTGCAATT
    TCAAGAAGAA
151 CAGGGTTCTG TGCCCTATAG TTTTTATTAT CCTTATGACT
    ATGGGTATTA
201 CTATCCAGAG ACTTATGGCT ATACTAAAAA TACAGGTCAA
    GAAAGTCGCG
251 AATGTTATAC CCGATTTGAA GATGGCACAA TTTTTTATGA
    ATGCGATTAG

The PSORT algorithm predicts inner membrane (0.143).

The protein was expressed in E. coli and purified as a GST-fusion (FIG. 100A) and a his-tag product. The proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 100B) and for FACS analysis (FIG. 100C).

These experiments show that cp7355 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 101

The following C. pneumoniae protein (PID 4377380) was expressed <SEQ ID 201; cp7380>:

1   VHYCERTLDP KYILKIALKL RQSLSLFFQN SQSLQRAYST
    PYSYYRIILQ
51  KENKEKQALA RHKCISILEF FKNLLFVHLL SLSKNQREGC
    STDMAVVSTP
101 FFNRNLWYRL LSSRFSLWKS YCPRFFLDYL EAFGLLSDFL
    DHQAVIKFFE
151 LETHFSYYPV SGFVAPHQYL SLLQDRYFPI ASVMRTLDKD
    NFSLTPDLIH
201 DLLGHVPWLL HPSFSEFFIN MGRLFTKVIE KVQALPSKKQ
    RIQTLQSNLI
251 AIVRCFWFTV ESGLIENHEG RKAYGAVLIS SPQELGHAFI
    DNVRVLPLEL
301 DQIIRLRFNT STPQETLFSI RHFDELVELT SKLEWMLDQG
    LLESIPLYNQ
351 EKYLSGFEVL CQ*

The cp7380 nucleotide sequence <SEQ ID 202> is:

1    GTGCACTACT GCGAGAGAAC CCTGGACCCA AAGTATATTC
     TGAAGATTGC
51   TCTAAAGCTG AGACAATCAC TTTCCCTGTT CTTCCAGAAC
     AGCCAATCAC
101  TCCAACGTGC ATACTCGACC CCATATTCCT ACTACCGAAT
     CATTCTACAA
151  AAGGATAATA AAGAGAAGCA AGCTTTAGCT CGACACAAAT
     GCATTTCTAT
201  TTTAGAATTT TTCAAAAACT TACTCTTTGT TCATCTTCTG
     TCATTATCAA
251  AGAATCAAAG GGAAGGTTGC TCCACTGATA TGGCTGTTGT
     AAGCACTCCC
301  TTTTTTAATC GGAATTTATG GTATCGACTC CTTTCCTCAC
     GGTTTTCTCT
351  ATGGAAAAGC TATTGTCCAA GATTTTTTCT TGATTACTTA
     GAAGCTTTCG
401  GTCTCCTTTC TGATTTCTTA GACCATCAAG CAGTCATTAA
     ATTCTTCCAA
451  TTAGAAACAC ATTTTTCCTA TTATCCCGTT TCAGGATTTG
     TAGCTCCCCA
501  TCAATACTTG TCTCTGTTGC AGGACCGTTA CTTTCCCATT
     GCCTCTGTAA
551  TGCGAACTCT CGATAAAGAT AGTTTCTCCT TAACTCCTGA
     TCTCATCCAT
601  GACCTTTTAG GGCACGTGCC TTGGCTTCTA CATCCCTCAT
     TTTCTGAATT
651  TTTCATAAAC ATGGGAAGAC TCTTCACTAA AGTCATAGAA
     AAAGTACAAG
701  CTCTTCCTAG TAAAAAACAA CGCATACAAA CCCTACAAAG
     CAATCTGATC
751  GCTATTGTAC GCTGCTTTTG GTTTACTGTT GAAAGCGGAC
     TTATTGAAAA
801  CCATGAAGGA AGAAAAGCAT ATGGAGCCGT TCTTATCAGT
     TCTCCTCAGG
851  AACTTGGACA CGCTTTCATT GATAACGTAC GTGTTCTCCC
     TTTAGAATTG
901  GATCAGATTA TTCGTCTTCC CTTCAATACA TCAACTCCAC
     AAGAGACTTT
951  ATTTTCAATA AGACATTTTG ATGAACTGGT AGAACTCACT
     TCAAAATTAG
1001 AATGGATGCT CGACCAAGGT CTGTTAGAAT CAATTCCCCT
     TTACAATCAA
1051 GAGAAATATC TTTCTGGTTT TGAGGTACTT TGCCAATGA

The PSORT algorithm predicts inner membrane (0.1362).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 101A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 101B) and for FACS analysis (FIG. 101C).

These experiments show that cp7380 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 102

The following C. pneumoniae protein (PID 4376904) was expressed <SEQ ID 203; cp6904>:

1   MMNYEDAKLR GQAVAILYQI GAIKFGKHIL ASGEETPLYV
    DMRLVISSPE
51  VLQTVATLIW RLRPSFNSSL LCGVRYTALT LATSISLKYN
    IPMVLRRKEL
101 QNVDRSDAIK VEGLFTPGQT CLVINDMVSS GKSIIETAVA
    LEENGLVVRE
151 ALVFLDRRKE ACQPLGPQGI KVSSVFTVPT LIKALIAYGK
    LSSGDLTLAN
201 KISEILEIES *

The cp6904 nucleotide sequence <SEQ ID 204> is:

1   ATGATGAACT ACGAAGATGC AAAATTACGC GGTCAAGCTG
    TAGCAATTCT
51  ATACCAAATC GGAGCTATAA AGTTCGGAAA ACATATTCTC
    GCTAGCGGAG
101 AAGAAACTCC TCTGTATGTA GATATGCGTC TTGTGATCTC
    CTCTCCAGAA
151 GTTCTCCAGA CAGTGGCAAC TCTTATTTGG CGCCTCCGCC
    CCTCATTCAA
201 TAGTAGCTTA CTCTGCGGAG TCCCTTATAC TGCTCTAACC
    CTAGCAACCT
251 CGATCTCTTT AAAATATAAC ATCCCTATGG TATTGCGAAG
    GAAGGAATTA
301 CAGAATGTAG ACCCCTCGGA CGCTATTAAA GTAGAAGGGT
    TATTTACTCC
351 AGGACAAACT TGTTTAGTCA TCAATGATAT GGTTTCCTCA
    GGAAAATCTA
401 TAATAGAGAC AGCAGTCGCA CTGGAAGAAA ATGGTCTGGT
    AGTTCGTGAA
451 GCATTGGTAT TCTTAGATCG TAGAAAAGAA GCGTGTCAAC
    CACTTGGTCC
501 ACAGGGAATA AAAGTCAGTT CGGTATTTAC TGTACCCACT
    CTGATAAAAG
551 CTTTGATCGC TTATGGGAAG CTAAGCAGTG GTGATCTAAC
    CCTGGCAAAC
601 AAAATTTCCG AAATTCTAGA AATTGAATCT TAA

The PSORT algorithm predicts cytoplasm (0.0358).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 102A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 102B) and for FACS analysis.

The cp6904 protein was also identified in the 2D-PAGE experiment.

These experiments show that cp6904 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 103

The following C. pneumoniae protein (PID 4376964) was expressed <SEQ ID 205; cp6964>:

1  MKKLIALIGI FLVPIKGNTN KEHDAHATVL KAARAKYNLF
   FVQVFPVHE
51 VIEPISPDCL VHYEGWV*

The cp6964 nucleotide sequence <SEQ ID 206> is:

1   ATGAAAAAAT TGATTGCTTT GATAGGGATA TTTCTTGTTC
    CAATAAAAGG
51  AAATACCAAT AAGGAACACG ACGCTCACGC GACTGTTTTA
    AAAGCGGCCA
101 GAGCAAAGTA TAATTTGTTC TTTGTTCAGG ATGTTTTCCC
    TGTACACGAA
151 GTTATCGAGC CTATTTCTCC CGATTGCCTG GTACATTATG
    AAGGGTGGGT
201 TTGA

The PSORT algorithm predicts inner membrane (0.091).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 103A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 103B) and for FACS analysis (FIG. 103C).

These experiments show that cp6964 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 104

The following C. pneumoniae protein (PID 4377387) was expressed <SEQ ID 207; cp7387>:

1   LNFAKIDHNH LYLTCLGDLG VACPILSTDC LPNYSEKASH
    EVLVYSKFRC
51  ISGFPSRLAT SGNDTYYSIV SLPIGLRYEV TSPSGRHDFN
    IDMHVAPKIG
101 AVLSHGTREA KEIPGSSKDY AFFSLTARES LMISEKLAMT
    FQVSEVIQNC
151 YSQCTKVTKT NLKEQYRHLS HNTGFELSVK SAF*

The cp7387 nucleotide sequence <SEQ ID 208> is:

1   TTGAATTTTG CAAAGATTGA TCACAATCAT CTCTACCTTA
    CATGTTTGGG
51  AGATCTTGGT GTAGCTTGTC CTATACTTTC TACAGATTGT
    CTACCTAATT
101 ATAGCGAGAA AGCATCTCAT GAGGTTCTTG TTTATAGTAA
    ATTTAGATGC
151 ATTTCTGGAG AGCCATCTCG ACTTGCAACT TCAGGAAATG
    ACACATATTA
201 TTCTATAGTA AGTTTACCTA TAGGACTCCG TTACGAAGTG
    ACTTCACCAT
251 CAGGACGTCA TGATTTCAAT ATTGATATGC ATGTAGCTCC
    AAAGATAGGT
301 GCAGTACTCT CTCATGGAAC ACGAGAGGCT AAAGAGATCC
    CAGGATCTTC
351 AAAAGACTAT GCATTTTTTA GCTTGACTGC TAGAGAAAGT
    TTAATGATTT
401 CTGAAAAGCT TGCGATGACT TTCCAAGTTA GCGAAGTTAT
    TCAGAATTGT
451 TATTCACAAT GTACTAAAGT AACGAAAACT AATTTAAAAG
    AACAGTATAG
501 GCACTTATCC CACAATACAG GGTTTGAGTT AAGCGTCAAG
    TCTGCATTCT
551 AA

The PSORT algorithm predicts inner membrane (0.043).

The protein was expressed in E. coli and purified as a his-tagged-fusion product (FIG. 104A) and also as a GST-fusion (FIG. 104B). The recombinant proteins were used to immunise mice, whose sera were used in a Western blot and for FACS analysis (FIG. 104C; his-tagged).

These experiments show that cp7387 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 105

The following C. pneumoniae protein (PID 4376281) was expressed <SEQ ID 209; cp6281>:

1   MFLQFFHPIV FSDQSLSFLP YLGKSSGIIE KCSNIVEHYL
    HLGGDTSVII
51  TGVSGATFLS VDHALPISKS EKIIKILSYI LILPLILALF
    IKIVLRIILF
101 FKYRGLILDV KKEDLKKTLT PDQENLSLPL PSPTTLKKIH
    ALHILVRSGK
151 TYNELIQEGF SFTKITDLGQ APSPKQDIGF SYNSLLPNFY
    FHSLVSVPNI
201 SGEERALNYH KEQQEEMAVK LKTMQACSFV FRSLHLPSMQ
    TKDKKAGFGL
251 LTFPPWKIYP I*

The cp6281 nucleotide sequence <SEQ ID 210> is:

1   ATGTTTCTTC AGTTTTTTCA TCCTATAGTC TTCTCGGATC
    AGTCCTTATC
51  TTTTCTTCCT TACCTAGGAA AAAGCTCTGG CATTATTGAA
    AAATGTTCCA
101 ATATCGTTGA ACACTATTTA CATTTGGGAG GAGACACTTC
    TGTTATCATC
151 ACAGGAGTTT CTGGAGCTAC CTTTCTATCT GTTGATCATG
    CCCTCCCAAT
201 CTCGAAATCT GAAAAAATAA TAAAAATTCT GCTTCATATT
    TTAATTCTTC
251 CTCTGATTCT AGCTCTCTTT ATTAAGATCG TTTTACGCAT
    TATCTTATTC
301 TTCAAGTATC GTGGTCTAAT CCTAGATGTT AAGAAGGAGG
    ATTTGAAAAA
351 AACACTTACA CCTGACCAAG AAAACCTCAG TCTTCCTTTA
    CCATCTCCTA
401 CAACATTAAA GAAAATCCAT GCGCTACACA TTTTAGTGCG
    TTCTGGAAAA
451 ACCTATAACG AGCTTATACA AGAAGGGTTT TCTTTCACTA
    AAATCACAGA
501 TCTTGGTCAA GCTCCTTCAC CAAAGCAAGA TATTGGCTTC
    TCTTATAATT
551 CCCTTCTCCC TAACTTCTAT TTTCATTCCT TGGTATCTGT
    TCCAAATATT
601 TCAGGCGAGG AACGGGCTCT TAATTATCAT AAAGAACAAC
    AAGAGGAAAT
651 GGCTGTTAAA TTAAAAACAA TGCAAGCGTG TTCTTTTGTC
    TTCCGATCCC
701 TGCATTTACC TTCAATGCAA ACGAAGGACA AAAAGGCTGG
    ATTTGGACTA
751 CTGACGTTTT TCCCTTGGAA AATCTACCCC CTATAA

The PSORT algorithm predicts inner membrane (0.5373).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 105A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 105B) and for FACS analysis.

These experiments show that cp6281 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 106 and Example 107

The following C. pneumoniae protein (PID 4376306) was expressed <SEQ ID 211; cp6306>:

1  MGNHETYIHP GVLPSSHAQD VSRSTVYPSR SFIMRRMLMG
   WNFNRVPSKS
51 SEQLMDGHRI PLIFFGKHHP TISILNVNRF SWLSIFYNGE
   RGF*

The cp6306 nucleotide sequence <SEQ ID 212> is:

1   ATGGGAAACC ATGAGACCTA TATACATCCA GGAGTGCTCC
    CGAGTAGTCA
51  TGCTCAGGAT GTTAGCAGAT CTACAGTTTA CCCCAGTCGA
    AGTTTTATCA
101 TGAGACGTAT GCTCATGGGC TGGAATTTCA ATCGTGTTCC
    CTCGAAGAGC
151 TCCGAGCAGT TAATGGATGG TCATCGCATA CCTCTTATAT
    TTTTTGGGAA
201 GCATCATCCT ACTATATCTA TTTTAAATGT CAATAGATTT
    TCTTGGCTCT
251 CCATTTTTTA CAATGGAGAA AGGGGGTTTT GA

The PSORT algorithm predicts cytoplasm (0.167).

The following C. pneumoniae protein (PID 4376434) was also expressed <SEQ ID 213; cp6434>:

1   MSESINRSIH LEASTPFFIK LTNLCESRLV LITSLVISLL
    ALVGAGVTLV
51  VLFVAGILPL LPVLILEIIL ITVLVLLFCL VLEPYLIEKP
    SKIKELPKVD
101 ELSVVETDST L*

The cp6434 nucleotide sequence <SEQ ID 214> is:

1   ATGTCTGAAA GTATTAACAG AAGCATTCAT TTAGAAGCCT
    CTACACCATT
51  TTTTATAAAA TTAACGAATC TCTGTGAAAG TAGATTAGTT
    AAGATCACTT
101 CTCTTGTTAT TTCTCTATTA GCTTTAGTGG GTGCGGGAGT
    CACTCTTGTG
151 GTTTTATTTG TAGCTGGGAT CCTTCCTTTA CTTCCTGTAC
    TCATCTTAGA
201 AATTATTTTA ATAACCGTCC TTGTCTTGCT TTTTTGTTTG
    GTATTGGAAC
251 CTTATTTAAT AGAAAAACCT AGTAAAATAA AGGAACTACC
    TAAAGTAGAC
301 GAGCTATCTG TAGTAGAAAC GGACAGTACT CTTTAA

The PSORT algorithm predicts inner membrane (0.6859).

The proteins were expressed in E. coli and purified as his-tag products (FIG. 106A; 6306=lanes 2-4; 6434=lanes 8-10). The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 106B & 107) and for FACS analysis.

These experiments show that cp6306 & cp6434 are surface-exposed and immunoaccessible proteins, and that they are useful immunogens. These properties are not evident from the sequences alone.

Example 108

The following C. pneumoniae protein (PID 4377400) was expressed <SEQ ID 215; cp7400>:

1  MRVMRFFCLF ELGFLGSFHC VAEDKGVDLF GVWDDNQITE
   CDDSYMTEGR
51 EEVEKVVDA

The cp7400 nucleotide sequence <SEQ ID 216> is:

1   GTGAGAGTTA TGAGATTTTT TTGTCTATTT TTTCTTGGGT
    TCCTAGGATC
51  TTTTCATTGT GTTGCTGAAG ACAAGGGCGT GGATTTATTT
    GGAGTCTGGG
101 ACGATAACCA AATTACAGAG TGTGACGATA GTTACATGAC
    AGAGGGTCGT
151 GAAGAGGTTG AAAAGGTAGT GGACGCTTAG

The PSORT algorithm predicts periplasmic space (0.924).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 108A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 108B) and for FACS analysis.

These experiments show that cp7400 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 109

The following C. pneumoniae protein (PID 4376395) was expressed <SEQ ID 217; cp6395>:

1   MENAMSSSFV YNGPSWILKT SVAQEVFKKH GKGIQVLLST
    SVMLFIGLGV
51  CAFIFPQYLI VFVLTIALLM LAISLVLFLL IRSVRSSMVD
    RLWCSEKGYA
101 LHQHENGPFL DVKRVQQILL RSPYIKVRAL WPSGDIPEDP
    SQAAVLLLSP
151 WTFFSSVDVE ALLPSPQEKE GKYIDPVLPK LSRIERVSLL
    VELSAFTLDD
201 LNEQGVNPLM NNEEFLFFIN KKAREHGIQD LKHEIMSSLE
    KTGVPLDPSM
251 SFQVSQAMFS VYRYLRQRDL TTSELRCFHL LSCFKGDVVH
    CLASFENPKD
301 LADSDFLEAC KNVEWGEFIS ACEKALLKNP QGISIKDLKQ
    FLVR*

The cp6395 nucleotide sequence <SEQ ID 218> is:

1    ATGGAGAATG CTATGTCATC ATCGTTTGTG TATAATGGGC
     CTTCGTGGAT
51   TTTAAAAACG TCAGTAGCTC AGGAGGTATT TAAAAAGCAC
     GGTAAGGGGA
101  TTCAGGTTCT CTTAAGTACT TCAGTGATGC TTTTTATAGG
     TCTTGGAGTC
151  TGTGCCTTTA TATTTCCTCA ATATCTGATT GTTTTTGTTT
     TGACTATAGC
201  TTTGCTTATG CTCGCTATAA GCTTGGTATT GTTTCTCTTA
     ATACGTTCTG
251  TACGCTCTTC AATGGTAGAT CGTTTGTGGT GTTCTGAAAA
     AGGATATGCT
301  CTTCATCAAC ATGAGAACGG GCCTTTTTTG GATGTGAAGC
     GTGTACAGCA
351  AATTCTTCTA AGATCACCCT ATATTAAAGT TCGGGCTTTA
     TGGCCGTCTG
401  GAGATATCCC TGAGGATCCT TCACAAGCTG CGGTTCTATT
     ACTTTCTCCT
451  TGGACTTTCT TTTCATCCGT GGATGTAGAG GCTTTATTAC
     CGAGTCCTCA
501  AGAAAAGGAG GGTAAGTATA TAGATCCTGT GCTGCCTAAG
     TTGTCTAGGA
551  TAGAGAGAGT CTCACTTTTA GTGTTTTTGA GTGCATTTAC
     TTTGGATGAC
601  TTAAACGAAC AGGGAGTCAA TCCTTTGATG AATAATGAGG
     AATTTTTATT
651  TTTTATAAAT AAGAAAGCGC GTGAGCATGG GATTCAGGAT
     TTAAAACACG
701  AGATTATGTC TTCGTTAGAG AAAACAGGAG TGCCATTAGA
     CCCCTCAATG
751  AGTTTTCAAG TTTCACAAGC GATGTTTTCT GTATATCGCT
     ACTTGAGACA
801  AAGGGATTTA ACGACTTCAG AATTAAGATG TTTTCACCTC
     TTAAGTTGTT
851  TTAAAGGGGA TGTGGTTCAT TGTTTAGCTT CATTTGAAAA
     CCCTAAAGAT
901  TTAGCAGATT CTGACTTTTT AGAAGCTTGT AAGAACGTGG
     AATGGGGTGA
951  GTTTATTTCG GCATGTGAGA AGGCTCTTTT AAAGAATCCG
     CAAGGAATTT
1001 CCATTAAGGA TCTAAAACAA TTTTTAGTGA GGTAA

The PSORT algorithm predicts inner membrane (0.6307).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 109A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 109B) and for FACS analysis.

These experiments show that cp6395 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 110

The following C. pneumoniae protein (PID 4376396) was expressed <SEQ ID 219; cp6396>:

1   MIEFAFVPHT SVTADRIEDR MACRMNKLST LAITSLCVLI
    SSVCIMIGIL
51  CISGTVGTYA FVVGIIFSVL ALVACVFFLY FFYFSSEEFK
    CASSQEFRFL
101 PIPAVVSALR SYEYISQDAI NDVIKDTMQL STLSSLLDPE
    AFFLEFPYFN
151 SLIVNHSMKE ADRLSREAFL ILLGEITWKD CEIKILPWLK
    DPNITPDDFW
201 KLLKDHFDLK DFKKRIATWI RKAYPEIRLP KKHCLDKSIY
    KGCCKFLLLS
251 ENDVQYQRLL HKVCYFSGEF PAMVLGLGSE VPMVLGLPKV
    PKDLTWEMFM
301 ENMPVLLQSK REGHWKISLE DVASL*

The cp6396 nucleotide sequence <SEQ ID 220> is:

1   ATGATCGAGT TTGCTTTTGT TCCTCATACC TCCGTGACAG
    CGGATCGGAT
51  TGAGGATCGC ATGGCCTGTC GCATGAACAA GTTGTCTACT
    TTAGCAATTA
101 CAAGTCTTTG TGTATTGATC AGTTCAGTTT GTATTATGAT
    TGGGATTTTA
151 TGCATTTCTG GAACGGTTGG GACCTATGCA TTTGTTGTAG
    GAATTATTTT
201 TTCTGTCCTT GCTTTGGTAG CATGTGTTTT CTTTCTTTAT
    TTCTTTTATT
251 TTTCTTCTGA GGAATTTAAG TGTGCTTCTT CGCAGGAGTT
    TCGTTTTTTG
301 CCTATACCAG CTGTGGTTTC TGCATTGCGT TCCTATGAAT
    ACATTTCTCA
351 GGACGCTATC AATGACGTTA TAAAAGATAC GATGCAGTTG
    TCTACCCTTT
401 CTTCTCTTTT AGATCCCGAA GCTTTTTTCT TAGAATTTCC
    TTATTTTAAC
451 TCTTTGATAG TGAATCATTC GATGAAGGAA GCGGATCGTT
    TGTCTCGAGA
501 GGCTTTTTTG ATTTTATTAG GTGAGATTAC TTGGAAGGAT
    TGTGAAACAA
551 AAATTTTGCC ATGGTTGAAA GATCCTAATA TCACTCCTGA
    TGATTTCTGG
601 AAGCTATTAA AAGACCATTT CGATTTAAAG GACTTTAAGA
    AGAGGATCGC
651 CACTTGGATA CGGAAGGCCT ATCCAGAAAT TAGATTACCG
    AAGAAGCATT
701 GTTTAGATAA GTCTATCTAT AAGGGGTGTT GTAAGTTTTT
    ATTACTTTCT
751 GAGAATGATG TGCAATATCA GAGGTTATTA CATAAGGTCT
    GTTATTTCTC
801 TGGGGAGTTT CCTGCCATGG TTTTAGGTTT GGGAAGTGAA
    GTGCCTATGG
851 TGTTAGGACT CCCTAAGGTT CCCAAGGATC TTACCTGGGA
    GATGTTTATG
901 GAAAATATGC CTGTTCTTCT GCAAAGCAAA AGAGAGGGGC
    ATTGGAAAAT
951 CTCCTTGGAA GACGTAGCCT CTCTTTAA

The PSORT algorithm predicts inner membrane (0.6095).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 110A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 110B) and for FACS analysis.

These experiments show that cp6396 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 111

The following C. pneumoniae protein (PID 4376408) was expressed <SEQ ID 221; cp6408>:

1   MNTSLKRPLK SHFDVVGSFL RPEHLKKTRE SLKEGSISLD
    QLMQIEDIAI
51  QDLIKKQKAA GLSFITDGEF RRATWHYDFM WGFEGVGHHR
    ATEGVFFDGE
101 RAMIDDTYLT DKISVSHHPF VDHFKFVKAL EDEFTTAKQT
    LPAPAQFLKQ
151 MIFPNNIEVT RKFYPTNQEL IEDIVAGYRK VIRDLYDAGC
    RYLQLDDCTR
201 GGLVDPRVCS WYGIDEKGLQ DLIQQYLLIN NLVIADRPDD
    LVVNLHVCRG
251 NYHSKFFASG SYDFIAKPLF EQTNVDGYYL EFDEERSGDF
    SPLTFISGEK
301 TVCLGLVTSK TPTLENKDEV IARIHQAADY LPLERLSLSP
    QCGFASCEIG
351 NKLTEEEQWA KVALVKEISE EVWK*

The cp6408 nucleotide sequence <SEQ ID 222> is:

1    ATGAATACTT CACTAAAAAG ACCTCTGAAA TCTCATTTTG
     ATGTTGTCGG
51   TAGTTTTTTG CGTCCTGAGC ATTTAAAAAA AACTAGAGAA
     AGCCTTAAAG
101  AAGGCTCTAT TTCTCTAGAT CAACTCATGC AAATTGAGGA
     TATCGCTATC
151  CAAGATTTGA TCAAAAAACA AAAAGCAGCA GGTCTTTCTT
     TTATTACTGA
201  TGGAGAATTC CGCAGAGCTA CGTGGCATTA CGACTTCATG
     TGGGGTTTTC
251  ATGGCGTAGG TCACCACAGA GCTACAGAAG GAGTTTTCTT
     TGATGGAGAA
301  CGCGCTATGA TCGATGATAC CTATCTGACA GACAAGATCT
     CTGTATCTCA
351  CCACCCATTT GTGGATCACT TTAAATTTGT AAAAGCTCTA
     GAAGATGAAT
401  TTACGACTGC AAAGCAAACT CTTCCTGCAC CGGCACAGTT
     TTTAAAGCAG
451  ATGATCTTCC CTAATAATAT AGAGGTCACA CGTAAATTCT
     ATCCTACAAA
501  TCAGGAGCTA ATTGAAGATA TTGTTGCAGG TTATCGTAAA
     GTCATTCGCG
551  ATCTTTATGA TGCTGGCTGC CGCTATCTCC AATTAGATGA
     CTGTACTCGG
601  GGAGGTTTAG TAGACCCTCG AGTCTGTTCG TGGTATGGTA
     TCGATGAAAA
651  AGGTCTTCAA GATCTGATTC AACAATATCT TCTGATTAAT
     AATCTTGTAA
701  TTGCAGATCG TCCCGATGAT CTAGTCGTTA ATTTACATGT
     ATGCCGTGGG
751  AACTACCACT CAAAATTCTT TGCTAGTGGT AGTTATGACT
     TTATTGCAAA
801  GCCCCTATTC GAACAAACAA ATGTAGACGG CTACTATTTA
     GAGTTTGATC
851  ATGAGCGTTC TGGAGACTTC TCTCCTCTCA CCTTCATTTC
     TGGAGAAAAA
901  ACTGTCTGCT TAGGTCTTGT TACCAGCAAA ACCCCTACAC
     TTGAAAATAA
951  GGATGAGGTC ATTGCTCGCA TACATCAAGC AGCAGACTAC
     CTGCCCTTGG
1001 AAAGACTCTC TCTAAGTCCA CAGTGTGGTT TTGCTTCATG
     TGAAATAGGA
1051 AATAAATTAA CAGAAGAAGA GCAATGGGCT AAAGTTGCTC
     TAGTAAAAGA
1101 AATTTCCGAA GAAGTTTGGA AATAA

The PSORT algorithm predicts cytoplasm (0.2171).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 111A) and also as a his-tagged product. The his-tag protein was used to immunise mice, whose sera were used in a Western blot (FIG. 111B) and for FACS analysis.

These experiments show that cp6408 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 112

The following C. pneumoniae protein (PID 4376430) was expressed <SEQ ID 223; cp6430>:

1   MKLYSISSDV DTPWIFQLMS KVDSYLFLGG NRIKVVSIVM
    QEPNLIIGKV
51  ENVRISTIVK ILKILSFLIF PLILIALALH YFLHAKYANH
    LLVSKILERA
101 PQYVPIPGRS GDTASHYKLT TLVPVSQKNL QAMGSNPLEV
    EAALRTTKPS
151 FFCVPAKYRQ IIISSHGIRF SLDLEQLADD INLDSVSWPT
    EYLNSTMDFC
201 SKADKRVIQN VQNLRTGTYI NSVGKRSLLK FMLQHLFIDG
    ITQENPEALP
251 NNTSGRLTLF PSVRYIYSHF TPQNPTIWPQ VFFRQGPLDE
    DRGGGFEILE
301 QLQELGVRFP ICPSQGPDNP NFQGFQGIRI YWEDSYQPNK EV*

The cp6430 nucleotide sequence <SEQ ID 224> is:

1    ATGAAACTTT ATAGCATCTC TTCAGATGTA GATACACCTT
     GGATATTTCA
51   GCTTATGTCA AAGGTAGATT CTTATCTTTT CTTAGGCGGG
     AATAGAATCA
101  AGGTTCTATC TATAGTTATG CAAGAACCTA ACTTAATTAT
     TGGAAAAGTA
151  GAAAACGTTC GGATCTCCAC AATAGTGAAA ATATTAGAGA
     TTTTATCCTT
201  CTTAATCTTC CCTCTGATTT TAATCGCTTT AGCCCTACAC
     TATTTTCTAC
251  ATGCTAAATA TGCTAATCAC TTACTTGTAT CTAGGATTTT
     AGAAAGAGCT
301  CCTCAGTATG TGCCTATTCC TGGTCGTTCA GGAGACACGG
     CGTCTCATTA
351  TAAATTAACA ACATTGGTTC CAGTATCCCA AAAAAATCTA
     CAAGCTATGG
401  GATCAAATCC TCTAGAAGTT GAAGCGGCTC TTCGAACTAC
     AAAACCCTCT
451  TTTTTCTGTG TACCTGCAAA ATACCGTCAG ATTATAATTT
     CAAGTCACGG
501  CATTCGCTTT TCTTTAGATC TTGAACAACT TGCTGATGAC
     ATTAATTTAG
551  ATTCGGTTTC CTGGCCTACG GAGTATCTTA ACTCTACTAT
     GGATTTTTGC
601  AGCAAGGCAG ATAAACGTGT TATACAGAAT GTACAAAATC
     TGCGGACAGG
651  AACTTACATA ACTTCTGTAG GAAAGCGTAG CCTTTTAAAA
     TTCATGTTAC
701  AGCACCTATT TATTGATGGG ATCACACAAG AAAACCCTGA
     AGCCCTTCCT
751  ACCAATACAT CTGGAAGACT GACTCTATTC CCTAGTGTTC
     GTTATATCTA
801  TTCTCATTTT ACTCCACAAA ATCCTACAAT ATGGCCGCAA
     GTCTTTTTCA
851  GACAAGGTCC TCTAGATGAA GATCGAGGAG GAGGATTTGA
     GATCTTAGAG
901  CAATTACAAG AGTTAGGAGT TAGGTTTCCA ATTTGCCCCT
     CTCAAGGACC
951  AGACAATCCT AATTTTCAAG GTTTTCAAGG GATTCGTATC
     TATTGGGAAG
1001 ATTCCTATCA ACCCAATAAG GAGGTTTAA

The PSORT algorithm predicts inner membrane (0.5140).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 112A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 112B) and for FACS analysis.

These experiments show that cp6430 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 113

The following C. pneumoniae protein (PID 4376439) was expressed <SEQ ID 225; cp6439>:

1   MSYDTLFKNL EKEDSVHKIC NEIFALVPRL NTIACTEAII
    KNLPKADIHV
51  HLPGTITPQL AWILGVKNGF LKWSYNSWTN HRLLSPKNPH
    KQYSNIFRNF
101 QDICHEKDPD LSVLQYNILN YDFNSFDRVM ATVQGHRFPP
    GGIQNEEDLL
151 LIFNNYLQQC LDDTIVYTEV QQNIRLAHVL YPSLPEKHAR
    MKFYQILYRA
201 SQTFSKHGIT LRFLNCFNKT FAPQINTQEP AQEAVQWLQE
    VDSTFPGLFV
251 GIQSAGSESA PGACPKRLAS GYRNAYDSGF GCEAHAGEGI
    ETRTIFSSAK
301 VNPEGLIEIT RVTFSSLKRK QPSSLPIRVT CQLG*

The cp6439 nucleotide sequence <SEQ ID 226> is:

1    ATGTCTTATG ATACGTTATT CAAGAATCTT GAAAAGGAAG
     ATTCTGTACA
51   TAAGATATGC AATGAGATCT TTGCATTAGT ACCACGACTC
     AATACAATCG
101  CTTGCACCGA AGCTATCATC AAAAACCTCC CCAAAGCAGA
     TATCCATGTA
151  CACCTTCCTG GGACCATAAC ACCTCAATTA GCTTGGATTT
     TAGGTGTGAA
201  AAATGGGTTC TTAAAATGGT CTTATAATTC TTGGACCAAT
     CATCGATTAC
251  TTTCTCCTAA GAATCCTCAT AAACAATACT CCAATATTTT
     CCGAAACTTT
301  CAAGATATCT GTCACGAAAA GGATCCGGAT TTAAGTGTAT
     TACAATATAA
351  TATCTTAAAT TACGATTTTA ATAGCTTTGA TAGAGTGATG
     GCTACAGTAC
401  AAGGACATCG CTTTCCTCCT GGAGGAATCC AAAATGAAGA
     AGACCTTCTT
451  CTCATTTTCA ATAACTATCT CCAGCAATGT CTGGACGATA
     CTATCGTGTA
501  TACTGAAGTA CAACAAAATA TCCGCCTTGC CCATGTTTTG
     TATCCTTCAT
551  TACCTGAAAA GCACGCGCGT ATGAAGTTTT ATCAAATCTT
     GTATCGTGCT
601  TCGCAAACGT TTTCAAAACA CGGGATTACT TTACGATTTT
     TAAACTGCTT
651  CAATAAAACA TTTGCTCCAC AAATAAACAC ACAAGAACCT
     GCCCAAGAAG
701  CTGTTCAATG GCTCCAAGAG GTTGATTCTA CATTTCCTGG
     TCTATTTGTA
751  GGGATACAAT CCGCAGGATC AGAATCTGCG CCCGGAGCCT
     GTCCTAAGCG
801  ATTAGCTTCT GGATATAGAA ATGCTTATGA CGCAGGGTTT
     GGTTGTGAAG
851  CTCATGCTGG AGAAGGCATA GAGACCCGGA CTATTTTTTC
     GTCAGCTAAG
901  GTAAATCCAG AGGGATTGAT CGAGATAACC CGAGTGACTT
     TCTCGTCTCT
951  TAAACGAAAA CAGCCATCTA GTTTACCCAT AAGAGTTACT
     TGCCAGTTAG
1001 GATAA

The PSORT algorithm predicts cytoplasm (0.1628).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 113A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 113B) and for FACS analysis.

These experiments show that cp6439 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 114

The following C. pneumoniae protein (PID 4376440) was expressed <SEQ ID 227; cp6440>:

1   LQSARRHLNT IFLLDFGSQY TYVLAKQVRK LFVYCEVLPQ
    NISVQCLKER
51  APLGIILSGG PHSVYENKAP HLDPEIYKLG IPILAICYGM
    QLMARDFGGT
101 VSPGVGEFGY TPIHLYPCEL FKHIVDCESL DTEIRMSHRD
    HVTTIPEGFN
151 VIASTSQCSI SGIENTKQRL YGLQFHPEVS DSTPTGNKIL
    ETFVQEICSA
201 PTLWNPLYIQ QDLVSKIQDT VIEVFDEVAQ SLDVQWLAQG
    TIYSDVIESS
251 RSGHASEVIK SHHNVGGLPK NLKLKLVEPL RYLFKDEVRI
    LGEALGLSSY
301 LLDRHPFPGP GLTIRVIGEI LPEYLAILRR ADLIFIEELR
    KAKLYDKISQ
351 AFALFLPIKS VSVKGDCRSY GYTIALRAVE STDFMTGRWA
    YLPCDVLSSC
401 SRRIINEIPE VSRVVYDISD KPPATIEWE*

The cp6440 nucleotide sequence <SEQ ID 228> is:

1    TTGCAGAGTG CAAGGAGACA TTTGAACACC ATATTTATTC
     TAGATTTTGG
51   ATCTCAATAT ACTTATGTAT TAGCAAAGCA AGTGCGGAAG
     TTATTTGTAT
101  ATTGCGAAGT TCTTCCCTGG AATATCTCTG TGCAATGTTT
     AAAAGAAAGA
151  GCGCCTTTGG GGATCATTCT CTCAGGAGGT CCTCACTCTG
     TCTATGAAAA
201  CAAGGCTCCA CATTTAGATC CTGAAATCTA TAAACTTGGC
     ATTCCAATTC
251  TAGCTATTTG CTATGGCATG CAGCTTATGG CTAGAGATTT
     TGGAGGGACT
301  GTAAGCCCTG GTGTAGGAGA ATTTGGATAT ACGCCCATCC
     ATCTGTATCC
351  TTGTGAGCTC TTCAAACACA TCGTCGACTG CGAATCTCTA
     GACACAGAGA
401  TTCGGATGAG CCATCGGGAT CATGTTAGCA CAATTCCTGA
     AGGATTTAAT
451  GTAATCGCAT CCACCTCACA ATGCTCGATC TCAGGAATAG
     AAAATACCAA
501  ACAACGGTTG TACGGGCTGC AATTTCATCC CGAGGTTTCT
     GACTCCACTC
551  CAACGGGAAA TAAGATTCTA GAAACTTTTG TTCAAGAGAT
     CTGTTCTGCT
601  CCCACACTAT GGAATCCCTT GTATATTCAG CAAGACCTTG
     TAAGTAAAAT
651  TCAAGATACC GTTATTGAAG TATTTGATGA AGTCGCTCAG
     TCATTAGACG
701  TACAATGGTT AGCTCAAGGA ACCATCTACT CAGATGTTAT
     TGAGTCCTCA
751  CGCTCTGGAC ATGCCTCCGA AGTAATAAAA TCACATCATA
     ATGTAGGGGG
801  GCTTCCAAAA AATCTTAAGC TGAAGTTAGT CGAGCCCTTA
     CGTTATTTAT
851  TTAAAGATGA AGTTCGAATT TTAGGAGAAG CCCTAGGACT
     TTCTAGCTAT
901  CTCTTGGACA GGCATCCTTT TCCTGGACCT GGCTTGACAA
     TTCGTGTGAT
951  TGGAGAGATC CTTCCTGAAT ATCTAGCCAT TTTACGACGG
     GCGGACCTCA
1001 TCTTTATAGA AGAGCTTAGG AAAGCAAAAC TCTACGATAA
     AATAAGCCAA
1051 GCCTTTGCTC TATTTCTTCC TATAAAATCA GTATCTGTAA
     AAGGAGATTG
1101 TAGAAGCTAT GGTTATACCA TAGCATTACG TGCTGTAGAA
     TCTACAGATT
1151 TCATGACAGG ACGATGGGCC TACCTTCCAT GCGATGTTCT
     CAGTTCTTGC
1201 TCATCGCGAA TTATTAATGA AATACCCGAG GTAAGCCGAG
     TGGTCTATGA
1251 TATTTCTGAC AAGCCACCAG CAACTATAGA ATGGGAATAG

The PSORT algorithm predicts cytoplasm (0.0481).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 114A) and also as a his-tagged product. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 114B) and for FACS analysis.

These experiments show that cp6440 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 115

The following C. pneumoniae protein (PID 4376475) was expressed <SEQ ID 229; cp6475>:

1   MNTYTFSPTL QKSFSLFLLE KLDSYFFFGG TRTQILVITP
    TNIRLAAKKR
51  GCKVSTIEKI IKILSFILLP LVIIAFILRY FLHKKFDKQF
    LCIPKVISNE
101 DEALLGSRPQ AVEKAVREIS PAFFSIPRKY QLIRIDTPKD
    DAPSILFPIG
151 IEIILKDLCI DTLKQSNLFL KREMDFLGHP EEKALFDSIC
    SIEKDQEWMS
201 LESKKLLITH FLKYLFVSGI EQLNPGFNPE NGRGYFSEIS
    TAKIHFHQHG
251 RYGPIRSSGP IMKEI*

The cp6475 nucleotide sequence <SEQ ID 230> is:

1   ATGAATACCT ATACCTTCTC TCCTACACTT CAGAAAAGCT
    TCAGCCTATT
51  TCTTTTAGAA AAATTAGACT CTTACTTTTT CTTTGGAGGG
    ACTCGTACAC
101 AAATCTTAGT CATCACACCA ACCAATATTA GATTAGCAGC
    TAAAAAAAGA
151 GGGTGTAAGG TTTCTACTAT AGAAAAGATA ATCAAGATCC
    TCTCTTTTAT
201 CCTGCTGCCC CTAGTTATCA TTGCCTTTAT ACTTCGCTAT
    TTCTTACATA
251 AGAAATTCGA TAAACAGTTC TTGTGTATCC CAAAAGTCAT
    TTCTAACGAA
301 GACGAAGCTC TTCTTGGATC TAGACCACAA GCAGTTGAAA
    AAGCAGTTCG
351 AGAAATATCT CCAGCCTTCT TCTCTATACC AAGAAAATAC
    CAACTTATTA
401 GAATCGACAC TCCTAAAGAT GACGCTCCCT CAATCCTTTT
    CCCTATAGGC
451 ATAGAGATCA TTCTCAAAGA TTTATGTATT GATACACTCA
    AGCAATCTAA
501 TCTTTTCCTT AAAAGAGAAA TGGATTTCTT AGGTCATCCA
    GAAGAAAAAG
551 CATTATTCGA CTCGATATGT TCTATAGAAA AAGATCAAGA
    ATGGATGAGC
601 TTGGAAAGTA AAAAACTTTT AATCACGCAC TTCCTAAAGT
    ATCTCTTTGT
651 CTCTGGAATC GAACAACTAA ATCCAGGCTT TAACCCAGAG
    AATGGGCGTG
701 GGTATTTTTC AGAAATAAGT ACAGCAAAGA TCCATTTTCA
    TCAGCACGGT
751 CGATATGGGC CAATCCGTTC TTCGGGACCC ATCATGAAGG
    AAATATAA

The PSORT algorithm predicts inner membrane (0.5373).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 115A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 115B) and for FACS analysis.

These experiments show that cp6475 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 116

The following C. pneumoniae protein (PID 4376482) was expressed <SEQ ID 231; cp6482>:

1   MLVELEALKR EFAHLKDQKP TSDQEITSLY QCLDHLEFVL
    LGLGQDKFLK
51  ATEDEDVLFE SQKAIDAWNA LLTKARDVLG LGDIGAIYQT
    IEFLGAYLSK
101 VNRRAFCIAS EIHFLKTAIR DLNAYYLLDF RWPLCKIEEF
    VDWGNDCVEI
151 AKRKLCTFEK ETKELNESLL REEHAMEKCS IQDLQRKLSD
    IIIELHDVSL
201 FCFSKTPSQE EYQKDCLYQS RLRYLLLLYE YTLLCKTSTD
    FQEQARAKEE
251 FIREKFSLLE LEKGIKQTKE LEFAIAKSKL ERGCLVMRKY
    EAAAKHSLDS
301 MFEEETVKSP RKDTE*

The cp6482 nucleotide sequence <SEQ ID 232> is:

1   ATGCTAGTAG AGTTAGAGGC TCTTAAAAGA GAGTTTGCGC
    ATTTAAAAGA
51  CCAGAAGCCG ACAAGTGACC AAGAGATCAC TTCACTTTAT
    CAATGTTTGG
101 ATCATCTTGA ATTCGTTTTA CTCGGGCTGG GCCAGGACAA
    ATTTTTAAAG
151 GCTACGGAAG ATGAAGATGT GCTTTTTGAG TCTCAAAAAG
    CAATCGATGC
201 GTGGAATGCT TTATTGACAA AAGCCAGAGA TGTTTTAGGT
    CTTGGGGACA
251 TAGGTCCTAT CTATCAGACT ATAGAATTCT TGGGTGCCTA
    TTTATCAAAA
301 GTGAATCGGA GGGCTTTTTG TATTGCTTCG GAGATACATT
    TTCTAAAAAC
351 AGCAATCCGA GATTTGAATG CATATTACCT GTTAGATTTT
    AGATGGCCTC
401 TTTCCAAGAT AGAAGAGTTT GTGGATTGGG GGAATGATTG
    TGTTGAAATA
451 GCAAAGAGGA AGCTATGCAC TTTTGAAAAA GAAACCAAGG
    AGCTCAATGA
501 GAGCCTTCTT AGAGAGGAGC ATGCGATGGA GAAATGCTCG
    ATTCAAGATC
551 TGCAAAGGAA ACTTAGCGAC ATTATTATTG AATTGCATGA
    TGTTTCTCTT
601 TTTTGTTTTT CTAAGACTCC CAGTCAAGAG GAGTATCAAA
    AGGATTGTTT
651 GTATCAATCA CGATTGAGGT ACTTATTGTT GCTGTATGAG
    TATACATTGT
701 TATGTAAGAC ATCCACAGAT TTTCAAGAGC AGGCTAGGGC
    TAAAGAGGAG
751 TTCATTAGGG AGAAATTCAG CCTTCTAGAG CTCGAAAAGG
    GAATAAAACA
801 ACCTAAAGAG CTTGAGTTTG CAGTTGCTAA AAGTAAGTTA
    GAACGGGGCT
851 GTTTAGTTAT GAGGAAGTAT GAAGCTGCCG CTAAACATAG
    TTTAGATTCT
901 ATGTTCGAAG AAGAAACTGT GAAGTCGCCG CGGAAAGACA
    CAGAATAA

The PSORT algorithm predicts cytoplasm (0.4607).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 116A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 116B) and for FACS analysis.

These experiments show that cp6482 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 117

The following C. pneumoniae protein (PID 4376486) was expressed <SEQ ID 233; cp6486>:

1   VVVVALFILG IFFLSGSLAF LVHTSCGVLL GAALPILCIG
    LVLLAVALIV
51  FLCHKHKTRQ DLDYYDQDLD SLVIHKKEIP NDISELRVTF
    EKLQNLFQFH
101 TKDFSDLSQE LQGKFINCME KWLTLEDEVT KFLIVRDRFL
    ETRRNFTTFG
151 EQVKGIQSNI FDLHEEKSSL YLELYRLRKD LQVLLNFFLL
    PPGILKVDYD
201 EIEAIKGLFI RLTSRLDKLD VKAQERKKFI NEMSREFKEV
    EKAFDIVDRA
251 TKKLMDRAKK ESPARLFMGR TESLLEMKKN EEALKNQGLD
    PENLSHPELF
301 SPYQQLLILN YLNSEIVLHH YEFLISGTVT SGLTLEECEN
    RMRAASTGLN
351 ALLVRKLQFR GAIKSAYFEK LTEIEKELRS LQDVIKSLEL
    ELIHKIKDIV
401 TEET*

The cp6486 nucleotide sequence <SEQ ID 234> is:

1    GTGGTGGTTG TCGCTTTATT TATCCTTGGG ATTTTCTTTT
     TATCTGGTTC
51   TCTTCCATTC CTTGTTCATA CGTCTTGCGG AGTTCTTTTA
     GGAGCGGCGC
101  TTCCCATACT TTGCATAGGT CTTGTTTTAT TGGCTGTAGC
     TCTTATTGTT
151  TTCTTATGTC ACAAACACAA GACTCGTCAA GATTTAGATT
     ATTATGATCA
201  AGATTTAGAT TCTTTGGTGA TTCATAAGAA AGAGATCCCC
     AATGACATCT
251  CTGAGTTGCG GGTAACATTT GAAAAGTTGC AAAATCTGTT
     TCAGTTCCAT
301  ACGAAAGATT TCTCTGATCT AAGCCAAGAG CTTCAGGGTA
     AATTTATCAA
351  TTGCATGGAG AAATGGCTAA CTTTAGAAGA CGAAGTGACT
     AAATTTCTTA
401  TTGTTCGAGA TAGATTTTTA GAAACCAGAA GAAATTTTAC
     CACTTTTGGA
451  GAACAGGTTA AAGGGATCCA AAGCAATATT TTTGATTTGC
     ATGAGGAAAA
501  GTCTTCATTA TATTTAGAAT TGTATAGGCT TAGGAAAGAC
     CTCCAAGTTC
551  TATTAAATTT TTTTCTGCTC CCCCCAGGTA TACTCAAGGT
     AGATTATGAT
601  GAAATTGAGG CTATCAAAGG TCTGTTTATA AGATTAACCT
     CTAGATTAGA
651  TAAGCTTGAT GTGAAAGCTC AGGAACGTAA GAAGTTCATT
     AATGAAATGA
701  GTAGGGAATT TAAAGAAGTA GAGAAAGCTT TTGATATTGT
     CGATAGGGCA
751  ACAAAAAAGC TTATGGATAG AGCCAAGAAA GAAAGTCCGG
     CACGTCTTTT
801  CATGGGTAGA ACTGAGTCTC TCTTAGAAAT GAAAAAAAAT
     GAAGAAGCCC
851  TTAAAAATCA GGGGCTAGAT CCTGAAAATC TTTCCCATCC
     TGAACTTTTT
901  AGTCCGTATC AACAGCTTTT AATTTTGAAT TATTTAAATA
     GCGAAATAGT
951  TCTGCATCAT TATGAGTTCC TTATTTCTGG AACAGTAACT
     TCTGGCCTAA
1001 CTCTTGAAGA ATGTGAAAAT CGAATGAGGG CGGCTTCTAC
     TGGGTTGAAC
1051 GCCCTTCTGG TGCGTAAGCT CCAGTTCAGA GGTGCTATAA
     AATCTGCGTA
1101 TTTTGAAAAA CTCACAGAGA TTGAAAAAGA GTTACGATCA
     CTTCAAGACG
1151 TAATAAAGTC ATTGGAACTA GAACTGATCC ATAAGATAAA
     AGATATAGTG
1201 ACAGAAGAAA CTTAG

The PSORT algorithm predicts inner membrane (0.7474).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 117A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 117B) and for FACS analysis.

These experiments show that cp6486 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 118

The following C. pneumoniae protein (PID 4376526) was expressed <SEQ ID 235; cp6526>:

1   MSPFKKIVNR LLCYISFQKE SRTLPIIIRE PRMTTKSLGS
    FNSVISKNKI
51  HFISLGCSRN LVDSEVMLGI LLKAGYESTN EIEDADYLIL
    NTCAFLKSAR
101 DEAKDYLDHL IDVKKENAKI IVTGCMTSNH KDELKPWMSH
    IHYLLGSGDV
151 ENILSAIESR ESGEKISAKS YIEMGEVPRQ LSTPKHYAYL
    KVAEGCRKRC
201 AFCIIPSIKG KLRSKPLDQI LKEFRILVNK SVKEIILIAQ
    DLGDYGDDLS
251 TDRSSQLESL LHELLKEPGD YWLRMLYLYP DEVSDGIIDL
    MQSNPKLLPY
301 VDIPLQHIND RILKQMRRTT SREQILGFLE KLRAKVPQVY
    IRSSVIVGFP
351 GETQEEFQEL ADFIGEGWID NLGIFLYSQE ANTPAAELPD
    QIPEKVKESR
401 LKILSQIQKR NVDKHNQKLI GEKIEAVIDN YHPETNLLLT
    ARFYGQAPEV
451 CPCIIVNEAK LVSHFGERCF IEITGTAGYD LVGRVVKKSQ
    NQALLKTSKA
501 *

The cp6526 nucleotide sequence <SEQ ID 236> is:

1    ATGAGTCCTT TTAAGAAAAT AGTAAATCGC TTACTATGCT
     ATATTTCTTT
51   TCAAAAAGAA TCAAGAACTC TCCCAATCAT TATTAGAGAA
     CCTAGGATGA
101  CAACAAAAAG TTTAGGATCT TTCAATTCAG TTATTTCCAA
     AAATAAAATT
151  CATTTTATTA GTTTGGGATG CTCTCGGAAC CTTGTAGATA
     GCGAAGTCAT
201  GCTAGGCATT CTTCTTAAGG CAGGTTACGA GTCTACTAAT
     GAAATTGAAG
251  ATGCTGACTA TTTAATTTTA AATACCTGTG CGTTTTTAAA
     AAGTGCTAGA
301  GATGAAGCTA AAGATTATCT AGACCATCTA ATTGATGTAA
     AAAAAGAGAA
351  CGCTAAAATT ATTGTAACTG GATGCATGAC TTCCAACCAC
     AAAGATGAGC
401  TTAAACCCTG GATGTCACAC ATCCATTACC TACTAGGTTC
     TGGGGATGTT
451  GAGAATATTC TTTCTGCTAT TGAGTCTCGT GAATCTGGAG
     AAAAAATCTC
501  TGCAAAGAGT TACATTGAGA TGGGAGAAGT TCCAAGACAG
     CTTTCCACAC
551  CAAAACACTA TGCCTATTTA AAAGTTGCTG AGGGCTGTAG
     AAAACGTTGT
601  GCTTTTTGTA TTATTCCTTC CATTAAAGGA AAGCTCCGCA
     GCAAACCTCT
651  GGATCAAATT CTTAAAGAAT TCCGCATCCT TGTAAACAAG
     AGTGTGAAAG
701  AGATTATATT GATAGCTCAA GACCTAGGAG ATTATGGAAA
     GGATCTCTCT
751  ACAGACCGCA GTTCGCAGCT AGAATCACTA TTACATGAGT
     TACTGAAAGA
801  GCCTGGTGAT TATTGGCTGC GGATGTTGTA TTTATATCCT
     GATGAAGTGA
851  GTGATGGCAT TATAGATCTT ATGCAATCTA ATCCCAAACT
     TCTTCCCTAT
901  GTAGATATTC CCTTACAGCA CATTAACGAC CGTATTTTAA
     AGCAAATGCG
951  AAGAACGACT TCTAGGGAGC AAATCCTAGG ATTCCTAGAA
     AAATTACGTG
1001 CCAAGGTTCC TCAGGTCTAT ATCCGTTCTT CTGTTATTGT
     GGGTTTCCCC
1051 GGTGAAACTC AGGAAGAATT CCAGGAGTTA GCTGATTTTA
     TTGGTGAGGG
1101 TTGGATTGAT AATCTCGGAA TTTTCTTGTA CTCTCAAGAA
     GCGAATACCC
1151 CGGCAGCAGA ACTCCCTGAC CAGATACCAG AAAAAGTTAA
     AGAATCGAGG
1201 TTGAAAATTC TATCTCAAAT TCAGAAACGC AATGTGGATA
     AACATAATCA
1251 GAAGCTCATT GGGGAAAAAA TAGAAGCAGT TATTGATAAC
     TATCATCCTG
1301 AAACGAATCT TTTACTCACT GCAAGGTTCT ATGGACAAGC
     TCCTGAAGTG
1351 GACCCTTGTA TTATTGTAAA TGAGGCGAAG CTTGTTTCTC
     ATTTTGGAGA
1401 AAGATGCTTT ATAGAAATCA CAGGGACTGC TGGTTACGAC
     CTTGTAGGGC
1451 GTGTTGTAAA AAAATCTCAG AACCAAGCTT TGCTAAAAAC
     TAGCAAAGCT
1501 TAG

The PSORT algorithm predicts cytoplasm (0.1296).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 118A) and also as a his-tagged product. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 118B) and for FACS analysis.

These experiments show that cp6526 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 119

The following C. pneumoniae protein (PID 4376528) was expressed <SEQ ID 237; cp6528>:

1   MKNNINNNEC YFKLDSTVDG DLLAANLKTF DTQAQGISST
    ETFSVQGNAT
51  FKDQVSATGL TSGTTYNLNA QNFTSSQISI DFKNNRLSNC
    ALPKFDCDPV
101 PANYVRSPEY FFCSKPLIGD FDFNSGESYL PLTGSEYTLY
    QSRNVNSIFR
151 FIGWKQSTRE LTVGGNTAIQ FLAAGTYIVS FTVGKRWGWN
    NGWGGAIYIN
201 NGLGQVQCES TIYSGGGYAT IGTLGTSIYR ASVDVAPNPN
    DPNASDRYRA
251 GIFYLSNGGS SAGIGNYSFS LLYYPDDRG*

The cp6528 nucleotide sequence <SEQ ID 238> is:

1   ATGAAAAACA ATATTAATAA TAATGAGTGC TATTTTAAAT
    TAGACTCAAC
51  TGTAGATGGT GATTTGTTAG CAGCCAATCT CAAGACCTTT
    GATACACAGG
101 CCCAAGGAAT CTCATCGACT GAAACATTTT CTGTTCAGGG
    GAATGCAACA
151 TTTAAAGATC AAGTTTCAGC AACTGGATTA ACTTCAGGAA
    CTACTTATAA
201 TTTAAATGCA CAAAACTTTA CTTCCTCCCA AATCTCTATA
    GATTTTAAAA
251 ATAATCGTCT GAGTAATTGT GCATTGCCAA AAGAAGACTG
    CGATCCGGTG
301 CCAGCGAATT ATGTTCGTTC TCCCGAATAT TTTTTCTGTT
    CCAAGCCTCT
351 GATCGGAGAT TTTGATTTTA ACTCAGGGGA ATCTTATTTG
    CCTCTGACTG
401 GTTCGGAATA TACTCTATAT CAGTCACGTA ATGTAAATAG
    TATATTTCGT
451 TTTATAGGAT GGAAGCAAAG TACACGAGAA TTAACTGTAG
    GGGGAAATAC
501 TGCGATACAA TTTCTTGCAG CAGGAACCTA TATCGTTTCA
    TTTACTGTTG
551 GTAAACGGTG GGGATGGAAT AATGGTTGGG GAGGAGCCAT
    TTATATCAAT
601 AATGGTTTAG GACAAGTCCA ATGTGAAAGC ACGATTTATA
    GTGGTGGAGG
651 GTATGCAACA ATAGGTACAC TGGGGACCTC AATATATAGA
    GCCTCTGTAG
701 ATGTAGCTCC TAATCCTAAT GATCCGAATG CTTCGGATCG
    CTATAGAGCG
751 GGTATTTTCT ATCTCAGTAA CGGTGGTTCT AGTGCAGGTA
    TAGGGAATTA
801 CTCCTTTTCT CTTCTCTATT ATCCGGACGA TAGAGGGTAG

The PSORT algorithm predicts cytoplasm (0.1668).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 119A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 119B) and for FACS analysis.

These experiments show that cp6528 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 120

The following C. pneumoniae protein (PID 4376627) was expressed <SEQ ID 239; cp6627>:

1   MKCSPLTLVP HIFLKNDCEC HRSCSLKIRT IARLILGLVL
    ALVSALSFVF
51  LAAPISYAIG GTLALAAIVI LIITLVVALL AKSKVLPIPN
    ELQKIIYNRY
101 PKEVFYFVKT HSLTVNELKI FINCWKSGTD LPPNLHKKAE
    AFGIDILKSI
151 DLTLFPEFEE ILLQNCPLYW LSHFIDKTES VAGEIGLNKT
    QKVYGLLGPL
201 AFHKGYTTIF HSYTRPLLTL ISESQYKFLY SKASKNQWDS
    PSVKKTCEEI
251 FKELPHNMIF RKDVQGISQF LELFFSHGIT WEQAQMIQLI
    NPDNWKMLCQ
301 FDKAGGHCSM ATFGGFLNTE TNMFDPVSSN YEPTVNFMTW
    KELKVLLEKV
351 KESPMHPASA LVQKICVNTT HHQNLLKRWQ FVRNTSSQWT
    SSLPQYAFHA
401 QTYKLEKKIE SSLPIRSSL*

The cp6627 nucleotide sequence <SEQ ID 240> is:

1    ATGAAGTGTA GTCCTTTAAC ACTAGTTCCC CATATATTTT
     TAAAAAATGA
51   CTGCGAATGT CATAGATCTT GTTCTTTAAA AATTAGGACA
     ATTGCCCGAC
101  TCATTCTTGG GCTTGTTCTA GCTCTTGTTA GCGCACTTTC
     TTTTGTTTTC
151  CTTGCTGCGC CGATTAGCTA TGCTATTGGA GGAACTTTAG
     CTTTAGCCGC
201  TATCCTAATC TTGATTATAA CGCTAGTCGT ACCACTCCTA
     GCTAAATCAA
251  AGGTTCTGCC CATCCCCAAC GAACTTCAGA AGATTATTTA
     CAATCGCTAT
301  CCTAAAGAAG TCTTTTATTT CGTGAAAACA CACTCCCTGA
     CTGTTAACGA
351  ATTAAAAATA TTTATTAATT GCTGGAAAAG CGGTACAGAC
     CTGCCTCCGA
401  ATTTACATAA AAAAGCAGAG GCTTTCGGGA TCCATATTCT
     AAAATCTATA
451  GATTTAACCC TGTTTCCAGA GTTCGAAGAG ATTCTTCTTC
     AAAACTGCCC
501  GTTATACTGG CTCTCCCATT TTATAGACAA AACTGAATCT
     GTTGCTGGGG
551  AAATCGGATT AAATAAAACA CAAAAAGTTT ATGGTTTACT
     TGGGCCCTTA
601  GCGTTTCATA AAGGATATAC AACTATTTTC CACTCTTATA
     CACGCCCTCT
651  ACTAACATTA ATCTCAGAAT CACAGTATAA GTTCCTATAT
     AGTAAAGCGT
701  CTAAGAATCA ATGGGATTCT CCTTCTGTGA AAAAAACCTG
     CGAAGAAATA
751  TTCAAGGAAC TCCCCCACAA TATGATTTTC CGGAAGGATG
     TTCAAGGAAT
801  CTCACAATTC TTATTTCTTT TCTTTTCTCA TGGTATCACT
     TGGGAACAGG
851  CTCAGATGAT TCAACTTATA AATCCTGATA ATTGGAAAAT
     GTTGTGTCAG
901  TTTGATAAAG CAGGAGGCCA CTGTTCCATG GCAACATTTG
     GAGGCTTTTT
951  GAATACTGAA ACAAATATGT TCGATCCAGT ATCCTCTAAC
     TATGAACCTA
1001 CAGTGAACTT CATGACGTGG AAAGAATTGA AGGTTTTACT
     AGAGAAAGTA
1051 AAAGAAAGTC CTATGCACCC AGCGAGTGCT CTTGTTCAGA
     AGATATGCGT
1101 AAATACAACG CACCATCAAA ATCTGTTAAA ACGATGGCAA
     TTTGTTCGTA
1151 ATACGAGTTC ACAATGGACA TCAAGCTTAC CTCAGTATGC
     TTTCCACGCC
1201 CAAACCTACA AACTAGAGAA AAAAATAGAA AGCAGTCTCC
     CTATACGATC
1251 TTCCCTATAA

The PSORT algorithm predicts inner membrane (0.7198).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 120A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 120B) and for FACS analysis.

These experiments show that cp6627 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 121

The following C. pneumoniae protein (PID 4376629) was expressed <SEQ ID 241; cp6629>:

1   MSNITSPVIQ NNRSCNYYFE LKNSTTIHIV ISAILLCGAL
    IAFLCVAAPV
51  SYILSGALLG LGLLIALIGV ILGIKKITPM ISSKEQVFPQ
    ELVNRIRAHY
101 PKFVSDFVSE AKPNLKDLIS FIDLLNQLHS EVGSSTNYNV
    SEELQQKIDT
151 FEGIARLKNE VRTASLKRLE SAASSRPLFP SLPKILQKVF
    PFFWLGEFIS
201 AGSKVVELHR VKKIGGSLEE DLSDYIKPEM LPTYWLIPLD
    FRPTNSSILN
251 LHTLVLARVL TRDVFQHLKY AALNGEWNLN HSDLNTMKQQ
    LFAKYHAAYQ
301 SYKHLSQPSL QEDEFYNLLL CIFKHRYSWK QMSLIKTVPA
    DLWENLCCLT
351 LDHTGRPQDM EFASLIGTLY TQGLIHKESE AFLESLTLLS
    LDQFKTIRRQ
401 STNIAMFLEN IATHNSTFRS LPPITVHPLK RSVFSQPEED
    ESSLLIG*

The cp6629 nucleotide sequence <SEQ ID 242> is:

1    ATGAGTAATA TAACCTCGCC AGTTATTCAA AATAATCGCT
     CTTGTAATTA
51   TTATTTTGAA TTAAAGAATT CAACCACTAT TCATATTGTT
     ATCAGTGCCA
101  TCTTACTCTG CGGAGCTTTG ATAGCTTTCT TGTGTGTAGC
     AGCTCCTGTT
151  TCCTATATTC TAAGTGGCGC ATTGTTAGGA TTAGGATTAT
     TAATAGCCTT
201  GATTGGTGTG ATTTTAGGAA TAAAAAAAAT CACGCCTATG
     ATTTCATCAA
251  AAGAACAAGT ATTCCCCCAA GAACTCGTAA ATAGAATCAG
     GGCGCACTAT
301  CCTAAATTTG TCTCTGATTT TGTTTCAGAA GCTAAACCAA
     ATCTTAAAGA
351  TCTCATAAGT TTTATTGATC TTCTAAATCA ATTGCACTCT
     GAAGTTGGAT
401  CATCTACAAA TTACAACGTA TCTGAAGAAC TACAACAGAA
     AATAGATACG
451  TTCGAGGGTA TCGCACGCTT AAAAAATGAA GTCCGTACTG
     CTTCTCTTAA
501  AAGACTTGAA AGCGCTGCTT CTTCCCGTCC CCTCTTCCCC
     TCTTTACCAA
551  AAATCTTACA AAAGGTATTT CCATTTTTCT GGTTAGGAGA
     GTTTATTTCT
601  GCAGGCAGCA AGGTTGTAGA GCTCCATCGA GTTAAGAAAA
     TTGGAGGCAG
651  CCTCGAAGAA GACCTTAGTG ATTATATAAA ACCAGAGATG
     CTTCCTACCT
701  ATTGGTTGAT TCCTTTAGAT TTTAGACCAA CAAATTCCTC
     TATTCTAAAT
751  CTACACACAT TAGTTTTAGC TAGAGTCTTA ACTCGTGATG
     TTTTTCAACA
801  TCTTAAGTAT GCAGCATTAA AGTTCGAGTG GAACCTGAAT
     CATAGTGATC
851  TAAATACTAT GAAACAGCAG CTCTTTGCTA AATATCATGC
     GGCGTATCAA
901  TCCTATAAAC ATCTATCTCA ACCCTCTCTT CAAGAGGATG
     AATTCTATAA
951  CCTGCTCTTG TGTATTTTTA AGCATAGGTA CTCGTGGAAG
     CAGATGTCCT
1001 TAATAAAAAC AGTCCCGGCT GATTTATGGG AAAACCTCTG
     TTGCTTGACT
1051 TTAGACCATA CAGGACGACC CCAAGACATG GAATTTGCCT
     CTCTAATTGG
1101 TACTCTCTAC ACACAAGGCC TAATTCATAA AGAAAGCGAA
     GCATTTCTTT
1151 CTTCATTGAC ACTCCTTAGT TTAGATCAGT TTAAAACGAT
     CCGTCGTCAG
1201 TCAACCAATA TAGCGATGTT CCTTGAGAAT TTAGCAACTC
     ATAATTCCAC
1251 CTTTAGAAGC TTACCACCTA TAACAGTCCA TCCACTCAAG
     AGAAGCGTCT
1301 TCTCCCAACC TGAAGAAGAC GAGTCCTCCC TGCTGATAGG
     TTAG

The PSORT algorithm predicts inner membrane (0.5776).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 121A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 121B) and for FACS analysis.

These experiments show that cp6629 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 122

The following C. pneumoniae protein (PID 4376732) was expressed <SEQ ID 243; cp6732>:

1   MEMMSPFQQP EQCHFDVVGS FLRPESLTRA RSDFEEGRIV
    YEQMRVVEDA
51  AIRNLIKKQT EAGLIFFTDG EFRRYSWDFD FMWGFHGVDR
    RRDSNDPEIG
101 VYLKDKISVS KHPFIEHFEF VKTFEKGNAK AKQTIPSPSQ
    FFHEMIFAPN
151 LKNTRKFYPT NQELIDDIVF YYRQVIQDLY AAGCRNLQLD
    DCAWCRLLDI
201 RAPSWYGVDS HDRLQEILEQ FLWIHNLVMK DRPEDLFVSL
    HVCRGDYQAE
251 FFSRRAYDSI EEPLFAKTDV DSYHYYWALD DKYSGGAEPL
    AYVSGEKHVC
301 LGLISSNHSC IEDRDAVVSR IYEAASYIPL ERLSLSPQCG
    FASCEGDHRM
351 TEEFQWKKIA FVKEIAKEIW G*

The cp6732 nucleotide sequence <SEQ ID 244> is:

1    ATGGAAATGA TGAGCCCATT CCAACAACCT GAGCAATGTC
     ATTTTGATGT
51   TGTGGGAAGT TTCTTACGTC CTGAAAGTCT TACACGAGCA
     CGCTCTGATT
101  TTGAAGAAGG AAGAATTGTC TATGAGCAGA TGCGAGTTGT
     CGAAGATGCT
151  GCTATTCGTA ATCTCATAAA AAAGCAAACA GAAGCAGGTC
     TTATCTTTTT
201  TACTGATGGG GAATTCCGTA GGTATAGTTG GGATTTCGAC
     TTTATGTGGG
251  GATTCCATGG CGTGGATCGT CGCAGGGACT CTAATGACCC
     TGAAATTGGA
301  GTGTATCTTA AAGATAAAAT CTCCGTATCA AAACATCCGT
     TTATAGAACA
351  TTTCGAGTTT GTCAAAACTT TTGAGAAGGG AAATGCAAAA
     GCAAAACAAA
401  CGATTCCTTC TCCATCACAA TTTTTCCATG AGATGATTTT
     TGCTCCTAAT
451  CTGAAAAATA CTCGGAAGTT TTATCCTACG AATCAAGAGC
     TAATTGATGA
501  TATTGTCTTT TATTATCGCC AAGTCATCCA AGATCTTTAT
     GCTGCAGGTT
551  GTCGTAATTT GCAGTTGGAC GATTGTGCTT GGTGTCGCCT
     CTTGGATATA
601  CGAGCGCCTT CTTGGTATGG TGTTGATTCT CATGACAGGT
     TGCAGGAAAT
651  TTTAGAACAG TTTTTATGGA TCCATAATTT AGTGATGAAG
     GATAGACCCG
701  AGGATCTTTT TGTAAGTCTG CATGTCTGTC GTGGTGATTA
     TCAGGCCGAG
751  TTTTTCTCTA GACGAGCTTA TGATTCTATA GAGGAGCCTT
     TATTTGCTAA
801  GACCGATGTG GATAGTTATC ACTATTATTG GGCTCTTGAT
     GATAAGTATT
851  CAGGAGGTGC TGAGCCTTTA GCTTACGTCT CTGGAGAGAA
     ACACGTCTGC
901  TTGGGATTGA TCTCCAGCAA CCATTCTTGT ATTGAAGATC
     GAGATGCTGT
951  GGTTTCTCGT ATTTATGAAG CTGCGAGCTA CATTCCCTTA
     GAGAGACTTT
1001 CTTTGAGCCC GCAATCTGGG TTTGCTTCTT GTGAGGGAGA
     CCATAGAATG
1051 ACTGAAGAAG AACAGTGGAA GAAGATCGCC TTTGTGAAAG
     AGATTGCTAA
1101 AGAGATCTGG GGATAA

The PSORT algorithm predicts cytoplasm (0.2196).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 122A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 122B) and for FACS analysis.

These experiments show that cp6732 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 123

The following C. pneumoniae protein (PID 4376738) was expressed <SEQ ID 245; cp6738>:

1   VWLRFLLLVS YDEKEKDVVV VCNHSEPNIL GLPPEAVSQL
    IEELSDEGYS
51  YLNVVRCDLS GETTVQQRLL LDANEGRSMT VVISELPEGH
    PDIRNLQLAS
101 ERIFVSREKE AADAYASGCK VVAFDDEHLP WVSSHIAYAE
    EIREKQEQTM
151 QGSLTEEQLG ALLCNTVSTE KNLAFALDAV IKQSVWRFRN
    PDLFAYEREA
201 LEASVTDALV SYVSNLDMIP YTSSQGIVIE DSSIVRTSQE
    HTLIVNCAAF
251 DKLASQIEFL CPSDVLPISG KDPLISDDED EELNPKVSSA
    ADSKDKT*

The cp6738 nucleotide sequence <SEQ ID 246> is:

1   GTGTGGCTGC GCTTTTTACT TTTAGTGTCC TATGATGAGA
    AGGAGAAAGA
51  CGTAGTTGTC GTTTGTAATC ATTCTGAACC TAATATCCTC
    GGCCTGCCTC
101 CTGAAGCAGT CTCTCAGCTT ATTGAAGAGC TTAGCGATGA
    AGGCTATAGC
151 TATCTGAATG TAGTGCGTTG TGATCTCTCC GGGGAGACTA
    CGGTTCAACA
201 ACGTCTGCTA TTGAATGCCG ATGAAGGGAG ATCTATGACG
    GTGGTGATCT
251 CAGAGCTTCC TGAAGGGCAC CCCGATATTC GGAATTTGCA
    GTTGGCATCC
301 GAAAGAATTT TTGTTTCTCG TGAAAAAGAA GCTGCTGATG
    CCTATGCTTC
351 AGGATGTAAA GTGGTCGCTT TCGATGATGA GCATCTCCCT
    TGGGTCTCCA
401 GTCATATTGC CTACGCGGAG GAGATCAGAG AGAAACAAGA
    ACAAACAATG
451 CAAGGGTCTT TAACTGAAGA GCAGTTAGGA GCACTCCTCT
    GCAACACAGT
501 CTCCACAGAG AAAAATCTAG CCTTTGCTCT AGACGCCGTG
    ATAAAACAGT
551 CTGTGTGGAG ATTCCGCAAT CCGGATCTTT TTGCTTATGA
    GAGAGAAGCT
601 CTAGAGGCTT CATGAACAGA TGCTTTAGTA TCTTACGTTT
    CAAATTTAGA
651 CATGATACCG TACACAAGTT CTCAGGGCAT AGTCATAGAA
    GATAGTAGTA
701 TCGTCCGTAC CTCTCAAGAG CATACACTCA TTGTGAACTG
    TGCAGCATTC
751 GATAAGTTAG CGAGCCAAAT AGAGTTCTTA TGCCCCAGTG
    ACGTGTTGCC
801 CATTTCTGGT AAAGACCCTT TGATTTCTGA TGATGAGGAT
    GAGGAACTGA
851 ATCCTAAAGT TTCATCTGCT GCAGACTCTA AAGATAAAAC
    CTAG

The PSORT algorithm predicts cytoplasm (0.1587).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 123A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 123B) and for FACS analysis.

These experiments show that cp6738 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 124

The following C. pneumoniae protein (PID 4376739) was expressed <SEQ ID 247; cp6739>:

1   MTHCLHGWFS VVRHHFVQAF NFSRPLYSRI THFALGVIKA
    IPIVGHLVMG
51  VDWLISHCFE RGVSHPGFPS DIAPILKVEK IAGRDHISRI
    ENQLKSLRKT
101 IEVEDLDKVH GQYQENPYAD MASSEVLKLD KGVHVSELGK
    AFSRVRNRIT
151 RSYSYAPTPQ LDSIAIVGID LVSPEEQENL VRLANEVIQL
    YPKSKTTLYL
201 LIDFNKEWVG DISSDKEKQL RSLGLHSEVQ CLSVLEPQGA
    EGEDTKHFDL
251 MVGCYGKDSY LREGKILQQA LGTSLGTVPW VNVMHTLPSR
    YRSRLSLPIN
301 IEKDKTELYK EISRTHHQLH TLGMGLGAQD SGLLLDRQRL
    HAPLSQGSHC
351 HSYLADLTHE ELKILLFSAF VDAKNISKKE LREVSLNFAN
    DTDVECECAF
401 YF*

The cp6739 nucleotide sequence <SEQ ID 248> is:

1    ATGACTCATT GCTTACATGG TTGGTTTTCT GTAGTTCGTC
     ATCACTTTGT
51   GCAGGCGTTT AGTTTCTCAC GTCCTTTATA TTCTCGAATT
     ACCCACTTCG
101  CTTTAGGGGT GATTAAGGCC ATCCCCATTG TAGGGCATCT
     TGTTATGGGA
151  GTCGATTGGT TGATCTCTCA TTGCTTCGAG AGGGGAGTCT
     CACACCCTGG
201  GTTCCCTTCA GATATTGCTC CTATACTGAA AGTAGAAAAG
     ATCGCGGGCC
251  GAGATCATAT TTCTAGAATC GAAAATCAGC TAAAGAGCCT
     TAGGAAAACT
301  ATCCAGGTTG AAGATCTAGA TAAAGTCCAC GGGCAATATC
     AAGAGAATCC
351  TTATGCAGAT ATGGCCTCTA GTGAGGTTCT TAAACTCGAT
     AAGGGAGTTC
401  ATGTTAGCGA GCTTGGCAAA GCCTTTTCTA GAGTTCGCAA
     TCGCATCACC
451  AGATCCTATA GTTATGCCCC TACTCCTCAG TTGGACTCTA
     TAGCTATTGT
501  TGGTATAGAT CTCGTCAGTC CTGAAGAACA AGAGAATTTA
     GTACGCTTGG
551  CGAATGAGGT CATTCAACTC TATCCCAAAT CAAAGACAAC
     TCTATATCTT
601  CTTATCGATT TTAATAAGGA GTGGGTAGGG GATATCTCCT
     CTGATAAGGA
651  AAAACAGCTC CGTTCTCTAG GTCTACATTC TGAAGTTCAG
     TGTCTTTCCG
701  TCTTGGAACC TCAGGGTGCC GAGGGCGAAG ATACGAAACA
     CTTTGACCTT
751  ATGGTCGGCT GTTATGGGAA GGATTCTTAC TTAAGGGAGG
     GTAAAATTTT
801  ACAGCAGGCC CTAGGGACTT CGTTAGGTAC TGTTCCCTGG
     GTGAATGTTA
851  TGCACACATT GCCATCTAGG TATAGATCTC GGCTTTCCTT
     ACCTATAAAT
901  ACCGAAAAGG ATAAGACAGA GCTTTATAAA GAGATTTCTC
     GTACACACCA
951  TCAGTTGCAT ACTTTGGGAA TGGGACTTGG AGCCCAGGAT
     TCAGGATTGC
1001 TCTTAGACCG GCAACGACTC CATGCTCCTT TATCTCAAGG
     GTCTCACTGC
1051 CATTCCTATC TTGCAGATCT CACCCATGAA GAGCTGAAAA
     TTTTGTTATT
1101 TTCAGCATTT GTGGATGCTA AGAACATAAG TAAGAAAGAG
     CTTCGTGAGG
1151 TATCTCTAAA TTTTGCTAAC GATACTTCCG TAGAGTGTGG
     CTGCGCTTTT
1201 TACTTTTAG

The PSORT algorithm predicts inner membrane (0.2190).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 124A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 124B) and for FACS analysis.

These experiments show that cp6739 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 125

The following C. pneumoniae protein (PID 4376741) was expressed <SEQ ID 249; cp6741>:

1   MASCLSAWFS IVREHFYRAF DFSLPFCARI TEFVLGVIKG
    IPVVGHIIVG
51  IEWLVSRYLE SFVTKPTFVS DVVSLLKTEK VAGRDHIARV
    VETLKRQRVA
101 VAPEDEDKVH GKIPVHPFGG IQPVEVLTLY PEVQDATLGL
    AFSKIRNRVR
151 QAYLQAPRPK LQKIYIIGND MNPFEVDDFL HLARLCNETQ
    RLYPDATISL
201 YLTASGGRNA MDKKNRKLLS DCELNPKIAC LDFNQGDVVK
    QATCDCWMVY
251 HGENDQGTLN QIQEELEKSG EETPWIHVGQ KPLSQSLWDF
    SPFSSLEMKG
301 DKEKALEYSE LEKEQLYSRL VYVGERSSVL SLGFGDSRSG
    ILMDPKRVHA
351 PLSEGHYCHS YLADIENPGL QKTILAAFLN PKELSSTILQ
    PISLNLILNS
401 KTYLRQHFGF FERMSRSDRN VVVVVCDSWW GTDWKEEPSF
    QHFIMELECR
451 GYSHFNIFAF RSNSMCVEER RILNESSQEK AFTMIFCEDS
    VSQGDIRCLH
501 LASEGMLCGK ECYAVDVYTS GCANFMMEEV LTLERESNLW
    NRKHGLWKRE
551 VRKQKQEAAL DQDESEIYVC NQLTAQQNFA GS*

The cp6741 nucleotide sequence <SEQ ID 250> is:

1    ATGGCTTCTT GTTTATCTGC CTGGTTTTCT ATAGTTCGTG
     AGCACTTTTA
51   TCGAGCCTTT GATTTTTCTT TGCCGTTTTG TGCTCGTATT
     ACGGAATTTG
101  TATTAGGGGT CATCAAGGGG ATCCCTGTTG TGGGTCACAT
     TATTGTTGGG
151  ATAGAGTGGC TCGTTTCTAG GTATTTAGAG AGTTTCGTGA
     CCAAGCCGAC
201  ATTTGTCTCT GATGTGGTGA GTCTTCTGAA AACAGAGAAA
     GTTGCTGGTC
251  GCGATCACAT TGCTCGTGTA GTGGAGACTT TGAAGAGGCA
     GAGAGTCGCT
301  GTGGCTCCTG AAGATGAGGA TAAGGTCCAT GGGAAGATTC
     CTGTGCATCC
351  TTTCGGGGGA ATCCAACCTG TAGAAGTTCT CACTCTCTAT
     CCCGAAGTTC
401  AAGATGCAAC GTTAGGGCTT GCCTTCTCTA AAATTCGTAA
     TCGTGTAAGA
451  CAGGCGTATT TGCAAGCTCC ACGGCCAAAA CTGCAGAAGA
     TTTACATCAT
501  AGGAAACGAT ATGAATCCTT TTGAAGTTGA CGACTTCTTG
     CATCTAGCCC
551  GTCTCTGTAA TGAAACTCAA AGACTCTATC CTGACGCTAC
     GATTTCTCTA
601  TATCTAACAG CTTCTGGTGG TCGCAATGCT ATGGACAAAA
     AGAATCGGAA
651  GTTACTTAGT GATTGCGAAC TAAACCCCAA GATTGCTTGT
     TTGGACTTTA
701  ATCAGGGTGA TGTAGTCAAA CAAGCAACTT GTGACTGTTG
     GATGGTGTAT
751  CATGGGGAGA ATGATCAAGG TACGTTGAAT CAGATTCAGG
     AAGAGTTAGA
801  AAAGTCAGGG GAGGAAACCC CTTGGATTCA TGTGGGGCAA
     AAGCCTCTTT
851  CACAATCCTT GTGGGATTTC TCTCCATTTT CATCTTTGGA
     GATGAAGGGA
901  CATAAAGAGA AAGCTCTAGA GTACTCTGAA TTAGAAAAAG
     AACAGCTATA
951  TTCTCGATTG GTATACGTAG GAGAGCGCTC TTCGGTTCTT
     AGTTTGGGGT
1001 TTGGAGATAG TCGGTCAGGG ATCTTGATGG ACCCAAAACG
     GGTGCATGCT
1051 CCCTTATCTG AAGGGCATTA TTGTCATTCC TACCTTGCAG
     ACTTAGAAAA
1101 TCCCGGGTTA CAAAAAACAA TTTTAGCGGC ATTTCTGAAT
     CCTAAGGAGT
1151 TGAGCAGTAC CATACTGCAA CCTATATCTC TAAATCTTAT
     CTTAAATAGC
1201 AAAACTTACT TAAGGCAGCA CTTTGGCTTT TTTGAGAGGA
     TGAGCAGAAG
1251 TGATCGCAAT GTGGTTGTCG TTGTATGTGA TTCTTGGTGG
     GGTACCGACT
1301 GGAAGGAGGA GCCAAGCTTC CAACACTTTA TTATGGAGCT
     AGAGTGTCGA
1351 GGGTATTCGC ACTTCAATAT TTTTGCCTTT AGATCTAATA
     GCATGTGTGT
1401 AGAAGAACGT AGGATCTTAA ATGAAAGTTC TCAAGAGAAA
     GCCTTTACCA
1451 TGATTTTCTG TGAGGATTCA GTATCTCAAG GAGATATCCG
     CTGTTTGCAT
1501 TTGGCGTCTG AAGGAATGCT TTGTGGTAAA GAGTGCTATG
     CTGTCGATGT
1551 CTATACGTCA GGATGCGCGA ACTTTATGAT GGAAGAAGTC
     TTAACTTTGG
1601 AGCGAGAATC TAATCTGTGG AATAGAAAGC ATGGTCTTTG
     GAAAAGAGAA
1651 GTTAGAAAAC AGAAACAAGA AGCTGCTTTG GATCAAGACG
     AGAGCGAGAT
1701 TTACGTTTGT AATCAGCTGA CGGCGCAACA GAACTTCGCT
     TGTTCTTGA

The PSORT algorithm predicts inner membrane (0.2869).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 125A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 125B) and for FACS analysis.

These experiments show that cp6741 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 126

The following C. pneumoniae protein (PID 4376742) was expressed <SEQ ID 251; cp6742>:

1   LFVSNFIFFV VMPIPYISSW ISTVRQHFVK AFDFSRPFCS
    RVTNFALGVI
51  KAIPIVGHIV MGMEWLVSSC VAGIITRSSF TSDVVQIVKT
    EKALGRDHIS
101 RVAFILQRER GTITPENQDK VHGKFPVCPF GRLKSEETLK
    LKPGEREGTL
151 DTVFSPIRTR VTRAYLQAPR PEIRTISIVG SKLKTPQDFS
    QFVSLANETQ
201 RLHPEALVCL YLTGLNRESQ MCDTTTAEKK QYLHNSGLDS
    RIQCKDSKFD
251 DAGSPENPEL WIGYYSREQQ HNIDGQYIQQ CLGKSADPIP
    WIHVTEDTKD
301 FYYPPNFTSY SHTRQSTDPT SPPRLPESEG DKDSLYGQLS
    RSYHHEYMLG
351 LGLKPEDAGL LMDPDRIYAP LSQGHYCHSY LADIENEDLR
    TLVLSPFLDP
401 GNLSSEDLRP VAFNIARLPL ELDSLFFRLV AGQQEGRNIV
    TLAHGTPRPE
451 DLDPDSMNIL TRRLQMSGYS YLNIFSYKSR KMIVKERQFF
    GDRSEGKSFT
501 LILFEDPISA ADFRCLQLAA EGMVAKDLPS VADICASGCS
    CIQFSEMQSP
551 QAIEYRQWEA RVEDEAGEEA REPVIYSQDQ LSSMLTTQQN
    FVFSLDAVVK
601 QAIWRFRSKG ILTMERKALG EEFLTAIFSY LGSQERNENM
    GKRRIEEHFV
651 VISFEELDRM VQVLPAEVPA DSGNDPTRPV PNPDSNPDSS
    QNEGS*

The cp6742 nucleotide sequence <SEQ ID 252> is:

1    TTGTTTGTTT CTAATTTTAT TTTTTTTGTT GTTATGCCAA
     TTCCCTATAT
51   TTCTTCTTGG ATTTCTACCG TTCGACAGCA TTTTGTTAAG
     GCGTTTGATT
101  TCTCTCGTCC CTTTTGTTCT AGGGTTACGA ATTTTGCTTT
     AGGGGTCATC
151  AAGGCCATCC CTATTCTAGG ACATATTGTC ATGGGGATGG
     AGTGGTTAGT
201  TTCTTCCTGT GTTGCCGGGA TTATTACTAG GTCCTCCTTT
     ACCTCAGATG
251  TCGTTCAGAT TGTAAAGACT GAGAAGGCGT TAGGTCGAGA
     TCATATATCT
301  CGAGTGGCGG AGATATTGCA AAGAGAAAGG GGGACCATAA
     CTCCTGAGAA
351  TCAAGATAAG GTGCATGGGA AGTTTCCTGT CTGTCCTTTT
     GGTCGTTTAA
401  AATCCGAGGA AACTTTAAAA CTTAAGCCGG GAGAAAGAGA
     GGGAACTTTA
451  GATACTGTAT TTTCTCCGAT TCGCACGCGC GTGACTCGTG
     CGTACTTACA
501  GGCCCCCCGA CCCGAAATAC GTACGATTTC TATTGTGGGT
     TCGAAACTTA
551  AAACTCCTCA AGATTTCTCG CAATTTGTGA GTCTCGCGAA
     TGAAACGCAG
601  AGACTGCATC CTGAAGCGTT AGTTTGTCTG TATTTGACAG
     GCTTGAATCG
651  CGAATCTCAG ATGTGCGATA CAACTACTGC AGAGAAGAAG
     CAGTACCTAC
701  ATAACTCAGG TCTCGACTCT AGAATCCAGT GCAAAGACAG
     TAAAGAAGAC
751  GACGCTGGCT CTCCTGAAAA TCCCGAACTT TGGATTGGCT
     ATTATTCACG
801  AGAGCAACAG CATAATATAG ACGGGCAGTA TATTCAGCAG
     TGTCTAGGGA
851  AGAGTGCAGA TCCAATTCCT TGGATTCATG TTACTGAAGA
     CACAAAGGAT
901  TTTTATTACC CACCAAACTT TACTTCATAC TCACATACAA
     GACAATCTAC
951  AGACCCAACA TCGCCACCAA GACTCCCTGA AAGTGAGGGG
     GATAAGGATT
1001 CCTTGTACGG ACAACTGAGT CGATCGTATC ACCATGAGTA
     TATGCTTGGT
1051 TTGGGATTAA AACCAGAGGA TGCAGGACTC CTGATGGACC
     CGGATAGAAT
1101 CTATGCTCCT CTATCCCAAG GGCATTATTG TCATTCCTAC
     CTTGCGGATA
1151 TAGAAAATGA GGATCTACGA ACTTTAGTCC TTTCGCCTTT
     CCTAGATCCT
1201 GGCAATCTTA GTAGCGAGGA TCTTCGTCCT GTAGCATTCA
     ATATCGCTAG
1251 ATTGCCATTA GAATTGGACT CGTTATTTTT CCGCCTTGTT
     GCGGGTCAGC
1301 AAGAAGGGAG AAACATAGTT ACCCTTGCCC ACGGAACTCC
     TCGTCCAGAA
1351 GATCTTGATC CTGACTCAAT GAACATTCTG ACCAGAAGAT
     TACAAATGTC
1401 TGGATATAGC TATTTGAACA TTTTCTCCTA TAAATCACGG
     AAAATGATTG
1451 TAAAAGAACG TCAGTTCTTT GGAGATCGTT CTGAAGGGAA
     GTCTTTCACA
1501 TTGATCTTAT TTGAGGATCC CATTAGTGCA GCAGATTTCC
     GTTGTTTGCA
1551 GCTAGCTGCA GAAGGTATGG TTGCTAAGGA TCTCCCCAGC
     GTAGCAGATA
1601 TTTGTGCCTC TGGATGTTCC TGCATTCAGT TTTCTGAGAT
     GCAGAGTCCT
1651 CAGGCTATTG AATATAGACA ATGGGAGGCA CGTGTCGAAG
     ATGAAGCAGG
1701 AGAAGAAGCC AGAGAACCAG TAATTTATTC TCAGGATCAA
     TTGAGCAGCA
1751 TGCTCACTAC ACAACAGAAT TTTGTATTTT CTCTAGATGC
     TGTGGTAAAA
1801 CAGGCGATCT GGAGATTCCG TTCGAAAGGT CTTCTTACTA
     TGGAAAGAAA
1851 GGCACTAGGC GAGGAGTTCT TAACTGCGAT ATTTTCCTAT
     TTAGGGAGTC
1901 AGGAGCGTAA TGAGAATATG GGGAAAAGAA CTACCGAAGA
     ACATGAGGTC
1951 GTTATCAGCT TCGAAGAGCT AGATCGCATG GTGCAAGTCC
     TCCCAGCCGA
2001 AGTCCCTGCA GATTCAGGCA ATGATCCTAC GCGTCCCGTT
     CCTAATCCAG
2051 ATAGTAACCC TGATTCCTCG CAAAATGAAG GCAGTTAG

The PSORT algorithm predicts inner membrane (0.2338).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 126A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 126B) and for FACS analysis.

These experiments show that cp6742 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 127

The following C. pneumoniae protein (PID 4376744) was expressed <SEQ ID 253; cp6744>:

1   VIQHLLNFAL EETPSISVQY QEQEKLSPCD HSPEIGKKKR
    WNKLESFSTY
51  CSLFMSVKDH YKLNLGIQNS LSGWLLDPYR VCAPLSSPYS
    CPSYLLDLQN
101 KELRRSLLST FLDPKNLTSE TFRSVSINFG NSSFGQRWSE
    FLSRVLHDEK
151 EKHVAVVCND AKLLEEGLSP EALSLLEEDL RESGYSYLNI
    LSVSPEGVSK
201 VQERQILRRD LQGRSFTVMI IDLPLGSEDI RSLQLASDRI
    LVSSSLDAAD
251 ACASGCKVLV YENPNASWAQ ELENFYKQVE RRR*

The cp6744 nucleotide sequence <SEQ ID 254> is:

1   GTGATACAAC ATCTTCTAAA CTTTGCTCTA GAAGAGACCC
    CTTCCATTTC
51  CGTGCAATAC CAAGAACAAG AGAAGCTCTC TCCGTGCGAT
    CATTCCCCAG
101 AAATAGGTAA AAAGAAAAGA TGGAATAAGC TGGAATCCTT
    CTCCACGTAT
151 TGTTCTCTGT TTATGTCTGT TAAGGATCAT TATAAGCTGA
    ATCTAGGAAT
201 TCAGAATTCC CTGTCAGGGT GGCTTCTGGA TCCCTATAGG
    GTTTGCGCGC
251 CTTTATCTTC ACCGTACTCG TGTCCTTCCT ATCTTTTAGA
    TTTGCAAAAC
301 AAAGAGCTAC GTCGTTCCCT TCTGTCAACG TTTCTAGACC
    CTAAAAATCT
351 CACTAGCGAA ACATTCCGTT CTGTCTCTAT AAACTTTGGC
    AACTCTTCGT
401 TTGGACAGAG ATGGTCAGAG TTTCTATCTC GTGTTCTGCA
    CGACGAGAAA
451 GAAAAGCACG TAGCTGTTGT TTGTAATGAT GCAAAACTTC
    TGGAAGAAGG
501 ATTGTCCCCA GAGGCATTGT CTCTATTAGA AGAAGACTTA
    AGAGAATCAG
551 GGTATTCGTA TCTAAACATT CTCTCGGTGA GCCCCGAAGG
    AGTCTCCAAG
601 GTTCAGGAAC GTCAGATTCT AAGGCGAGAT CTCCAAGGAC
    GGTCCTTTAC
651 TGTCATGATT ACAGATCTTC CTTTAGGTAG CGAAGATATC
    CGTAGTTTAC
701 AATTAGCCTC GGATAGGATT TTAGTCTCCA GTTCTCTTGA
    TGCCGCGGAT
751 GCATGTGCTT CGGGATGTAA AGTCTTAGTC TACGAAAATC
    CAAATGCATC
801 CTGGGCTCAG GAATTGGAGA ACTTCTACAA ACAAGTTGAG
    AGAAGAAGGT
851 AG

The PSORT algorithm predicts cytoplasm (0.3833).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 127A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 127B) and for FACS analysis.

These experiments show that cp6744 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 128

The following C. pneumoniae protein (PID 4376745) was expressed <SEQ ID 255; cp6745>:

1   VACPSISSWF TVVRQHFVNA FDFTHPVCSR ITNFALGIIK
    AIPVLGHIVM
51  GIEWLISWIP RHTVRHGMFT SDVSSAIEVE QTRGHNCLAP
    LEAYLSSLRV
101 PISQEDLGKV HGRTPEDPFV DITPTEIVQL LPDEELSTVD
    EALQGVRSRL
151 TYAYRSVEKP MIQDLALVGF GLRDSADLIN FVRLANGVQN
    HYPHTKVKLY
201 LAKNLADVWD CEISEEEKGQ LRALGLDPKI ESISLTSAGL
    PSVPEVATVD
251 FMITCYGKDQ EVQDP*

The cp6745 nucleotide sequence <SEQ ID 256> is:

1   GTGGCTTGTC CAAGTATTTC TTCTTGGTTT ACTGTCGTTC
    GACAGCATTT
51  TGTAAACGCC TTTGATTTCA CCCATCCCGT TTGTTCTCGG
    ATTACAAATT
101 TTGCTTTGGG GATCATTAAG GCAATTCCCG TATTAGGACA
    CATTGTCATG
151 GGAATCGAGT GGTTGATTTC CTGGATTCCC AGACACACCG
    TTCGTCATGG
201 AATGTTTACT TCTGATGTCT CTAGTGCTAT TAAAGTAGAA
    CAAACACGGG
251 GTCATAATTG TTTAGCTCCC CTAGAAGCCT ATTTAAGTAG
    CTTGAGAGTC
301 CCCATTTCCC AAGAAGATCT AGGCAAAGTA CACGGGAGAA
    CCCCAGAAGA
351 TCCCTTCGTA GATATCACAC CCACAGAAAT TGTCCAACTT
    CTCCCTGATG
401 AAGAACTCTC TACTGTAGAT GAGGCACTGC AAGGCGTTCG
    TAGTAGGTTA
451 ACCTATGCCT ATAGGTCCGT AGAGAAACCT ATGATTCAAG
    ATCTTGCTCT
501 TGTGGGTTTT GGTCTCCGAG ATTCTGCGGA CCTCATAAAT
    TTCGTGCGTC
551 TTGCTAATGG CGTGCAGAAT CACTATCCCC ATACTAAAGT
    GAAGCTCTAT
601 TTAGCGAAGA ACTTGGGAGA TGTCTGGGAC TGTGAAATTT
    CTGAAGAGGA
651 AAAAGGGCAA CTCCGAGCTC TAGGTTTAGA CCCTAAAATA
    GAGAGTATAT
701 CCCTTACGAG TGCAGGTCTT CCTTCAGTGC CAGAAGTCGC
    TACTGTCGAT
751 TTTATGATTA CCTGTTACGG GAAAGATCAG GAAGTCCAAG
    ATCCCTAG

The PSORT algorithm predicts inner membrane (0.2253).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 128A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 128B) and for FACS analysis.

These experiments show that cp6745 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 129

The following C. pneumoniae protein (PID 4376747) was expressed <SEQ ID 257; cp6747>:

1   MMKQGVGQDA KELYTFLSRG NEHYQPCLWF SLEEELGFLF
    DEKMLCAPLS
51  EDHYCHSYLV DLVDQHLKDL ILSMFLDPQN ISAGELLKVS
    INVGDSFSPL
101 QQKDFLSMVL RDETGKNVVV VFKGVLSLPA TQVCKLVEEL
    NSKDYSYLNI
151 FSCHGDSSPQ LLFRKELEGT SGRYFTVICA LYLGDTDMRS
    LQLASERIMV
201 SREFDLVDAY AARCKLLKID HTNWRPGTFS RHADFADAVD
    VSAGFNSREF
251 KLITQANQGI LESGELPLPS KTFWEGFLAF CDRVTVTRHF
    IPMLDAAIKQ
301 AVWTHKHPSL IDKECEALDL KTQCLPSIVS YLEYVTNSHE
    KTSKGPFIQK
351 EIIADCSPLK EALFPGSDED VPSTSEDPSD DHPSDLEDS*

The cp6747 nucleotide sequence <SEQ ID 258> is:

1    ATGATGAAAC AAGGAGTCGG GCAGGATGCT AAAGAGCTAT
     ACACATTTCT
51   ATCTCGTGGG AATGAGCATT ACCAACCGTG TCTATGGTTC
     AGTCTCGAAG
101  AGGAACTCGG ATTCCTTTTC GATGAAAAAA TGCTCTGCGC
     CCCTCTATCT
151  GAGGATCACT ATTGCCACTC GTATCTTGTA GATCTAGTGG
     ATCAACATTT
201  AAAGGATTTA ATATTATCGA TGTTTTTAGA TCCTCAGAAT
     ATCTCAGCAG
251  GAGAACTCCT GAACCTCTCT ATAAACGTTG GAGATTCTTT
     TTCTCCTCTA
301  CAACAGAAAG ATTTCCTCTC GATGGTCTTA GGTGATGAAA
     CGGGAAAAAA
351  CGTCGTCGTG GTTTTTAAAG GAGTTCTCTC CTTACCCGCA
     ACCCAAGTCT
401  GCAAATTAGT AGAGGAATTG AACTCTAAGG ACTACTCCTA
     CCTCAATATA
451  TTTTCTTGTC ACGGAGATAG TAGTCCTCAG CTTTTATTCC
     GTAAGGAATT
501  AGAGGGAACT TCAGGGCGTT ATTTTACAGT GATTTGCGCT
     TTATATCTAG
551  GGGATACAGA CATGCGTAGT TTACAACTTG CTTCTGAAAG
     GATCATGGTC
601  TCTAGAGAGT TTGATCTTGT AGATGCCTAT GCTGCAAGAT
     GCAAGCTCTT
651  GAAAATCGAT CATACAAATT GGAGACCTGG AACTTTCAGT
     CGCCACGCCG
701  ATTTCGCAGA TGCTGTAGAC GTATCAGCAG GATTTAACTC
     AAGAGAATTT
751  AAACTGATTA CGCAGGCGAA TCAAGGGATC CTAGAGTCTG
     GAGAACTCCC
801  GCTCCCTTCA AAAACCTTCT GGGAAGGATT CTTAGCATTC
     TGTGATCGAG
851  TGACTGTCAC GAGACACTTC ATTCCAATGT TAGACGCCGC
     TATAAAGCAA
901  GCGGTATGGA CTCATAAACA TCCCAGCTTG ATAGATAAAG
     AGTGTGAAGC
951  CCTAGACTTG AAAACACAGT GCTTGCCATC TATCGTATCG
     TACCTTGAAT
1001 ATGTCACAAA CTCTCACGAA AAAACATCGA AAGGCCCGTT
     CATACAAAAA
1051 GAGATTATCG CAGACTGTTC TCCTCTTAAA GAGGCGCTCT
     TCCCAGGTTC
1101 TGATGAAGAT GTTCCCTCTA CCTCTGAGGA TCCTTCAGAT
     GATCATCCTT
1151 CGGATCTTGA AGACTCTTAA

The PSORT algorithm predicts inner membrane (0.1447).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 129A) and also as a his-tagged product. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 129B) and for FACS analysis.

These experiments show that cp6747 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 130

The following C. pneumoniae protein (PID 4376756) was expressed <SEQ ID 259; cp6756>:

1   MASGIGGSSG LGKIPPKDNG DRSRSPSPKG ELGSHEISLP
    PQEHGEEGAS
51  GSSHIHSSSS FLPEDQESQS SSSAASSPGF FSRVRSGVDR
    ALKSFGNFFS
101 AESTSQARET RQAFVRLSKT ITAGERRDVD SSSAAATEAR
    VAEDASVSGE
151 NPSQGVPETS SGPEPQRLFS LPEVKKQSGL GRLVQTVRDR
    IVLPSGAPPT
201 DSEPLSLYEL NLRLSSLRQE LSDIQSNDQL TPEEKAEATV
    TIQQLIQITE
251 FQCGYMEATQ SSVSLAEARF KGVETSDEIN SLCSELTDPE
    LQELMSDGDS
301 LQNLLDETAD DLEAALSHTR LSFSLDDNPT PIDNNPTLIS
    QEEPIYEEIG
351 GAADPQRTRE NWSTRLWNQI REALVSLLGM ILSILGSILH
    RLRIARHAAA
401 EAVGRCCTCR GEECTSSEED SMSVGSPSEI DETERTGSPH
    DVPRRNGSPR
451 EDSPLMNALV GWAHKHGAKT KESSESSTPE ISISAPIVRG
    WSQDSSVSFI
501 VMEDDHIFYD VPRRKDGIYD VPSSPRWSPA RELEEDVFGD
    YEVPITSAEP
551 SKDKNIYMTP RLAIPAIYDL PSRPGSSGSS RSPSSDRVRS
    SSPNRRGVPL
601 PPVPSPAMSE EGSIYEDMSG ASGAGESDYE DMSRSPSPRG
    DLDEPIYANT
651 PEDNPFTQRN IDRILQERSG GASASPVEPI YDEIRWIHGR
    PPATLPRPEN
701 TLTNVSLRVS PGFGPEVRAA LLSESVSAVM VEAESIVPPT
    EPGDGESEYL
751 EPLGGLVATT KILLQKGWPR GESNA*

The cp6756 nucleotide sequence <SEQ ID 260> is:

1    ATGGCATCAG GAATCGGAGG ATCTAGTGGA TTAGGAAAGA
     TTCCACCTAA
51   AGATAATGGG GATAGAAGTC GATCGCCCTC TCCTAAGGGA
     GAACTTGGCA
101  GCCACGAGAT TTCCCTGCCT CCTCAAGAAC ATGGAGAGGA
     AGGAGCTTCA
151  GGATCTTCGC ATATACATAG CAGTTCCTCT TTTCTACCAG
     AAGATCAGGA
201  GTCTCAGAGC TCTTCTTCGG CAGCTTCTAG CCCGGGATTT
     TTTTCTCGCG
251  TACGTTCTGG GGTAGACAGG GCCTTAAAAT CATTTGGCAA
     CTTTTTTTCC
301  GCAGAGTCTA CGAGTCAAGC GCGTGAAACG CGACAAGCTT
     TTGTTAGATT
351  ATCAAAAACC ATCACCGCGG ATGAGAGACG GGATGTCGAT
     TCATCAAGTG
401  CTGCTGCTAC AGAAGCCCGA GTGGCAGAGG ACGCGAGTGT
     TTCAGGCGAA
451  AATCCTTCTC AGGGGGTTCC AGAAACCTCT TCTGGACCAG
     AACCTCAGCG
501  TTTATTTTCT CTTCCTTCAG TAAAAAAACA GAGCGGTTTG
     GGTCGGTTGG
551  TACAGACAGT TCGCGATCGC ATAGTACTTC CTAGTGGGGC
     TCCACCTACA
601  GACAGCGAGC CTTTAAGTCT CTACGAGCTA AACCTCCGTT
     TGAGTAGTTT
651  ACGTCAGGAG CTCTCTGACA TACAAAGTAA TGATCAGTTG
     ACTCCAGAGG
701  AAAAAGCAGA AGCCACAGTT ACCATACAAC AGCTGATCCA
     AATTACAGAA
751  TTCCAATGCG GCTATATGGA GGCAACACAA TCTTCGGTAT
     CTCTAGCAGA
801  AGCTCGTTTT AAGGGGGTAG AAACTAGTGA TGAGATCAAT
     TCCCTCTGTT
851  CAGAACTGAC AGATCCTGAG CTTCAAGAAC TCATGAGTGA
     TGGAGACTCT
901  CTTCAAAACC TATTAGATGA GACTGCCGAC GATTTAGAAG
     CTGCTTTGTC
951  CCATACTCGA TTGAGTTTTT CTTTAGACGA TAATCCAACT
     CCGATAGACA
1001 ATAATCCAAC TCTGATTTCT CAAGAAGAGC CTATTTATGA
     GGAAATCGGA
1051 GGAGCTGCAG ATCCTCAAAG AACTCGGGAA AACTGGTCTA
     CAAGATTATG
1101 GAATCAGATT CGCGAGGCTC TGGTTTCTCT TTTAGGAATG
     ATTTTAAGCA
1151 TTCTAGGGTC CATCTTGCAC AGGTTGCGTA TTGCTCGTCA
     TGCAGCTGCT
1201 GAAGCAGTGG GTCGTTGTTG CACGTGCCGA GGAGAAGAGT
     GTACTTCTTC
1251 TGAAGAGGAC TCGATGTCGG TGGGGTCTCC TTCAGAAATT
     GATGAAACTG
1301 AAAGAACGGG CTCTCCGCAT GACGTTCCAC GCAGAAATGG
     AAGTCCACGT
1351 GAAGATTCTC CATTGATGAA TGCCTTAGTA GGATGGGCAC
     ATAAGCACGG
1401 TGCTAAAACC AAGGAGAGTT CAGAATCAAG TACCCCGGAA
     ATTTCGATTT
1451 CTGCTCCCAT AGTGAGAGGT TGGAGTCAAG ACAGTTCCGT
     CAGTTTTATT
1501 GTTATGGAAG ATGATCATAT TTTCTATGAT GTTCCTCGTA
     GAAAAGATGG
1551 AATCTATGAC GTTCCTAGTT CCCCTAGATG GAGTCCTGCG
     CGAGAGTTGG
1601 AAGAGGATGT TTTTGGAGAT TATGAAGTTC CTATAACCTC
     TGCTGAACCA
1651 TCTAAAGACA AGAACATCTA CATGACACCT AGATTAGCAA
     CTCCTGCTAT
1701 CTATGATCTT CCTTCACGTC CAGGATCGTC TGGAAGCTCA
     CGTTCTCCGT
1751 CTTCAGATCG CGTACGAAGC AGCTCACCAA ATAGACGGGG
     TGTGCCTCTT
1801 CCTCCAGTTC CTTCACCTGC TATGAGTGAG GAGGGGAGCA
     TTTATGAGGA
1851 TATGAGCGGT GCTTCAGGTG CAGGTGAAAG TGATTATGAA
     GATATGAGCC
1901 GTTCCCCCTC TCCTAGAGGC GACTTGGATG AACCCATATA
     TGCTAATACT
1951 CCTGAAGATA ATCCATTTAC TCAGAGAAAT ATAGATAGAA
     TTTTACAGGA
2001 GAGGTCAGGC GGTGCTTCCG CTTCTCCTGT AGAGCCTATT
     TATGATGAGA
2051 TCCCATGGAT TCATGGCAGG CCCCCTGCTA CACTTCCAAG
     ACCCGAGAAT
2101 ACATTGACTA ATGTTTCGCT TAGAGTGAGC CCAGGGTTTG
     GACCAGAAGT
2151 AAGAGCCGCT TTGCTTAGCG AGAGCGTGAG TGCTGTTATG
     GTCGAAGCAG
2201 AGAGTATTGT TCCTCCAACA GAGCCGGGGG ACGGAGAATC
     AGAATATCTA
2251 GAGCCCTTAG GGGGACTTGT AGCTACAACG AAAATCTTAC
     TACAAAAAGG
2301 ATGGCCTCGT GGAGAGTCGA ATGCTTAG

The PSORT algorithm predicts inner membrane (0.3994).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 130A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 130B) and for FACS analysis.

These experiments show that cp6756 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 131

The following C. pneumoniae protein (PID 4376761) was expressed <SEQ ID 261; cp6761>:

1   MTVAEVKGTF KLVCLGCRVN QYEVQAYRDQ LTILGYQEVL
    DSEIPADLCI
51  INTCAVTASA ESSGRHAVRQ LCRQNPTAHI VVTGCLGESD
    KEFFASLDRQ
101 CTLVSNKEKS RLIEKIFSYD TTFPEFKIHS FEGKSRAFIK
    VQDGCNSFCS
151 YCIIPYLRGR SVSRPAEKIL AEIAGVVDQG YREVVIAGIN
    VGDYCDGERS
201 LASLIEQVDR IPGIERIRIS SIDPDDITED LHRAITSSRH
    TCPSSHLVLQ
251 SGSNSILKRM NRKYSRGDFL DCVEKFRASD PRYAFTTDVI
    VGFPGESDQD
301 FEDTLRIIED VGFIDVHSFP FSARRRTKAY TFDNQIPNQV
    IYERKKYLAE
351 VAKRVGQKEM MKRLGETTEV LVEKVTGQVA TGHSPYFEKV
    SFPVVGTVAI
401 NTLVSVRLDR VEEEGLIGEI V*

The cp6761 nucleotide sequence <SEQ ID 262> is:

1    ATGACGGTTG CGGAAGTCAA AGGAACATTT AAGCTGGTCT
     GTTTAGGCTG
51   TCGGGTGAAT CAGTATGAGG TCCAAGCATA TCGCGACCAG
     TTGACTATCT
101  TAGGTTACCA AGAGGTCCTG GATTCTGAAA TCCCTGCAGA
     TTTATGCATA
151  ATCAATACGT GTGCTGTCAC AGCTTCTGCT GAGAGTTCGG
     GTCGTCATGC
201  TGTGCGTCAG TTATGTCGTC AGAACCCTAC AGCACATATT
     GTTGTCACAG
251  GTTGTTTGGG GGAATCTGAC AAAGAGTTTT TTGCTTCTTT
     GGATCGGCAA
301  TGCACACTTG TTTCCAATAA AGAAAAATCC CGACTTATAG
     AAAAAATTTT
351  TTCCTATGAT ACGACCTTCC CTGAGTTCAA GATCCATAGT
     TTTGAGGGAA
401  AGTCTCGAGC TTTTATTAAA GTTCAAGATG GCTGTAATTC
     TTTTTGCTCG
451  TACTGCATTA TTCCTTATTT GCGGGGGCGT TCGGTTTCTC
     GTCCTGCTGA
501  GAAGATTTTA GCTGAAATCG CAGGGGTTGT AGACCAAGGA
     TATCGCGAAG
551  TTGTAATTGC AGGAATTAAT GTTGGAGATT ATTGCGATGG
     AGAGCGTTCA
601  TTAGCCTCTT TGATTGAACA GGTGGACCGG ATTCCTGGAA
     TTGAGAGGAT
651  TCGAATTTCC TCTATAGATC CTGATGATAT CACTGAAGAT
     CTGCACCGTG
701  CCATCACCTC ATCGCGTCAC ACTTGTCCTT CGTCACACCT
     TGTTCTTCAA
751  TCGGGGTCGA ATTCAATTTT AAAGAGAATG AACCGGAAGT
     ATTCTCGCGG
801  AGATTTTTTA GATTGTGTAG AGAAGTTCCG TGCTTCTGAT
     CCTCGCTATG
851  CCTTTACTAC AGATGTGATT GTCGGATTTC CTGGAGAGAG
     TGATCAAGAT
901  TTTGAAGATA CTTTGAGAAT TATTGAAGAT GTAGGCTTTA
     TTAAAGTGCA
951  TAGTTTCCCT TTCAGTGCTC GTCGTCGTAC TAAGGCATAT
     ACTTTTGATA
1001 ATCAGATTCC CAATCAGGTG ATCTATGAGA GGAAGAAGTA
     TCTTGCTGAG
1051 GTTGCTAAGA GGGTAGGCCA GAAAGAGATG ATGAAGCGTT
     TAGGAGAGAC
1101 TACAGAGGTG CTTGTTGAGA AAGTAACGGG GCAGGTTGCT
     ACGGGTCACT
1151 CTCCTTATTT TGAAAAGGTT TCTTTCCCTG TTGTAGGAAC
     GGTAGCTATC
1201 AACACTCTAG TTTCTGTGCG TCTTGATAGG GTAGAGGAAG
     AAGGGCTGAT
1251 TGGGGAGATT GTATGA

The PSORT algorithm predicts inner membrane (0.1574).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 131A) and also as a his-tagged product. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 131B) and for FACS analysis.

These experiments show that cp6761 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 132

The following C. pneumoniae protein (PID 4376766) was expressed <SEQ ID 263; cp6766>:

1   MATSVPVTSS TSVGEANSSN ERFTERTSRM YYAALVLGAL
    SCLIFIAMIV
51  IFPQVGLWAV VLGFALGCLL LSLAIVFAVS GLVLGKTLEP
    SREATPPEIV
101 AQKEWTTQQD VLGNEYWRSE LISLFLRGDL HESLIVDSKD
    RSLDIDQSLQ
151 NILKLEPLST TLSLLKKDCV HINIILHLVR QWNLLGVDLS
    PEVTAHAEEL
201 LLFLIEEQYY SPDILKLIRY GDALQATSPL MDWADSGSFS
    VDADGVFSCR
251 REECSPEDAL AQFDLLLALE NPDRRFLKDS FLTYIWSSSF
    FEKFLHRHLE
301 SLQRKLPETA IDVARYFAQI QTFLSRYFQK LDLINAMSLD
    WGYNCAEGEK
351 CYESANQRLD NLFIAFSSSV PAMKRLFDKY GSVVRVDRRQ
    IREQILSNTE
401 ILENESGFLC SLYEYPLSYL IDWAVLLDCV RGTEISLEDQ
    ADYTVCLQGL
451 DSMLSQFASR LQSGQKVLNP RDVLSEQAAV MLVEGLAAQG
    VSFQGLKALM
501 YLIAVPQRMW LGALPLFESF PVFNRMKEFL GESLGD*

The cp6766 nucleotide sequence <SEQ ID 264> is:

1    ATGGCAACCT CTGTTCCTGT AACTTCATCT ACTTCTGTAG
     GAGAGGCTAA
51   CTCCTCCAAC GAAAGATTTA CTGAACGAAC ATCGCGAATG
     TATTACGCAG
101  CTTTAGTCCT AGGGGCTTTG AGCTGTTTAA TTTTTATTGC
     TATGATTGTC
151  ATTTTCCCAC AGGTCGGATT GTGGGCTGTG GTCCTCGGGT
     TTGCTCTTGG
201  ATGTTTACTT TTAAGCTTAG CTATCGTTTT TGCTGTCTCC
     GGTCTCGTTT
251  TAGGCAAGAC TTTAGAACCT AGTCGAGAAG CGACTCCTCC
     AGAAATTGTT
301  GCGCAAAAGG AGTGGACTAC ACAACAAGAT GTCTTAGGGA
     ATGAGTATTG
351  GCGTTCCGAG TTGATTTCCT TGTTCTTACG AGGGGATCTC
     CACGAATCTC
401  TGATTGTTGA TTCTAAGGAT CGATCTTTAG ATATTGATCA
     GAGTTTACAA
451  AATATATTGA AACTTGAGCC CCTATCTACG ACACTTTCGC
     TGTTAAAGAA
501  AGATTGTGTC CACATCAATA TCATTTTACA TTTAGTGAGA
     CAGTGGAACT
551  TACTGGGAGT GGATCTTAGT CCTGAAGTCA CTGCGCACGC
     CGAGGAACTT
601  CTACTCTTTT TGATAGAAGA GCAGTATTAC TCTCCTGATA
     TTTTGAAATT
651  GATTCGCTAC GGAGATGCTT TACAAGCAAC GTCTCCTTTG
     ATGGATTGGG
701  CAGATTCAGG TTCCTTTAGT GTAGACGCAG ACGGGGTATT
     TAGCTGTCGC
751  AGAGAAGAAT GTTCTCCTGA GGATGCTTTG GCGCAATTCG
     ATCTTCTTTT
801  GGCGTTGGAA AATCCCGACA GACGCTTCTT AAAGGATTCT
     TTTCTTACCT
851  ACATTTGGTC GTCTTCATTT TTTGAGAAGT TTTTACATCG
     CCATCTAGAG
901  AGCTTGCAAA GAAAGCTCCC AGAGACAGCG ATCGATGTCG
     CCCGCTATGA
951  AGCACAAATA CAAACATTTC TCTCTCGCTA TTTTCAGAAG
     CTCGATTTGA
1001 TAAACGCAAT GTCCTTAGAT TGGGGATATA ACTGTGCTGA
     GGGAGAAAAA
1051 TGTTATGAGA GCGCAAATCA AAGATTAGAC AACCTATTTA
     TTGCTTTTTC
1101 TTCTTCTGTT CCTGCTATGA AGCGGCTCTT TGACAAATAT
     GGTTCTGTGG
1151 TACGGGTAGA TCGTAGGCAG ATTCGTGAGC AGATTCTTTC
     GAACACTGAA
1201 ATCTTAGAAA ATGAGTCAGG GTTCCTCTGC AGTTTGTATG
     AATATCCTTT
1251 ATCCTATTTG ATAGATTGGG CTGTTTTGCT AGACTGTGTT
     CGCGGTACCG
1301 AAATCTCTCT AGAAGATCAG GCCGATTACA CCGTTTGTTT
     GCAAGGCTTG
1351 GATTCTATGT TATCTCAATT TGCGAGTCGT TTACAGTCTG
     GACAAAAAGT
1401 ATTGAATCCT AGAGATGTTT TAAGTGAACA GGCTGCGGTT
     ATGCTTGTTC
1451 ATGGCTTGGC AGCACAGGGC GTGTCGTTTC AAGGATTGAA
     AGCTTTGATG
1501 TATTTGACAG CCGTTCCCCA AAGAATGTGG TTAGGAGCAT
     TGCCTTTATT
1551 TGAATCTTTT CCTGTCTTTA ATCGGATGAA AGAATTTCTT
     GGGGAATCTC
1601 TGGGAGACTA G

The PSORT algorithm predicts inner membrane (0.6158).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 132A) and also as a his-tagged product. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 132B) and for FACS analysis.

These experiments show that cp6766 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 133

The following C. pneumoniae protein (PID 4376804) was expressed <SEQ ID 265; cp6804>:

1   MSNQLQPCIS LGCVSYINSF PLSLQLIKRN DIRCVLAPPA
    DLLNLLIEGK
51  LDVALTSSLG AISHNLGYVP GFGIAANQRI LSVNLYAAPT
    FFNSPQPRIA
101 ATLESROSIG LLKVLCRHLW RIPTPHILRF ITTKVLRQTP
    ENYDGLLLIG
151 DAALQHPVLP GFVTYDLASG WYDLTKLPFV FALLLHSTSW
    KEHPLPNLAM
201 EEALQQFESS PEEVLKEAHQ HTGLPPSLLQ EYYALCQYRL
    GEEHYESFEK
251 FREYYGTLYQ QARL

The cp6804 nucleotide sequence <SEQ ID 266> is:

1   ATGTCTAACC AACTCCAGCC ATGTATAAGC TTAGGCTGCG
    TAAGTTATAT
51  TAATTCCTTT CCGCTGTCCC TACAACTCAT AAAAAGAAAC
    GATATTCGCT
101 GTGTTCTTGC TCCCCCTGCA GACCTCCTCA ACTTCCTAAT
    CGAAGGGAAA
151 CTCGATGTTG CTTTGACCTC ATCCCTAGGA GCTATCTCTC
    ATAACTTGGG
201 GTATGTCCCC GGCTTTGGAA TTGCAGCAAA CCAACGTATC
    CTCAGTGTAA
251 ACCTCTATGC AGCTCCCACT TTCTTTAACT CACCGCAACC
    TCGGATTGCC
301 GCAACTTTAG AAAGTCGCTC CTCTATAGGA CTCTTAAAAG
    TGCTTTGTCG
351 TCATCTCTGG CGCATCCCAA CTCCTCATAT CCTAAGATTC
    ATAACTACAA
401 AAGTACTCAG ACAAACCCCT GAAAATTATG ATGGCCTCCT
    CCTAATCGGA
451 GATGCAGCGC TACAACATCC TGTACTTCCT GGATTTGTAA
    CCTATGACCT
501 TGCCTCGGGG TGGTATGATC TTACAAAGCT ACCTTTTGTA
    TTTGCTCTTC
551 TTCTACACAG CACCTCTTGG AAAGAACATC CCCTACCCAA
    CCTTGCGATG
601 GAAGAAGCCC TCCAACAGTT CGAATCTTCA CCCGAAGAAG
    TCCTTAAAGA
651 AGCTCATCAA CATACAGGTC TGCCCCCTTC TCTTCTTCAA
    GAATACTATG
701 CCCTATGCCA GTACCGTCTA GGAGAAGAAC ACTACGAAAG
    CTTTGAAAAA
751 TTCCGGGAAT ATTATGGAAC CCTCTACCAA CAAGCCCGAC
    TGTAA

The PSORT algorithm predicts inner membrane (0.060).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 133A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 133B) and for FACS analysis.

These experiments show that cp6804 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 134

The following C. pneumoniae protein (PID 4376805) was expressed <SEQ ID 267; cp6805>:

1   MSSLLSCGRI EPTRVTCSLK TYLEDTSQNQ LSTRLVRASV
    IFLCALLIIL
51  VCVALSSIIP SIMALATSFT VAGLILFVMS LLGDVAIISY
    LTYSTVTSYR
101 QNKRAFEIHK PARSVYYEGV RHWDLGRSSL GTGEIPIVRT
    LFSPFQNHGL
151 NHALAAKIFL FMEHFSPEPP NEPLVDWACL IRDFRPHVSS
    LCFVIEKQGS
201 SLRTKEGNTI CEAFRSDYDA HFAMVDCYRL IHSKLIIEKM
    GLKNIDIIPS
251 VMVREDYPSR PGEGYREGLL RMYGGKGAL*

The cp6805 nucleotide sequence <SEQ ID 268> is:

1   ATGTCATCAC TACTGAGCTG CGGAAGAATA GAGCCGACTC
    GGGTTACCTG
51  TAGCTTAAAG ACGTATCTTG AGGATACGAG TCAGAATCAG
    TTGAGCACAC
101 GTCTAGTTCG GGCAAGTGTC ATCTTTTTAT GCGCATTGTT
    GATCATTTTG
151 GTTTGTGTGG CCCTCTCTAG TTTGATTCCA AGCATTATGG
    CCTTGGCGAC
201 CTCTTTTACG GTAATGGGGT TAATTCTTTT TGTGATGTCA
    CTTCTTGGTG
251 ACGTTGCAAT TATAAGTTAT CTTACTTATA GCACTGTTAC
    GAGTTACCGG
301 CAAAATAAGA GAGCTTTTGA GATTCACAAG CCCGCTCGCT
    CCGTTTACTA
351 CGAGGGGGTC CGCCATTGGG ATTTAGGACG ATCATCTTTA
    GGCACAGGCG
401 AGATTCCTAT AGTAAGGACG TTATTCTCTC CATTTCAGAA
    CCATGGTCTT
451 AACCATGCCT TAGCTGCTAA AATTTTCCTA TTTATGGAGC
    ATTTCAGCCC
501 TGAGCCACCG AACGAGCCTT TGGTGGATTG GGCCTGTTTG
    ATTCGGGATT
551 TTAGGCCTCA CGTCAGTTCT TTGTGCTTTG TTATTGAAAA
    ACAAGGGTCA
601 TCGCTGAGGA CTAAGGAAGG CAATACGATT TGTGAGGCTT
    TCCGCTCTGA
651 TTACGACGCC CATTTTGCTA TGGTAGATTG CTACCGGTTG
    ATCCACTCTA
701 AGTTGATTAT AGAGAAAATG GGATTGAAGA ATATCGATAT
    CATTCCGAGT
751 GTCATGGTTC GTGAAGATTA TCCTAGCCGT CCTGGGGAGG
    GCTATCGCGA
801 AGGCCTATTA CGTATGTATG GTGGCAAGGG GGCTCTGTGA

The PSORT algorithm predicts inner membrane (0.711).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 134A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 134B) and for FACS analysis.

These experiments show that cp6805 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 135

The following C. pneumoniae protein (PID 4376813) was expressed <SEQ ID 269; cp6813>:

1   MSGPSRTESS QVSVLSYVPR DKEIAPKKQF TIAKISTLAI
    LASLALGALV
51  AGISLTIVLG NPVFLALLIT TALFSVVTFL VYHQMTSKVS
    SNWQKVLEQN
101 FKPLGKAWQE KNVDCYSNEM QFYNNHLNPK FKVAIQTDAS
    QPFQPTFLTG
151 LRVIEKNQST GIIFNPVGPT NLIDNTATNL STILYSTLKD
    KSVWDTCKQR
201 EGGPAKGEDP ESPTEVRVVK LPNEALDQTF NLNLSSAEKK
    SILPTFLGHV
251 CGPKSEELPN QQEYYRQALL AYENCLKAAI ESHAAIVALP
    LFTSVYEVPP
301 EEILPKEGTF YWDNQTQAFC KRALLDAIQN TALRYPQRSL
    LVILQDPFNT
351 IESQSRSEE*

The cp6813 nucleotide sequence <SEQ ID 270> is:

1    ATGTCAGGAC CCTCACGTAC TGAGAGCTCT CAAGTTTCTG
     TACTATCCTA
51   TGTGCCTCGG GATAAAGAAA TTGCTCCTAA AAAACAGTTT
     ACCATAGCAA
101  AAATATCCAC TCTTGCAATC CTAGCTTCTT TAGCTTTAGG
     AGCTTTGGTG
151  GCTGGAATCT CTTTAACGAT AGTATTAGGG AACCCTGTAT
     TTTTGGCTCT
201  TCTCATTACC ACGGCCCTCT TCTCAGTTGT AACCTTCTTA
     GTCTACCACC
251  AAATGACCTC AAAGGTATCT TCTAACTGGC AGAAAGTTCT
     AGAGCAAAAC
301  TTCAAGCCTT TGGGAAAAGC GTGGCAAGAA AAAAACGTAG
     ACTGCTACTC
351  AAACGAGATG CAATTTTACA ATAATCACCT GAACCCTAAG
     TTCAAGGTAG
401  CGATACAAAC AGATGCGTCT CAACCATTTC AGCCTACTTT
     CTTAACTGGA
451  CTTAGAGTGA TCGAAAAAAA TCAATCCACA GGGATCATCT
     TTAATCCCGT
501  AGGCCCAACG AATCTGATCG ACAACACTGC AACGAACCTC
     TCTACTATCC
551  TTTACTCCAC CCTAAAAGAT AAAAGCGTGT GGGATACATG
     CAAGCAACGC
601  GAAGGGGGTC CCGCAAAAGG AGAAGACCCC TTTTCCCCTA
     CCGAAGTGAG
651  AGTAGTAAAA CTTCCAAACG AAGCTCTAGA TCAAACGTTT
     AATCTAAATT
701  TAAGCTCTGC AGAAAAGAAA AGTATTCTTC CGACCTTTTT
     AGGCCACGTA
751  TGCGGCCCTA AATCTGAAGA GTTACCAAAT CAGCAAGAAT
     ATTATCGCCA
801  AGCTTTACTA GCGTACGAGA ACTGCCTTAA AGCAGCTATA
     GAAAGTCATG
851  CAGCAATCGT TGCTCTTCCT CTCTTTACTT CGGTCTATGA
     AGTGCCTCCA
901  GAAGAGATTC TTCCTAAAGA AGGCACTTTC TATTGGGACA
     ACCAAACTCA
951  AGCGTTTTGC AAACGCGCTT TATTGGACGC TATTCAAAAT
     ACGGCCCTAC
1001 GCTATCCTCA AAGATCCTTA CTTGTTATAC TCCAAGATCC
     TTTTAATACT
1051 ATAGAATCAC AAAGTCGTTC TGAGGAGTAA

The PSORT algorithm predicts inner membrane (0.4291).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 135A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 135B) and for FACS analysis.

These experiments show that cp6813 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 136

The following C. pneumoniae protein (PID 4376844) was expressed <SEQ ID 271; cp6844>:

1   MWRVVLRFLI IFILGRAVFP LRASESFSWE TSTCLTVLGI
    PFIDIILTIN
51  EDFVAQCGLQ IGTISSTNNA KIKEIFLIYK EKFPEASISF
    KRKEPLNLSQ
101 SHLSDLGILC MRNGETYAEG MANKENGPAL KQPKDLRLVL
    RCPNQPDTLL
151 YSEKEAEKGI ETNTCLCNQG YTLLDGQLIL YGDSIEKFLK
    ETKRKNNHTL
201 VDLCDSQVVT TFLGRFWSLL NYVQVLFLSE DSAKILAGIP
    DLAQATQLLS
251 HTVPLLFIYT NDSIHIIEQG KESSFTYNQD LTEPILGFLF
    GYINRGSMEY
301 CFNCAQSSLG ET*

The cp6844 nucleotide sequence <SEQ ID 272> is:

1   ATGTGGCGCG TTGTCCTCAG ATTCCTTATA ATTTTTATCT
    TGGGAAGAGC
51  CGTCTTCCCT CTAAGAGCTT CAGAAAGCTT CTCCTGGGAA
    ACATCGACCT
101 GTTTACCAGT GCTAGGGATT CCTTTCATAG ATATTATCCT
    CACAACGAAT
151 GAGGACTTTG TTGCCCAGTG CGGCCTGCAA ATAGGAACCA
    TTTCTTCGAC
201 TAATAACGCA AAAATAAAAG AAATTTTTTT GATATATAAG
    GAAAAATTTC
251 CAGAAGCCTC TATCAGTTTC AAACGAAAAG AACCTCTAAA
    CCTTTCCCAA
301 TCCCATCTCT CCGATTTAGG TATTTTATGT ATGCGTAACG
    GAGAAACTTA
351 CGCTGAGGGA ATGGCAAATA AAGAAAACGG ACCCGCTCTA
    AAACAACCCA
401 AGGATCTAAG ATTAGTTTTA CGTTGTCCTA ACCAACCAGA
    TACCCTGCTC
451 TACTCGGAAA AAGAAGCAGA AAAGGGCATA GAAACAAATA
    CTTGCCTATG
501 CAATCAGGGA TACACACTCC TGGATGGGCA ATTGATTCTC
    TACGGGGATA
551 GTATAGAAAA GTTTCTGAAA GAGACCAAAA GAAAGAATAA
    CCACACGCTT
601 GTTGATCTTT GTGACTCACA AGTCGTGACC ACGTTCCTCG
    GTCGCTTTTG
651 GTCTCTTCTA AACTACGTTC AAGTTCTTTT CCTATCTGAA
    GACTCCGCTA
701 AAATTCTTGC GGGCATCCCA GACCTAGCTC AAGCTACGCA
    ATTGCTTTCC
751 CACACCGTAC CTTTGCTTTT TATTTATACC AACGATTCTA
    TTCACATCAT
801 AGAACAAGGC AAAGAAAGTA GTTTTACCTA TAACCAAGAT
    TTAACAGAGC
851 CCATTTTAGG ATTTCTCTTT GGTTACATAA ATCGCGGCTC
    TATGGAATAC
901 TGCTTTAATT GTGCACAGTC TTCATTAGGA GAAACCTAA

The PSORT algorithm predicts inner membrane (0.1786).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 136A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 136B) and for FACS analysis.

These experiments show that cp6844 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 137

The following C. pneumoniae protein (PID 4377201) was expressed <SEQ ID 273; cp7201>:

1   VLVGICRSLY PEHPRSFYYR VSGDIGSRFD DRGFVNSGVE
    TLPYSSGSFG
51  IFWISFTDPT FNFAIVNTFM RTAGINEVSR PMTQDTETSL
    IEMRDLSEQQ
101 EANNTDSLEQ EESLMGIVGH TVGGVSMTVT SSPNIFYRIQ
    TLLGLPETLA
151 EAEENPTFPN STIDDLAEIM NNLVRISDAV SIFWIFPIVD
    TTYNGVLLAV
201 CIGFFGINGI CSTFLMLTNP RSRRDRWRNL RIMVLCYRSL
    GSGMNLFDLS
251 NNVRMAARRH VTSCTVALYA MVTLFGWTVA IQDALQYGFP
    SVRDAFYRYC
301 LRHRYCLTQR NEDSLQTTGT RFQVTRTHLE DQQMVASILN
    LSVFGLFFGF
351 VGLMTTFGGL EISPSCRWDA ANNRTVGIF*

The cp7201 nucleotide sequence <SEQ ID 274> is:

1    GTGCTCGTTG GTATCTGTCC TTCTCTATAT CCAGAACATC
     CTCGCTCCTT
51   TTATTATCGT GTTTCTGGAG ATATAGGCTC CCGATTCGAC
     GATAGAGGAT
101  TTGTAAACTC TGGAGTCGAA ACCCTGCCAT ACTCTTCAGG
     CAGCTTTGGG
151  ATTTTTTGGA TCTCGTTTAC GGATCCCACA TTTAATTTTG
     CTATCGTAAA
201  TACCTTTATG CGAACTGCAG GGATCAATGA AGTCTCTAGA
     CCCATGACAC
251  AAGATACAGA AACTTCATTG ATAGAAATGA GAGACCTAAG
     TGAACAACAA
301  GAAGCGAATA ACACAGATTC TTTAGAGCAA GAAGAGAGCT
     TAATGGGTAT
351  TGTAGGACAT ACTGTGGGAG GAGTTTCCAT GACCGTGACC
     TCCAGTCCAA
401  ATATCTTTTA TCGTATACAA ACACTTCTGG GACTGCCAGA
     GACTCTTGCA
451  GAAGCTGAAG AAAATCCTAC CTTCCCAAAT TCTACTATAG
     ATAGCCTTGC
501  AGAAATAATG ATGAACCTCG TAAGGATCTC TGATGCTGTC
     TCTATTTTCT
551  GGATTTTTCC TATCGTAGAT ACTACATATA ATGGAGTTTT
     ATTAGCCGTC
601  TGTATCGGCT TCTTCGGAAT CAATGGGATT TGTTCCACGT
     TCCTTATGCT
651  TACGAATCCA CGCTCTCGTC GAGATAGATG GAGGAATTTA
     CGCATCATGG
701  TTCTTTGCTA TCGTTCTTTG GGAAGCGGAA TGAATCTCTT
     TGATCTTAGC
751  AATAATGTGC GCATGGCAGC ACGTAGGCAT GTGACATCAT
     GTACAGTAGC
801  TCTCTATGCT ATGGTCACTC TATTTGGATG GACAGTAGCA
     ATACAAGATG
851  CTTTGCAATA TGGTTTCCCT AGCGTTCGGG ATGCCTTCTA
     TAGATATTGC
901  TTACGCCACA GATATTGCTT AACTCAAAGA AACGAAGACT
     CTCTGCAAAC
951  TACAGGAACG CGCTTTCAGG TTACCCGTAC ACATCTAGAA
     GATCAACAGA
1001 TGGTGGCTTC TATTTTGAAT TTGAGTGTTT TTGGGCTCTT
     TTTTGGATTC
1051 GTAGGGCTAA TGACCACGTT TGGAGGATTA GAAATCTCAC
     CATCTTGTCG
1101 GTGGGATGCA GCAAATAACC GAACGGTAGG TATTTTTTAG

The PSORT algorithm predicts inner membrane (0.3102).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 137A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 137B) and for FACS analysis.

These experiments show that cp7201 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 138

The following C. pneumoniae protein (PID 4377251) was expressed <SEQ ID 275; cp7251>:

1   MAPIHGSNAF VEDILHSHPS PQATYFSSTR AQKLHEFKDR
    HPVLTRIASV
51  IIKIFKVLIG LIILPLGIYW LCQTLCTNSI LPSKNLLKIF
    KKQPNTKTLK
101 TNYLHALQDY SSKNRVASMR RVPILQDNVL IDTLEICLSQ
    APTNRWMLIS
151 LGSDCSLEEI ACKEIFDSWQ RFAKLIGANI LVYNYPGVMS
    STGSSSLKDL
201 ASAHNICTRY LKDKEQGPGA KEIITYGYSL GGLIQAEALR
    DQKIVANDDT
251 TWIAVKDRCP LFISPEGFHS CRRIGKLVAR LFGWGTKAVE
    RSQDLPCLEI
301 FLYPTDSLRR STVRQNKLLA PELTLAHAIK NSPYVQNKEF
    IEVRLSSDID
351 PIDSKTRVAL ATPILKKLS*

The cp7251 nucleotide sequence <SEQ ID 276> is:

1    ATGGCTCCAA TTCACGGAAG TAATGCGTTT GTTGAGGATA
     TTTTACATTC
51   CCACCCTTCT CCACAAGCGA CTTATTTTTC TTCAACACGC
     GCCCAAAAAC
101  TTCATGAGTT TAAAGACAGG CATCCCGTGC TTACACGGAT
     TGCTTCTGTA
151  ATTATTAAAA TTTTTAAAGT TCTGATAGGG CTGATCATCC
     TTCCCTTAGG
201  AATCTACTGG CTATGTCAAA CGCTTTGTAC AAACTCGATT
     CTCCCTTCCA
251  AGAATTTATT AAAAATTTTC AAGAAGCAAC CCAACACTAA
     AACCTTAAAA
301  ACTAATTATT TGCATGCTTT GCAAGATTAT TCCTCGAAAA
     ACCGCGTTGC
351  TTCCATGAGA CGAGTTCCTA TCCTCCAGGA TAATGTTCTC
     ATCGACACTT
401  TGGAAATATG CCTTTCACAA GCACCTACGA ATCGTTGGAT
     GCTCATTTCT
451  TTAGGAAGTG ACTGTAGCTT GGAAGAAATC GCTTGTAAGG
     AGATCTTTGA
501  TTCTTGGCAA AGATTTGCCA AGTTGATAGG GGCCAATATA
     CTCGTTTATA
551  ACTACCCCGG AGTCATGTCC AGCACAGGGA GCAGCAGCCT
     AAAGGACCTA
601  GCATCAGCTC ATAATATTTG TACAAGATAC CTTAAAGATA
     AAGAACAGGG
651  CCCTGGAGCA AAAGAAATCA TTACCTATGG GTACTCCCTA
     GGAGGTTTGA
701  TACAAGCAGA AGCATTGCGA GACCAGAAGA TTGTTGCAAA
     CGATGATACT
751  ACTTGGATAG CAGTCAAAGA TAGGTGTCCT CTCTTTATAT
     CTCCAGAAGG
801  TTTCCACAGT TGCAGACGCA TAGGAAAGCT AGTAGCTCGT
     CTTTTTGGCT
851  GGGGGACCAA AGCCGTAGAG AGAAGCCAAG ACCTTCCCTG
     CCTAGAAATT
901  TTTCTCTATC CTACGGATTC CTTACGAAGA TCAACAGTCA
     GACAGAACAA
951  GCTCTTAGCA CCTGAACTTA CTCTCGCTCA TGCGATAAAA
     AATAGTCCCT
1001 ATGTTCAAAA TAAAGAATTT ATAGAAGTAC GATTATCGTC
     TGATATCGAT
1051 CCCATCGACA GCAAAACAAG AGTGGCTCTT GCCACACCAA
     TTTTGAAAAA
1101 GCTCTCTTAG

The PSORT algorithm predicts inner membrane (0.4545).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 138A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 138B) and for FACS analysis.

These experiments show that cp7251 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 139

The following C. pneumoniae protein (PID 4377288) was expressed <SEQ ID 277; cp7288>:

1   MHMSNPISLP SPAELIAKYN LIPKTSPIYP RRTELIILEE
    NACQTRLTNV
51  AQVLHPSSLF SMSKKILNPC GCSGGPLCWV ILNILAFIIT
    SVLFIILLPV
101 NLIVAGLRLF MPLPPKKIVE DLSEPTTEET NEVIQPFIFA
    LQALLFEDNK
151 LRSFKIVEQS VGKAPLPNPF LNRLVAISPQ ESQEAMRKIP
    DLCSQLKKVL
201 KSLGVLTPEW KHNLKYFEGL KNEHDSNPKD KTFPILIKLL
    IEALTGKSSL
251 PKTPSTKERM QAALFIASSC KTCKPTWGEV ITRSLNRLYS
    IANEGDNQLL
301 IWVQEFKERE LMSIQDGDDA EEYRFAAQQH GERYTEAIEQ
    VLRNESAAKL
351 QWHVINTMKF FHGKNLGLVT EHLQDTLGAL TLRQTTVDTH
    QGREDADLSA
401 ALFLNKYLNS GNQLVNSVFK SMQKADPETK ALIREFALDI
    LYASLRLPQT
451 SAHTEVFSTL LMDPETYEPN KACIAYLLYV LKIIEL*

The cp7288 nucleotide sequence <SEQ ID 278> is:

1    ATGCATATGT CTAACCCCAT CTCTTTGTTT TCCCCTGCAG
     AGTTAATAGC
51   AAAGTACAAT TTAATTCCAA AAACTTCGCC GATTTATCCT
     CGGAGGACGG
101  AACTTATTAT CTTGGAAGAA AATGCGTGTC AAACACGCCT
     AACCAACGTG
151  GCTCAGGTCC TACATCCTTC TAGCCTATTC AGTATGTCAA
     AAAAAATACT
201  GAATCCCTGC GGGTGCTCTG GTGGTCCCTT ATGTTGGGTG
     ATTCTCAACA
251  TCCTAGCATT TATTATTACT TCAGTACTGT TTATCATTCT
     TTTACCGGTG
301  AATCTCATCG TAGCAGGTCT TCGTCTCTTC ATGCCTCTTC
     CCCCTAAAAA
351  AATCGTAGAG GATTTAAGTG AACCTACTAC TGAAGAAACG
     AATGAGGTCA
401  TTCAACCCTT CATTTTCGCT TTGCAAGCGT TGCTTTTTGA
     GGATAACAAA
451  CTTCGCTCTT TTAAAATTGT TGAACAAAGT GTAGGCAAAG
     CACCCTTACC
501  TAATCCCTTT TTAAATAGAC TAGTAGCAAT TTCGCCGCAA
     GAAAGCCAAG
551  AAGCCATGCG GAAGATTCCG GATCTATGCT CACAACTGAA
     AAAAGTATTA
601  AATGCTCTAG GCGTGCTAAC TCCAGAATGG AAGCACATGC
     TGAAGTACTT
651  TGAGGGACTG AAAAACGAAC ATGATAGTAA TCCTGATAAA
     AAGACGTTCC
701  CAATATTGAT CAAGCTCCTC ATAGAAGCTC TTACTGGAAA
     GTCCTCTTTA
751  CCCAAAACTC CTAGTACAAA GGAAAAAATG CAAGCGGCCT
     TATTTATTGC
801  AAGTTCTTGC AAGACTTGTA AGCCGACTTG GGGAGAAGTC
     ATAACCAGAT
851  CTCTTAACAG ACTCTATAGT ATAGCTAATG AAGGAGACAA
     TCAGCTTCTG
901  ATTTGGGTTC AAGAGTTTAA AGAACGAGAG CTGATGTCCA
     TCCAAGATGG
951  TGATGATGCT GAAGAGTATC GGTTTGCGGC TCAGCAACAC
     GGTGAGCGTT
1001 ACACAGAGGC AATAGAACAA GTTCTACGAA ACGAGTCAGC
     AGCCAAACTA
1051 CAATGGCATG TGATCAACAC TATGAAATTC TTCCATGGGA
     AAAATCTCGG
1101 TCTAGTTACA GAACACCTAC AAGATACTCT CGGCGCCCTA
     ACTTTACGTC
1151 AAACTACAGT GGACACACAT CAAGGCAGAG AAGACGCTGA
     TTTGTCAGCT
1201 GCTCTTTTCC TAAATAAGTA TTTAAATTCT GGAAATCAAC
     TTGTTAATAG
1251 CGTCTTTAAA TCCATGCAAA AAGCAGATCC AGAAACCAAA
     GCTTTAATCC
1301 GTGAGTTTGC TCTAGATATA TTATATGCAT CCTTACGGCT
     TCCTCAAACT
1351 TCCGCTCATA CCGAGGTCTT TTCTACACTC TTAATGGACC
     CAGAGACCTA
1401 TGAACCTAAT AAAGCTTGTA TCGCCTACTT GCTCTATGTA
     TTAAAGATCA
1451 TCGAACTATA A

The PSORT algorithm predicts inner membrane (0.5989).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 139A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 139B) and for FACS analysis.

These experiments show that cp7288 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 140

The following C. pneumoniae protein (PID 4377359) was expressed <SEQ ID 279; cp7359>:

1 MPGSVSSPPL SPVIVRERVP SSSGSDLIQP HAVLKISILI FALVTILGIV
51 LVVLSSALGA IPSLVLTVSG CIAIAVGLIG LGILVTRLIL STIRKVDAMG
101 YDAAVKEEQY ISRIRELESE NREIRDRNRA VEDQCAHLSE ENKDLRDPFY
151 LHGMTERLIA SLEIENQALV AENILLKDWN ASLSRDFRAY KQKFPLGALE
201 PWKFDIACIM EQNLFLKPEC IAMVKSLPLE TQRLFLYPKG FQSLVNRFAP
251 RSRFFQTPKY EYNSRNENED GKVAAVCARL KKEFFSAVLG ACSYEELGGI
301 CERAVALKET LPLPEAVYDT LVQEFPNLLT AESLWKEWCF YSYPYLRPYL
351 SVDYCKRIFV QLFEELCLKL FTTGSPEDQA LVRLFSYYRN HIPAVLASFG
401 LPPPETGGSV FVLLPKQENL LWSQIEVLAT RYLKDTFVRN SEWTGSFEMM
451 FSYNEMCKEI SEGRIRFAED YETRHSEEFP PSPLSEEGEG EEFLPPCSEE
501 EVSVLERPDL DVDSMWVWHP PVPKGPL*

The cp7359 nucleotide sequence <SEQ ID 280> is:

1 ATGCCAGGTT CTGTGTCATC ACCTCCTTTG TCTCCTGTAA TTGTCCGTGA
51 AAGGGTCCCA TCCTCTTCAG GATCCGACCT CATACAGCCT CATGCTGTTT
101 TAAAGATCTC CATCCTAATT TTTGCGCTTG TGACAATTTT AGGAATTGTT
151 CTTGTAGTGT TGTCTAGTGC TTTAGGAGCT CTTCCTAGTT TAGTTTTGAC
201 GGTTTCTGGT TGTATTGCAA TAGCTGTAGG CCTGATTGGT TTAGGGATTC
251 TTGTGACACG GCTGATTCTC TCTACGATCA GAAAAGTAGA TGCCATGGGT
301 TATGATGCTG CGGTCAAAGA AGAGCAGTAT TTGTCACGTA TCAGAGAATT
351 AGAGTCTGAA AATAGAGAGA TTAGAGATAG AAATCGTGCT GTCGAAGATC
401 AGTGTGCCCA TTTATCCGAA GAGAACAAGG ACCTTAGGGA TCCCGAATAT
451 CTACATGGAA TGACTGAAAG GCTCATTGCG AGCTTAGAAA TAGAGAATCA
501 AGCTCTCGTA GCTGAGAACA TTCTTCTCAA AGACTGGAAT GCAAGCCTAT
551 CTAGAGATTT CCGCGCATAT AAGCAAAAAT TTCCTCTTGG GGCATTAGAA
601 CCCTGGAAAG AAGATATTGC ATGTATCATG GAACAAAATC TCTTTTTAAA
651 ACCGGAATGT ATCGCGATGG TTAAGTCTCT TCCATTAGAG ACGCAACGGC
701 TGTTTTTATA TCCAAAAGGA TTTCAGTCTT TAGTTAATCG ATTTGCTCCG
751 CGGTCTCGCT TTTTCCAGAC TCCAAAGTAT GAATATAACA GTAGGAATGA
801 AAATGAGGAC GGAAAGGTAG CCGCAGTGTG CGCCCGTTTG AAAAAAGAAT
851 TCTTCAGTGC TGTTTTAGGA GCCTGTAGTT ACGAAGAACT AGGGGGCATT
901 TGTGAAAGAG CAGTAGCACT TAAAGAGACG TTGCCATTGC CTGAAGCTGT
951 CTATGATACC CTAGTTCAGG AGTTCCCAAA TCTTCTTACT GCTGAGAGTT
1001 TATGGAAAGA ATGGTGCTTC TATTCCTATC CCTACCTTCG TCCCTATCTT
1051 TCTGTGGATT ACTGTAAGAG GTTATTTGTA CAACTTTTTG AGGAACTCTG
1101 CCTAAAGCTT TTTACAACGG GATCTCCAGA AGACCAAGCT TTGGTTCGCC
1151 TTTTCTCTTA CTATAGGAAT CATATTCCCG CAGTCTTGGC CTCATTTGGT
1201 TTGCCCCCGC CTGAGACAGG GGGGTCTGTA TTTGTATTGC TACCAAAACA
1251 AGAAAACCTT CTTTGGAGTC AAATTGAGGT GCTGGCTACA AGGTATCTCA
1301 AAGATACCTT CGTGAGAAAC TCAGAATGGA CGGGCTCTTT CGAGATGATG
1351 TTTTCTTATA ACGAGATGTG TAAGGAGATC TCCGAAGGAA GGATTCGTTT
1401 TGCTGAAGAC TATGAAACGA GGCATTCCGA AGAATTCCCT CCTTCCCCTC
1451 TCTCTGAAGA AGGAGAGGGC GAAGAATTCC TTCCTCCTTG CTCTGAAGAA
1501 GAGGTTTCGG TTCTTGAGCG CCCAGATCTA GATGTAGACT CTATGTGGGT
1551 CTGGCATCCG CCGGTCCCTA AGGGACCTCT TTAA

The PSORT algorithm predicts inner membrane (0.7453).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 140A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 140B) and for FACS analysis.

These experiments show that cp7359 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 141

The following C. pneumoniae protein (PID 4377374) was expressed <SEQ ID 281; cp7374>:

1 MDKQSSGNSG CIWHPFTQSA LDSTPIKIVR GEGAYLYAES GTRYLDAISS
51 WWCNLHGHGH FYITKKLCEQ AQKLEHVIFA NFTHEPALEL VSKLAPLLPE
101 GLERFFFSDN GSTSIEIAMK IAVQYYYNQN KAKSHFVGLS NAYHGDTFGA
151 MSIAGTSPTT VPFHDLFLPS STIAAPYYGK EELAIAQAKT VFSESNIAAF
201 IYEPLLQGAG GMLMYNPEGL KEILKLAKHY GVLCIADEIL TGFGRTGPLF
251 ASEFTDIPPD IICLSKGLTG GYLPLALTVT TKEIHDAFVS QDRMKALLHG
301 HTFTGNPLGC SAALASLDLT LSPECLQQRQ MIERCHQEFQ EAHGSLWQRC
351 EVLGTVLALD YPAEATGYFS QYRDHLNRFF LERGVLLRPL GNTLYVLPPY
401 CIQEEDLRII YSHLQDALCL QPQ*

The cp7374 nucleotide sequence <SEQ ID 282> is:

1 ATGGACAAGC AATCATCAGG GAATTCAGGG TGTATCTGGC ACCCCTTCAC
51 TCAATCTGCA TTAGATTCTA CACCCATAAA GATTGTAAGG GGAGAAGGTG
101 CTTACCTCTA TGCGGAATCA GGAACAAGAT ATCTTGATGC GATATCTTCA
151 TGGTGGTGCA ACCTCCACGG TCATGGGCAT CCCTACATTA CAAAAAAATT
201 ATGTGAGCAA GCACAGAAGT TAGAACATGT GATCTTCGCA AATTTCACCC
251 ATGAACCGGC TCTAGAGCTC GTATCGAAAC TCGCTCCCCT CCTTCCTGAA
301 GGTCTAGAAC GTTTCTTTTT CTCTGACAAC GGATCAACGT CTATCGAAAT
351 AGCAATGAAA ATTGCTGTGC AATATTACTA CAATCAAAAC AAGGCTAAGA
401 GCCATTTTGT TGGACTCAGC AATGCCTATC ACGGAGATAC ATTTGGAGCT
451 ATGTCGATAG CTGGCACGAG CCCTACTACA GTTCCCTTTC ATGATCTTTT
501 TCTTCCTTCC AGTACAATTG CTGCTCCCTA TTATGGCAAG GAAGAGCTTG
551 CCATTGCCCA AGCAAAAACA GTCTTTTCTG AAAGCAATAT CGCAGCGTTT
601 ATCTATGAGC CGCTATTGCA AGGTGCTGGA GGGATGTTAA TGTATAATCC
651 CGAAGGCCTA AAGGAGATTC TCAAGCTTGC CAAGCATTAC GGGGTTCTCT
701 GTATTGCTGA TGAAATTCTT ACTGGCTTTG GCCGTACGGG TCCACTGTTT
751 GCTTCTGAAT TTACAGACAT TCCTCCTGAC ATTATCTGTC TTTCTAAAGG
801 TCTTACAGGA GGCTATCTCC CTCTAGCCTT GACAGTAACC ACTAAAGAAA
851 TTCATGATGC CTTTGTCTCC CAAGATCGGA TGAAGGCACT GCTTCATGGC
901 CATACCTTCA CAGGAAATCC TTTAGGCTGT AGTGCTGCCC TCGCTTCTTT
951 GGATCTCACC CTATCTCCAG AATGCCTACA ACAAAGGCAA ATGATAGAAC
1001 GGTGTCATCA AGAGTTCCAA GAAGCTCATG GTTCCCTATG GCAACGGTGT
1051 GAGGTTCTGG GCACGGTACT CGCTCTAGAT TACCCTGCAG AAGCTACAGG
1101 ATATTTTTCA CAATATAGAG ACCATCTCAA TCGCTTTTTC TTAGAACGTG
1151 GAGTCCTTCT TCGTCCTTTA GGGAACACAC TGTATGTGCT GCCCCCCTAC
1201 TGTATCCAAG AAGAAGATCT CCGGATTATT TATTCTCACC TACAGGATGC
1251 CCTATGTCTA CAACCACAGT AA

The PSORT algorithm predicts cytoplasm (0.2930).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 141A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 141B) and for FACS analysis.

These experiments show that cp7374 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 142

The following C. pneumoniae protein (PID 4377377) was expressed <SEQ ID 283; cp7377>:

1 MREFTVSWSL EDIREIYHTP VFELIHKANA ILRSNFLHSE LQTCYLISIK
51 TGGCVEDCAY CAQSSRYHTH VTPEPMMKIV DVVERAKRAV ELGATRVCLG
101 AAWRNAKDDR YFDRVLAMVK SITDLGAEVC CALGMLSEEQ AKKLYDAGLY
151 AYNHNLDSSP EFYETIITTR SYEDRLNTLD VVNKSGISTC CGGIVGMGES
201 EEDRIKLLHV LATRDHIPES VPVNLLWPID GTPLQDQPPI SFWEVLRTIA
251 TARVVFPRSM VRLAAGRAFL TVEQQTLCFL AGANSIFYGD KLLTVENNDI
301 DEDAEMIKLL GLIPRPSFGI ERGNPCYANN S*

The cp7377 nucleotide sequence <SEQ ID 284> is:

1 ATGCGTGAAG AAACTGTATC CTGGTCATTA GAAGACATCC GCGAAATTTA
51 TCACACTCCC GTATTTGAGC TGATTCACAA AGCCAATGCC ATATTGCGTA
101 GTAATTTCCT CCATTCAGAA CTGCAGACTT GCTATCTGAT TTCGATTAAA
151 ACTGGTGGAT GCGTTGAAGA TTGCGCCTAC TGTGCCCAAT CTTCCCGCTA
201 TCATACCCAC GTCACACCAG AACCTATGAT GAAAATTGTA GACGTTGTGG
251 AAAGGGCAAA ACGTGCTGTA GAGCTAGGCG CCACTCGTGT GTGTCTTGGG
301 GCTGCCTGGC GCAATGCTAA GGACGATCGA TACTTTGATA GAGTCCTCGC
351 TATGGTGAAA AGTATCACAG ATCTCGGAGC CGAGGTTTGT TGTGCTTTAG
401 GCATGCTCTC CGAAGAGCAA GCTAAAAAAC TGTATGATGC AGGACTTTAT
451 GCCTACAATC ATAATTTAGA CTCTTCTCCG GAATTCTATG AAACTATAAT
501 CACAACACGT TCTTATGAAG ATCGCCTCAA CACTCTTGAT GTAGTAAATA
551 AATCTGGCAT TAGTACATGC TGCGGTGGTA TTGTAGGTAT GGGAGAATCT
601 GAAGAAGACC GTATAAAGCT TCTTCATGTT CTTGCAACAA GAGATCATAT
651 CCCAGAATCC GTACCTGTAA ATTTACTTTG GCCGATTGAC GGCACGCCTT
701 TGCAAGACCA GCCTCCGATT TCTTTCTGGG AAGTCTTGCG AACCATAGCA
751 ACGGCACGGG TTGTTTTCCC CAGATCCATG GTACGACTTG CTGCAGGACG
801 CGCTTTCCTC ACAGTAGAAC AACAAACCTT ATGTTTTCTA GCCGGTGCCA
851 ACTCCATATT CTATGGAGAT AAACTGTTGA CTGTAGAAAA CAATGATATA
901 GATGAAGATG CTGAAATGAT CAAACTTTTA GGCTTAATCC CTCGCCCTTC
951 ATTTGGAATA GAAAGAGGTA ACCCATGTTA TGCCAACAAT TCCTAA

The PSORT algorithm predicts cytoplasm (0.2926).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 142A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 142B) and for FACS analysis.

These experiments show that cp7377 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 143

The following C. pneumoniae protein (PID 4377407) was expressed <SEQ ID 285; cp7407>:

1 MVCPNNSWFR MCGNFNCEWV EVTTTEETTR QSASDISEEA GSSGGAAPIT
51 TQPTKITKVE KRVQFNTAQG DEFTIHMIQE AGELVDSILS HRRTQGCTFY
101 CYDSYATGCG QRCGSFGRLI CGTYKACCLD REDNQVAGLV HECEQTHGPI
151 AVALAAKTMG LNLMELVEKN TILSEEQKNE FRQHCSEAKT QLYGTMQSLS
201 QNFFLEGVNS IRERFLDDSL VQAVLSFIAT RSWEKTIESE EASGTSSASN
251 STRIPACYIL NTSPITTSRL SCGSRDARRP SSVGAEPQYV AKKYNDNGMA
301 RQLGKIQVTN LKTGDFSALG PRGLLIVKML NSFLLSASQS TSSILKHTGG
351 EICYTCPNFR DIVVLLMLAI GYCPANTDET SVVDIHMIDD PIMTIFYRLQ
401 YSYRTGKTSA SFLKKKPSLV RQESLDCPTP AESVPLMSSL EEEDENEDDD
451 EDGNLAYQQR ILECSGHLQT LFLGIKINKE *

The cp7407 nucleotide sequence <SEQ ID 286> is:

1 ATGGTTTGCC CAAATAATTC TTGGTTCAGA ATGTGTGGAA ATTTCAACTG
51 CGAATGGGTT GAAGTAACAA CAACAGAAGA AACAACGCGG CAATCGGCTT
101 CAGATATAAG CGAAGAAGCT GGTTCGAGTG GAGGAGCTGC TCCTATAACT
151 ACGCAACCTA CTAAAATTAC AAAAGTAGAG AAACGTGTCC AATTTAATAC
201 TGCTCAAGGT GATGAAAGTA CAATACACAT GATCCAAGAA GCAGGAGAAT
251 TGGTAGACTC CATTCTATCA CATAGACGAA CGCAAGGATG TACAGAGTAT
301 TGTTATGACA GTTACGCAAC TGGATGTGGT CAGCGTTGCG GATCTTTTGG
351 AAGACTCATT TGTGGAACGT ATAAAGCGTG TTGCTTAGAC AGAGAGGATA
401 ATCAGGTTGC TGGACTTGTC CATGAATGCG AACAGACCCA TGGTCCTATT
451 GCCGTTGCTT TAGCTGCTAA AACTATGGGC CTCAACTTAA TGGAACTTGT
501 AGAAAAAAAC ACTATTTTGT CTGAAGAACA GAAAAATGAA TTTAGACAGC
551 ATTGCTCGGA AGCTAAAACC CAACTCTATG GAACGATGCA GAGCCTTTCT
601 CAAAACTTTT TCCTTGAAGG AGTCAACAGC ATTAGAGAAC GCGGTCTAGA
651 CGATTCACTA GTCCAAGCCG TGCTAAGCTT TATTGCTACA AGGTCTTGGG
701 AAAAAACTAT AGAATCAGAG GAAGCCTCAG GAACATCTTC TGCTTCTAAT
751 TCTACACGCA TTCCTGCGTG CTATATCTTA AATACGAGCC CCTTAACGAC
801 GTCACGCCTA TCCTGTGGAT CAAGAGATGC GCGACGCCCA TCTTCAGTCG
851 GTGCAGAGCC CCAGTACGTA GCAAAAAAAT ACAATGACAA TGGCATGGCC
901 AGACAATTAG GAAAAATCCA AGTCACCAAT CTAAAAACAG GAGATTTTTC
951 AGCTTTAGGT CCTTTTGGTC TCCTGATTGT GAAAATGCTG AATAGCTTTC
1001 TCTTATCTGC ATCACAAAGC ACATCTTCTA TTCTAAAGCA CACAGGTGGA
1051 GAAATATGTT ATACGTGCCC AAATTTTCGT GATATCGTCG TTTTATTGAT
1101 GTTAGCGATT GGCTATTGCC CTGCAAATAC CGATGAGACA TCTGTCGTAG
1151 ATATACACAT GATAGATGAT CCGATTATGA CCATCTTCTA TCGACTACAA
1201 TACAGCTATA GAACAGGGAA AACTTCAGCA TCGTTTTTAA AAAAGAAACC
1251 CTCATTAGTA AGACAGGAAA GTCTTGATTG TCCTACCCCT GCAGAATCTG
1301 TCCCTCTCAT GTCAAGTCTC GAAGAAGAAG ATGAAAATGA AGATGATGAT
1351 GAGGATGGGA ATTTGGCGTA TCAACAGCGT ATCCTTGAAT GCTCGGGTCA
1401 TTTACAAACT CTATTTTTAG GGATAAAAAT AAACAAAGAA TAA

The PSORT algorithm predicts inner membrane (0.1319).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 143A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 143B) and for FACS analysis.

These experiments show that cp7407 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 144

The following C. pneumoniae protein (PID 4376432) was expressed <SEQ ID 287; cp6432>:

1 MTRSTIESSD SLCSRSFSQK LSVQTLKNLC ESRLMKITSL VIAFLTLIVG
51 GALIALAGGG VLSFPLGLIL GSVLVLFSSI YLVSCCKFFT LKEMTMTCSV
101 KSKINIWFEK QRNKDIEKAL ENPDLFGENK RNVGNRSARN QLEMILHETD
151 GIILKRYMKG AKMYFYL*

The cp6432 nucleotide sequence <SEQ ID 288> is:

1 ATGACTAGAA GTACTATTGA AAGCAGTGAT TCGCTATGCT CAAGGTCTTT
51 TTCTCAAAAA TTAAGTGTCC AGACATTAAA AAATCTCTGT GAAAGTAGAT
101 TAATGAAGAT CACTTCTCTT GTGATTGCTT TCCTAACTCT AATTGTGGGG
151 GGTGCTCTTA TAGCTTTAGC AGGAGGGGGG GTTCTTTCTT TCCCTCTTGG
201 GCTAATCTTA GGAAGCGTAC TCGTTTTGTT TTCTTCTATC TATTTAGTCT
251 CTTGTTGTAA ATTTTTCACT TTAAAAGAGA TGACAATGAC CTGTAGTGTC
301 AAATCTAAAA TCAATATATG GTTTGAAAAG CAACGAAACA AAGACATCGA
351 AAAGGCATTA GAGAATCCAG ATCTCTTTGG AGAAAATAAG AGAAATGTTG
401 GAAATCGTTC GGCAAGAAAT CAACTAGAAA TGATCTTACA CGAGACTGAC
451 GGAATTATTT TGAAAAGATA TATGAAAGGA GCTAAAATGT ACTTTTATTT
501 ATGA

The PSORT algorithm predicts inner membrane (0.5394).

The protein was expressed in E. coli and purified as a his-tagged product (FIG. 144A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 144B) and for FACS analysis.

These experiments show that cp6432 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 145

The following C. pneumoniae protein (PID 4376433) was expressed <SEQ ID 289; cp6433>:

1 MNWVPKTIDH VDPESEIDIR KVVSCYKLIK ECQPEFRSLI SELLGVIRCG
51 LRLLKRSKYQ EQARTVSDED APLFCLTRSY YQDGYLTPLR AGPRDLINHY
101 IHLRRRENPK HFFSPKHPCY YARLAFNESV CVYRELFDIE RLTKMYVEGD
151 YSKEQEKNLQ AILSFVKTLD EGKDFLIEHK DTDLIGRGFT DVFCT*

The cp6433 nucleotide sequence <SEQ ID 290> is:

1 ATGAATTGGG TTCCAAAAAC AATAGACCAT GTAGATCCAG AATCAGAGAT
51 AGATATACGT AAAGTCGTCT CCTGCTATAA GTTGATAAAA GAATGTCAAC
101 CTGAATTTCG ATCTCTTATA AGTGAATTAC TAGGAGTGAT TCGGTGTGGC
151 TTAAGACTAT TAAAACGTTC TAAGTATCAA GAACAGGCTA GAACTGTATC
201 TGATGAAGAT GCACCTCTTT TCTGCCTGAC TCGTTCTTAT TATCAAGATG
251 GTTATCTCAC GCCATTAAGA GCAGGACCTC GTGATCTTAT AAATCACTAT
301 ATACACTTGC GTCGCCGAGA GAATCCTAAG CATTTTTTCA GTCCTAAGCA
351 TCCATGTTAT TATGCTCGAT TGGCTTTTAA TGAGTCAGTG TGTGTCTATA
401 GAGAACTCTT TGATATAGAG CGACTTACAA AAATGTATGT CGAGGGTGAT
451 TATTCTAAAG AACAAGAGAA AAACCTACAG GCTATTCTTA GTTTTGTGAA
501 AACTCTAGAT GAAGGAAAGG ACTTTCTTAT TGAACATAAA GATACCGATC
551 TCATTGGGAG AGGTTTTACT GATGTGTTCT GCACTTAA

The PSORT algorithm predicts cytoplasm (0.4068).

The protein was expressed in E. coli and purified as a his-tagged product (FIG. 145A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 145B) and for FACS analysis.

These experiments show that cp6433 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 146

The following C. pneumoniae protein (PID 4376643) was expressed <SEQ ID 291; cp6643>:

1 MGYLPVSATD VLFESPAAPL INSANTQNQK LIELKGKQQA ESSPRTITSV
51 ILEVLLVIGC CLIVLSLLAI RPALQFTLET GHPAAIAVLA VSGTILLVAV
101 IILFCFLAAV PFAAKKTYKY VKTVDDYASW HSHQQTPTLG TIFSGIVYAE
151 SQAQL*

The cp6643 nucleotide sequence <SEQ ID 292> is:

1 ATGGGATATC TTCCAGTATC TGCTACGGAC GTTCTTTTTG AAAGTCCAGC
51 CGCTCCCTTA ATCAATAGCG CAAACACACA AAATCAGAAA CTCATAGAAC
101 TCAAGGGGAA GCAGCAAGCT GAGTCTTCTC CACGGACAAT CACTTCTGTC
151 ATATTGGAAG TTCTCCTAGT GATCGGATGC TGCCTCATAG TTCTTAGTTT
201 ATTGGCAATC CGCCCTGCTC TGCAATTCAC TCTAGAAACT GGACATCCAG
251 CTGCCATTGC AGTCCTTGCT GTCTCAGGAA CAATTCTATT GGTGGCTGTT
301 ATCATCTTGT TTTGCTTTCT AGCAGCTGTG CCATTCGCTG CTAAGAAAAC
351 TTATAAATAT GTTAAGACGG TTGATGACTA TGCTTCTTGG CATTCTCATC
401 AGCAAACACC GACCCTAGGC ACTATCTTTT CAGGTATCGT CTATGCAGAA
451 TCCCAGGCGC AATTATAG

The PSORT algorithm predicts inner membrane (0.6859).

The protein was expressed in E. coli and purified as a his-tagged product (FIG. 146A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 146B) and for FACS analysis.

These experiments show that cp6643 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 147

The following C. pneumoniae protein (PID 4376722) was expressed <SEQ ID 293; cp6722>:

1 VSSTLNGVFP SSLPEESADL FITNKEIVAL GEKGNVFLTH SIPMHIAAIT
51 ILVIVALAGI AIICLGCYSQ SILLIAVGIV LTILTLLCLQ ALVGFIFKIR
101 QLPQQLHTTV QFIREKIRPE SSLQLVTNAQ RKTTQDTLKL YEELCDLSQK
151 EFKLQSTLYQ KRFELSHKNE KTNQN*

The cp6722 nucleotide sequence <SEQ ID 294> is:

1 GTGTCTAGTA CTTTAAACGG GGTATTTCCC TCATCCCTTC CGGAAGAGTC
51 TGCTGATTTA TTCATTACGA ATAAGGAGAT CGTAGCTTTG GGGGAGAAGG
101 GCAATGTTTT TCTCACCCAC TCCATTCCTA TGCATATTGC TGCGATTACG
151 ATCTTAGTGA TTGTAGCTCT TGCTGGAATC GCTATTATCT GTTTGGGTTG
201 CTATAGCCAA AGCATTCTGT TGATTGCCGT TGGCATTGTT CTTACTATTT
251 TGACTCTTCT CTGCCTACAA GCCTTGGTAG GATTTATTAA ATTCATCCGG
301 CAGCTCCCTC AGCAGCTCCA TACGACAGTA CAATTTATCA GGGAGAAGAT
351 TCGACCTGAA TCCTCTCTAC AGCTTGTAAC CAATGCACAG AGAAAAACCA
401 CTCAAGATAC GCTAAAGTTA TACGAAGAAC TCTGCGACCT CTCACAAAAA
451 GAGTTCAAAC TGCAATCAAC TCTTTATCAA AAACGTTTTG AGCTTTCTCA
501 CAAGAATGAA AAGACAAATC AAAACTAG

The PSORT algorithm predicts inner membrane (0.6668).

The protein was expressed in E. coli and purified as a his-tagged product (FIG. 147A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 147B) and for FACS analysis.

These experiments show that cp6722 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 148

The following C. pneumoniae protein (PID 4377253) was expressed <SEQ ID 295; cp7253>:

1 MSELAPCSTG LQMVPHTQVH HALDTRRVIL TIAACLSLIA GIVLVGLGAA
51 AILPSLFGVI GGMILILFSS IALIYLYKKT REVDQIALEP LPEMISKDQS
101 IIDFVKTRDY ASLEKKATFA YTHTHYYDGS MVFYREIPRF MLGSYLALRK
151 DMDRQALF*

The cp7253 nucleotide sequence <SEQ ID 296> is:

1 ATGAGCGAGC TCGCCCCCTG CTCGACAGGA TTGCAGATGG TCCCCCATAC
51 GCAGGTCCAT CATGCCCTTG ATACGCGGAG AGTCATTCTA ACGATAGCCG
101 CCTGTCTGTC TTTAATTGCA GGAATCGTGT TGGTTGGCTT AGGTGCTGCA
151 GCAATCCTGC CCTCGCTTTT TGGAGTCATT GGAGGAATGA TTCTTATTCT
201 GTTTTCTTCG ATCGCCCTCA TTTATTTATA CAAGAAGACA AGGGAGGTGG
251 ATCAGATTGC TCTGGAGCCT CTTCCTGAGA TGATTTCTAA AGATCAAAGC
301 ATTATAGATT TTGTAAAGAC ACGAGACTAT GCATCTTTAG AAAAGAAAGC
351 GACCTTTGCT TATACTCATA CTCATTATTA CGATGGAAGC ATGGTCTTCT
401 ATAGGGAGAT CCCTAGATTT ATGTTAGGCT CTTATCTCGC GCTTCGCAAA
451 GACATGGACC GCCAAGCTCT TTTTTGA

The PSORT algorithm predicts inner membrane (0.5394).

The protein was expressed in E. coli and purified as a his-tagged product (FIG. 148A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 148B) and for FACS analysis.

These experiments show that cp7253 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 149

The following C. pneumoniae protein (PID 4376264) was expressed <SEQ ID 297; cp6264>:

1 VISGLLFLLV RREVPTVRSE EIPRGVSVTP SEEPALEKAQ KEPETKKILD
51 RLPKELDQLD TYIQEVFACL ERLKDPKYED RGLLTEAKEK LRVFDVVEKD
101 MMSEFLDIQR VLNEEAYYVE HCQDPLENIA YEIFSSQELR DYYCAGVCGY
151 LPSGDARADR LKRSVKEVMD RFMRVIWKSW EASVMLDESY GVARELFKKA
201 VGVLEESVYK ILFKSYRDAF YECEKAKIQR DGRFKWL*

The cp6264 nucleotide sequence <SEQ ID 298> is:

1 GTGATTTCGG GACTTCTATT CCTTCTAGTA AGACGAGAGG TTCCGACAGT
51 ACGTTCAGAG GAAATTCCCA GAGGGGTTTC TGTGACCCCT TCTGAAGAGC
101 CTGCTCTAGA GAAGGCTCAA AAAGAACCGG AGACAAAGAA AATTTTAGAT
151 CGGTTGCCGA AGGAATTGGA TCAGTTAGAT ACGTATATTC AGGAAGTGTT
201 TGCATGTTTA GAGAGGCTGA AGGATCCTAA GTACGAAGAT CGAGGTCTTT
251 TAACAGAGGC GAAGGAGAAA CTTCGAGTTT TTGACGTTGT TGAGAAAGAT
301 ATGATGTCAG AGTTTTTAGA CATACAACGA GTGTTGAATG AGGAAGCATA
351 TTATGTAGAA CATTGTCAAG ATCCCCTAGA GAATATAGCC TACGAGATTT
401 TCTCTTCCCA AGAGCTTCGT GATTACTACT GTGCAGGGGT GTGTGGGTAT
451 TTGCCTTCTG GGGATGCTCG AGCGGATCGA TTAAAGAGAT CAGTTAAGGA
501 GGTAATGGAT CGCTTTATGA GGGTGACCTG GAAATCTTGG GAGGCATCAG
551 TCATGTTGGA TCATAGCTAT GGGGTAGCGC GAGAGTTATT CAAGAAGGCA
601 GTAGGAGTAC TAGAGGAGAG TGTCTATAAA ATTCTGTTTA AGAGCTATAG
651 AGATGCGTTT TATGAATGTG AGAAGGCAAA GATCCAGAGG GATGGGCGTT
701 TCAAATGGTT ATAG

The PSORT algorithm predicts cytoplasm (0.2817).

The protein was expressed in E. coli and purified as a his-tagged product (FIG. 149A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 149B) and for FACS analysis.

These experiments show that cp6264 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 150

The following C. pneumoniae protein (PID 4376266) was expressed <SEQ ID 299; cp6266>:

1 MLLLISGALF LTLGIPGLSA AISFGLGIGL SALGGVLMIS GLLCLLVKRE
51 IPTVRPEEIP EGVSLAPSEE PALQAAQKTL AQLPKELDQL DTDIQEVFAC
101 LRKLKDSKYE SRSFINDAKK ELRVFDFVVE DTLSEIFELR QIVAQEGWDL
151 NFLINGGRSL MMTAESESLD LFHVSKRLGY LPSGDVRGEG LKKSAKEIVA
201 RLMSLHCEIH KVAVAFDRNS YAMAEKAFAK ALGALEESVY RSLTQSYRDK
251 FLESERAKIP WNGHITWLRD DAKSGCAEKK LGMPRNVGRN LGKQSFG*

The cp6266 nucleotide sequence <SEQ ID 300> is:

1 ATGCTCTTAC TGATTTCAGG AGCTCTCTTT CTGACGTTAG GGATTCCAGG
51 ATTGAGTGCA GCAATTTCTT TTGGATTAGG CATCGGTCTC TCCGCATTAG
101 GAGGAGTGCT GATGATTTCG GGACTACTAT GTCTTTTAGT AAAACGAGAG
151 ATTCCGACAG TACGACCAGA AGAAATTCCT GAAGGGGTTT CGCTGGCTCC
201 TTCTGAGGAG CCAGCTCTAC AGGCAGCTCA GAAGACTTTA GCTCAGCTGC
251 CTAAGGAATT GGATCAGTTA GATACAGATA TTCAGGAAGT GTTCGCATGT
301 TTAAGAAAGC TGAAAGATTC TAAGTATGAA AGTCGAAGTT TTTTAAACGA
351 TGCTAAGAAG GAGCTTCGAG TTTTTGACTT TGTGGTTGAG GATACCCTCT
401 CGGAGATTTT CGAGTTGCGG CAGATTGTGG CTCAAGAGGG ATGGGATTTA
451 AACTTTTTGA TCAATGGGGG ACGAAGCCTC ATGATGACTG CAGAATCTGA
501 ATCGCTTGAT TTGTTTCATG TATCGAAGCG GCTAGGGTAT TTACCTTCTG
551 GGGATGTTCG AGGGGAGGGG TTAAAGAAAT CTGCGAAGGA GATAGTCGCT
601 CGTTTGATGA GCTTGCATTG CGAGATTCAC AAGGTGGCGG TAGCGTTTGA
651 TAGGAATTCC TATGCGATGG CAGAAAAGGC GTTTGCGAAA GCGTTGGGAG
701 CTTTAGAAGA GAGTGTGTAT CGGAGTCTGA CGCAGAGTTA TAGAGATAAA
751 TTTTTGGAGA GCGAGAGGGC GAAGATCCCA TGGAATGGGC ATATAACCTG
801 GTTAAGAGAT GATGCGAAGA GTGGGTGTGC TGAAAAGAAG CTCGGGATGC
851 CGAGGAACGT TGGAAGAAAT TTAGGAAAGC AGTCTTTTGG GTAG

The PSORT algorithm predicts inner membrane (0.3590).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 150A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 150) and for FACS analysis.

These experiments show that cp6266 is a surface-exposed and immunoaccessible protein and that they it is a useful immunogen. These properties are not evident from the sequence alone.

Example 151

The following C. pneumoniae protein (PID 4376895) was expressed <SEQ ID 301; cp6895>:

1 MKIKKSFQYS LCQAKRFQNM LPNHFDPCLQ PVNLQLKQDR LAYGELIILL
51 SKYQQKTFSS LLKEETCSLN RAKQHLLYKI LRDFNTMQHL RSLGLNGWGE
101 IPMSPCL*

The cp6895 nucleotide sequence <SEQ ID 302> is:

1 ATGAAGATTA AAAAATCTTT TCAATACAGT TTATGCCAAG CAAAGAGATT
51 TCAGAACATG CTGCCAAACC ACTTTGATCC ATGTTTGCAG CCAGTGAATT
101 TACAACTCAA ACAAGACAGA TTGGCATACG GGGAGCTCAT CATATTGCTA
151 TCTAAATATC AACAAAAGAC CTTTTCCTCT TTGTTGAAGG AAGAAACATG
201 TTCTCTTAAT CGTGCGAAGC AGCACTTATT GTATAAGATT TTGAGAGATT
251 TTAATACTAT GCAGCATCTA AGGTCCCTCG GATTAAATGG TTGGGGAGAG
301 ATCCCTATGA GTCCTTGCCT CTAA

The PSORT algorithm predicts cytoplasm (0.3264).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 151A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 151B) and for FACS analysis.

These experiments show that cp6895 is a surface-exposed and immunoaccessible protein and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 152 and Example 153

The following C. pneumoniae protein (PID 4376282) was expressed <SEQ ID 303; cp6282>:

1 MSLLNLPSSQ DSASEDSTSQ SQIFDPIRNR ELVSTPEEKV RQRLLSFLMH
51 KLNYPKKLII IEKELKTLFP LLMRKGTLIP KRRPDILIIT PPTYTDAQGN
101 THNLGDPKPL LLTECKALAV NQNALKQLLS YNYSIGATCI AMAGKHSQVS
151 ALFNPKTQTL DFYPGLPEYS QLLNYFISLN L*

The cp6282 nucleotide sequence <SEQ ID 304> is:

1 ATGTCCTTAT TGAACCTTCC CTCAAGCCAG GATTCTGCAT CTGAGGACTC
51 CACATCGCAA TCTCAAATCT TCGATCCCAT TAGAAATCGG GAGTTAGTTT
101 CTACTCCCGA AGAAAAAGTC CGCCAAAGGT TGCTCTCCTT CCTAATGCAT
151 AAGCTGAACT ACCCTAAGAA ACTCATCATC ATAGAAAAAG AACTCAAAAC
201 TCTTTTTCCT CTGCTTATGC GTAAAGGAAC CCTAATCCCA AAACGCCGCC
251 CAGATATTCT CATCATCACT CCCCCCACAT ACACAGACGC ACAGGGAAAC
301 ACTCACAACC TAGGCGACCC AAAACCCCTG CTACTTATCG AATGTAAGGC
351 CTTAGCCGTA AACCAAAATG CACTCAAACA ACTCCTTAGC TATAACTACT
401 CTATCGGAGC CACCTGCATT GCTATGGCAG GGAAACACTC TCAAGTGTCA
451 GCTCTCTTCA ATCCAAAAAC ACAAACTCTT GATTTTTATC CTGGCCTCCC
501 AGAGTATTCC CAACTCCTAA ACTACTTTAT TTCTTTAAAC TTATAG

The PSORT algorithm predicts cytoplasm (0.362).

The following C. pneumoniae protein (PID 4377373) was also expressed <SEQ ID 305; cp7373>:

1 MSTTTVKHFI HTASRWEPVL KEIVASNYWH AQWINTLSFL ENSGAKKISA
51 SEHPTEVKEE VLKHAAEEFR HGHYLKTQIS RISETSLPDY TSKNLLGGLL
101 TKYYLHLIDL RTCRVLENEY SLSGQTLKTA AYILVTYAIE LRASFLYPLY
151 HDILKEAQSK ITVKSIILEE QGHLQEMERE LKDLPHGEEL LGYACQFEGE
201 LCLQFVERLE QMIFDPSSTF TKF*

The cp7373 nucleotide sequence <SEQ ID 306> is:

1 ATGTCTACAA CCACAGTAAA ACACTTTATC CACACAGCCT CTCGTTGGGA
51 GCCCGTTCTC AAAGAGATCG TAGCTTCCAA CTATTGGCAT GCACAATGGA
101 TAAATACCCT GTCCTTTTTA GAAAATAGTG GAGCAAAAAA AATCTCCGCA
151 AGTGAACATC CTACGGAGGT AAAGGAAGAA GTTTTAAAAC ATGCTGCTGA
201 AGAATTTCGT CATGGTCACT ATCTAAAAAC TCAGATTTCT AGAATCTCAG
251 AGACTTCTCT CCCTGACTAT ACATCTAAAA ATCTTCTGGG AGGCTTACTT
301 ACAAAATATT ACCTCCATCT TCTAGATTTA AGGACGTGCC GAGTACTGGA
351 AAATGAATAC TCCCTATCGG GACAAACGTT AAAAACTGCA GCGTATATTT
401 TAGTTACCTA CGCAATCGAA CTTCGTGCTT CTGAACTTTA TCCTCTGTAT
451 CACGATATTC TGAAAGAAGC TCAAAGTAAA ATAACGGTAA AATCCATTAT
501 CTTAGAAGAG CAAGGCCATC TGCAAGAGAT GGAACGTGAA CTTAAAGATC
551 TCCCCCACGG GGAGGAACTC TTAGGCTATG CTTGCCAATT CGAAGGGGAG
601 CTTTGCTTGC AGTTTGTAGA GAGATTAGAA CAAATGATCT TCGATCCTTC
651 CTCGACTTTT ACAAAGTTCT AG

The PSORT algorithm predicts cytoplasm (0.1069).

The proteins were expressed in E. coli and purified as his-tag products (FIG. 152A; 6282=lanes 8 & 9; 7373=lanes 2-4). The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 152B & 153) and for FACS analysis.

These experiments show that cp6282 & cp7373 are surface-exposed and immunoaccessible proteins and that they are useful immunogens. These properties are not evident from the sequence alone.

Example 154, Example 155, Example 156, Example 157 and Example 158

The following C. pneumoniae protein (PID 4376412) was expressed <SEQ ID 307; cp6412>:

1 MSSSEVVFQT VHGLGFGGLS SKSVVPFKKS LSDAPRVVCS ILVLTLGLGA
51 LVCGIAITCW CVPGVILMGG ICAIVLGAIS LALSLFWLWG LFSNCCGSKR
101 VLPGEGLLRD KLLDGGFSRA APSGMGLPGD GSPRASTPSC LEELQAEIQA
151 VTQAIDQMSD D*

The cp6412 nucleotide sequence <SEQ ID 308> is:

1 ATGAGCAGTT CGGAAGTTGT TTTCCAGACA GTTCATGGCC TTGGCTTTGG
51 TGGATTGTCT TCAAAAAGTG TTGTCCCTTT TAAGAAAAGT CTTTCGGATG
101 CGCCCCGTGT TGTGTGCTCG ATTTTAGTTT TGACTCTGGG GTTGGGAGCG
151 CTTGTTTGTG GTATTGCCAT TACTTGTTGG TGTGTCCCGG GAGTTATTTT
201 AATGGGGGGA ATTTGCGCTA TAGTTTTAGG TGCAATTTCT TTAGCTTTAA
251 GTCTATTTTG GTTGTGGGGT TTATTTTCTA ATTGTTGTGG TTCTAAGAGA
301 GTTTTACCGG GTGAGGGATT GCTACGGGAT AAGCTTTTAG ATGGTGGATT
351 TTCAAGAGCG GCACCTTCAG GAATGGGACT TGGCCCTGAT GGATCTCCAA
401 GAGCGTCAAC GCCATCTTGC CTAGAGGAAC TTCAAGCAGA GATACAGGCA
451 GTTACTCAAG CTATCGATCA GATGTCAGAT GATTGA

The PSORT algorithm predicts inner membrane (0.4864).

The following C. pneumoniae protein (PID 4376431) was also expressed <SEQ ID 309; cp6431>:

1 LRAGGSLVTT YPKEGQRLRS PEQLRVLDDL VQSYPNHLHA IELDCGAIPQ
51 DLIGATYIIT FADFSTYILS LRSYQANSPS DDTWGIWFGS IDDPVQAVIS
101 FLKDHGFALP STLAQDPLLC TNK*

The cp6431 nucleotide sequence <SEQ ID 310> is:

1 TTGCGAGCAG GAGGTAGTCT TGTTACAACA TACCCTAAGG AAGGTCAGAG
51 ATTGCGCTCC CCAGAACAGT TAAGAGTTCT GGATGATTTA GTGCAAAGCT
101 ATCCAAATCA CCTACATGCG ATTGAACTTG ATTGTGGTGC AATCCCTCAA
151 GATTTGATCG GAGCCACCTA TATCATCACG TTCGCCGATT TTTCCACCTA
201 TATTCTCTCT TTAAGAAGCT ACCAAGCCAA TTCTCCCTCC GATGATACAT
251 GGGGGATTTG GTTTGGATCT ATTGACGATC CTGTTCAAGC AGTCATATCA
301 TTTTTAAAAG ATCATGGATT TGCTCTTCCC TCGACCTTAG CTCAAGATCC
351 TTTGCTTTGT ACTAACAAGT AA

The PSORT algorithm predicts cytoplasm (0.2115).

The following C. pneumoniae protein (PID 4376443) was also expressed <SEQ ID 311; cp6443>:

1 MIMTTISNSP SPALNPELSL IPPPTLVSSG TQTSLAYTIP AQGRRSTLRI
51 ILDIFIIILG LATIISTFIV IFFLNGLNLL STPSIISSSC LIIVGLLFLI
101 NGLYFMISSL DQGLVGLLQK ELSQAEEREE EYIQEIEALR GAPRAESPTE
151 SPSTWL*

The cp6443 nucleotide sequence <SEQ ID 312> is:

1 ATGATTATGA CTACTATATC TAACTCACCC TCCCCTGCAT TGAATCCCGA
51 ACTTTCCCTT ATTCCTCCAC CAACACTTGT ATCTTCAGGT ACGCAAACAT
101 CTCTAGCTTA TACGATCCCC GCACAAGGAC GAAGATCCAC CCTACGTATT
151 ATATTAGATA TATTCATTAT CATTCTTGGT TTAGCTACGA TCATTTCTAC
201 CTTTATTGTT ATTTTCTTTT TAAATGGGCT GAACTTGCTC TCGACCCCAT
251 CTATTATCTC TTCGTCATGT TTAATCATTG TTGGATTGCT TTTTTTGATT
301 ATGGGGTTAT ATTTCATGAT CTCGAGTTTG GATCAGGGGC TTGTAGGCCT
351 TCTGCAAAAG GAACTCTCTC AAGCCGAAGA AAGAGAAGAA GAGTATATCC
401 AGGAAATCGA AGCTTTAAGA GGAGCTCCTA GAGCAGAATC TCCCACAGAG
451 TCTCCTAGTA CCTGGTTATG A

The PSORT algorithm predicts inner membrane (0.5585).

The following C. pneumoniae protein (PID 4376496) was also expressed <SEQ ID 313; cp6496>:

1 MLIGRYSSDD QFTEATKNTP TIIKLGFVRD NLEGLTNPIS EIVSETSSSI
51 KDSVLRSIPI LGSILGCARL YSTLSTNDPL DETQEKIWHT IFGALETLGL
101 GILILLFKII FVILHCIFHL VIGFCK*

The cp6496 nucleotide sequence <SEQ ID 314> is:

1 ATGCTAATAG GCAGATACAG TAGTGATGAC CAATTCACTG AAGCAACAAA
51 AAACACCCCA ACCATAATTA AGCTAGGTTT TGTTAGAGAT AATCTCGAGG
101 GATTAACGAA CCCTATCTCT GAAATCGTCT CGGAAACCTC CTCTTCTATT
151 AAAGATTCCG TTCTTCGCTC TCTTCCTATT TTAGGGTCCA TTTTAGGATG
201 CGCCCGACTT TACAGCACAC TCTCTACAAA TGATCCTCTT GACGAAACTC
251 AAGAAAAGAT TTGGCACACT ATATTTGGAG CCTTAGAAAC CTTAGGCTTA
301 GGGATTCTCA TCCTCTTATT TAAAATTATT TTTGTTATAT TACACTGCAT
351 ATTTCATCTA GTTATTGGGT TCTGCAAATA A

The PSORT algorithm predicts inner membrane (0.5989).

The following C. pneumoniae protein (PID 4376654) was also expressed <SEQ ID 315; cp6654>:

1 MKTKMNSRKK AGQWAIFNSP TPGVSSTLVL AWTPWGYYDK DVQDILERKD
51 PMSSSLSEKD SKEFLKNLFV DLLENGFTSV HIHAEEAFTP LDHTGKPHFK
101 RDNVYLPGKL LGALNEAAVQ ANVSADTQFT LFLTQDECNP FHDKKRG*

The cp6654 nucleotide sequence <SEQ ID 316> is:

1 ATGAAAACTA AAATGAACTC TAGAAAAAAA GCAGGTCAAT GGGCAATTTT
51 CAATTCTCCA ACTCCTGGTG TCAGTTCAAC TTTAGTTTTA GCATGGACTC
101 CTTGGGGTTA TTACGACAAG GATGTACAAG ATATCTTAGA AAGAAAAGAT
151 CCGATGAGCT CTTCGCTTTC TGAAAAAGAC TCAAAGGAGT TCTTGAAAAA
201 TCTGTTTGTA GATCTCTTAG AAAATGGCTT CACATCAGTA CATATTCACG
251 CAGAAGAAGC TTTCACTCCT CTTGATCATA CCGGGAAACC TCACTTTAAA
301 AGAGACAATG TGTACTTACC CGGAAAGTTG TTAGGCGCCT TGAATGAGGC
351 TGCGGTACAA GCCAATGTAA GTGCGGATAC TCAATTTACA TTGTTCCTTA
401 CTCAAGATGA GTGCAATCCT TTTCATGATA AGAAAAGAGG TTAA

The PSORT algorithm predicts cytoplasm (0.0730).

The proteins were expressed in E. coli and purified as his-tag products (FIG. 154A; 6412=lanes 2-3; 6431=lanes 11-12; 6443=lanes 5-6; 6496=lanes 8-9; 6654=lane 10; markers in lanes 1, 4, 7). The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 154B, 155, 156, 157 & 158) and for FACS analysis.

These experiments show that cp6412, cp6431, cp6443, cp6496 & cp6654 are surface-exposed and immunoaccessible proteins and that they are useful immunogens. These properties are not evident from their sequences alone.

Example 159 and Example 160

The following C. pneumoniae protein (PID 4376477) was expressed <SEQ ID 317; cp6477>:

1 LLKFFLVCEE LCILTVATHR ALLETPLALS FFKELKTKYV YRAKDILQLH
51 NYKGFTIINT SPLCS*

The cp6477 nucleotide sequence <SEQ ID 318> is:

1 TTGCTAAAGT TCTTTCTAGT ATGTGAAGAG TTATGTATAC TTACTGTTGC
51 TACACATAGA GCTCTCTTAG AAACTCCTTT AGCTCTATCA TTTTTTAAAG
101 AACTTAAGAC AAAATATGTC TACAGGGCGA AAGACATACT ACAACTACAT
151 AACTATAAAG GATTTACTAT CCTTAATACA TCACCGTTAT GTTCTTAA

The PSORT algorithm predicts inner membrane (0.128).

The following C. pneumoniae protein (PID 4376435) was also expressed <SEQ ID 319; cp6435>:

1 LWSHFPRGFF MLPFCPTILL AKPFLNSENY GLERLAATVD SYFDLGQSQI
51 VFLSKQDQGI TVEELSAKDR KFKPGSMNCT LYTEDPILPA HNSFSNCSDI
101 QMRTPISPIH *

The cp6435 nucleotide sequence <SEQ ID 320> is:

1 TTGTGGTCGC ATTTCCCAAG AGGATTTTTT ATGCTCCCTT TTTGCCCTAC
51 CATCCTTCTT GCTAAACCTT TTTTAAATAG CGAGAATTAC GGCTTAGAAC
101 GTTTAGCTGC AACCGTAGAT TCTTATTTTG ATCTGGGACA GTCTCAAATA
151 GTCTTCCTAA GCAAACAGGA TCAAGGAATC ACTGTGGAAG AATTGAGTGC
201 TAAAGATAGG AAATTCAAGC CAGGCTCTAT GAACTGTACA CTGTACACTG
251 AAGATCCTAT CTTACCTGCT CATAATTCCT TTAGTAATTG CTCTGATATT
301 CAAATGCGTA CTCCGATTAG CCCTATACAT TAA

The PSORT algorithm predicts periplasmic space (0.4044).

The proteins were expressed in E. coli and purified as his-tag products (FIG. 159A; 6435=lanes 2-4; 6477=lanes 5-7). The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 159B & 160) and for FACS analysis.

These experiments show that cp6477 & cp6435 are surface-exposed and immunoaccessible proteins and that they are useful immunogens. These properties are not evident from the sequences alone.

Example 161 and Example 162 and Example 163

The following C. pneumoniae protein (PID 4376441) was expressed <SEQ ID 321; cp6441>:

1 VEAGANVLVI DTAHAHSKGV FQTVLEIKSQ FPQISLVVGN LVTAFAAVSL
51 AEIGVDAVKV GIGPGSICTT RIVSGVGYPQ ITAITNVAKA LKNSAVTVIA
101 DGRIRYSGDV VKALAAGADC VMLGSLLAGT DEAPGDIVSI DEFLFKRYRG
151 MGSLGAMKQG SADRYFQTQG QKKLVPGGVE GLVAYKGSVH DVLYQILGGI
201 RSGMGYVGAE TLKDIKTKAS FVRITESGRA ESHIHNIYKV QPTLNY

The cp6441 nucleotide sequence <SEQ ID 322> is:

1 GTGGAAGCTG GAGCAAATGT TCTAGTCATT GACACAGCTC ATGCACACTC
51 TAAAGGAGTA TTCCAAACAG TTTTAGAAAT AAAATCCCAG TTCCCACAAA
101 TTTCTTTAGT TGTAGGGAAT CTTGTTACAG CTGAAGCCGC AGTTTCCTTA
151 GCTGAGATTG GAGTTGACGC TGTAAAGGTA GGTATTGGCC CAGGATCTAT
201 CTGTACAACT AGAATCGTTT CAGGGGTCGG TTATCCACAA ATTACTGCCA
251 TTACAAACGT AGCAAAAGCT CTTAAAAACT CTGCCGTGAC TGTAATTGCT
301 GATGGGAGAA TCCGCTATTC TGGAGATGTG GTAAAAGCAT TAGCAGCAGG
351 AGCAGACTGT GTCATGCTAG GAAGTTTGCT TGCAGGGACT GATGAAGCTC
401 CTGGGGATAT CGTTTCTATC GATGAGAAGC TTTTTAAAAG GTACCGCGGC
451 ATGGGATCTT TAGGCGCTAT GAAACAAGGA AGTGCTGACC GGTATTTTCA
501 AACACAGGGA CAGAAAAAGC TGGTTCCTGG GGGAGTTGAA GGACTAGTCG
551 CTTATAAAGG CTCTGTCCAC GATGTCCTCT ATCAAATTTT AGGAGGAATA
601 CGCTCAGGTA TGGGGTATGT TGGAGCTGAA ACTCTCAAAG ATTTAAAAAC
651 TAAGGCTTCC TTTGTTCGAA TTACTGAATC TGGAAGAGCT GAAAGTCATA
701 TTCATAATAT TTACAAAGTT CAACCAACCT TAAATTATTA A

The PSORT algorithm predicts bacterial inner membrane (0.132).

The following C. pneumoniae protein (PID 4376748) was also expressed <SEQ ID 323; cp6748>:

1 LFSFGTAINL FRIFAPLRNR VTTEYSRARQ PDLHRIAIVY IGVLDSESSK
51 ILERLISYMS CIMSESQMYL RFFMGKNVNQ SAVLSKLEVE NLHIRCGFFS
101 EDAVPESEPF DLSIYVHTDR SCPLPTKKRS SSWELQTVEL PESIYPQSFF
151 LLMRPRMLS*

The cp6748 nucleotide sequence <SEQ ID 324> is:

1 TTGTTCTCTG AGGGGACAGC TCTAAATTTA TTTCGTATAT TTGCTCCACT
51 ACGCAACCGT GTGACTACAG AATACAGTCG TGCTAGGCAA CCCGACCTAC
101 ATAGAATTGC CATCGTCTAT ATAGGAGTTC TCGATTCAGA AAGTTCCAAG
151 ATCCTAGAGC GGCTAATCTC TTATATGAGT TGTATCTATT CTGAATCGCA
201 AATGTATTTA AGATTCTTTA TGGGCAAGAA TGTAAATCAA AGTGCTGTAC
251 TCTCAAAATT ACATGTAGAA AATCTGCACA TCCGTTGTGG GTTTTTCAGC
301 GAGGATGCTG TTCCAGAGAG TGAGCCCTTC GATCTCTCCA TCTACGTGCA
351 CACAGATCGT AGCTGTCCTC TCCCTACGAA AAAACGGAGC AGCTCCTGGG
401 AACTCCAAAC TGTAGAACTC CCAGAGTCAA TATATCCACA GTCGGAATTC
451 CTATTGATGA GACCTCGAAT GCTTTCGTAG

The PSORT algorithm predicts cytoplasm (0.170).

The following C. pneumoniae protein (PID 4376881) was also expressed <SEQ ID 325; cp6881>:

1 MRPHRKHVSS KSLALKQSAS THVEITTKAF RLSMPLKQLI LEKSDHLPPM
51 ETIRVVLTSH KDKLGTEVHV VASHGKEILQ TKVENANPYT AVINAFKKIR
101 TMANKHSNKR KDRTKHDLGL AAKEERIAIQ EEQEDRLSNE WLPVFGLDAW
151 DSLKTLGYVP ASAKKKISKK KMSIRMLSQD EAIRQLESAA ENFLIFLNEQ
201 EHKIQCIYKK HDGNYVLIEP SLKPGFCI*

The cp6881 nucleotide sequence <SEQ ID 326> is:

1 ATGAGACCTC ATCGTAAACA CGTATCATCT AAAAGCTTAG CTTTAAAGCA
51 ATCTGCATCA ACTCATGTAG AGATCACAAC AAAAGCCTTT CGTCTCTCTA
101 TGCCTCTAAA ACAGCTGATC CTAGAGAAAA GCGACCACCT CCCCCCTATG
151 GAAACAATCC GTGTGGTGCT AACCTCTCAT AAAGATAAGC TAGGCACCGA
201 GGTGCATGTT GTAGCTTCTC ATGGCAAAGA AATCCTTCAA ACTAACCTTC
251 ATAACGCAAA CCCATACACT GCAGTGATCA ATGCTTTTAA GAAAATCCGC
301 ACCATGGCAA ATAAGCACTC CAATAAACGT AAAGACAGGA CAAAACATGA
351 TCTAGGTCTT GCAGCAAAAG AAGAACGTAT CGCAATACAG GAAGAACAAG
401 AAGATCGCCT TAGCAACGAG TGGCTTCCTG TCGAAGGCCT CGATGCCTGG
451 GATTCTCTAA AAACTCTTGG GTATGTTCCC GCATCAGCGA AAAAGAAGAT
501 CTCCAAGAAA AAGATGAGCA TTCGTATGCT ATCTCAAGAC GAGGCTATCC
551 GCCAGCTAGA GTCTGCCGCA GAAAACTTCC TGATCTTCTT GAACGAGCAA
601 GAGCATAAAA TCCAATGCAT TTATAAAAAA CATGACGGCA ACTATGTCCT
651 TATTGAACCT TCCCTCAAGC CAGGATTCTG CATCTGA

The PSORT algorithm predicts cytoplasm (0.249).

The proteins were expressed in E. coli and purified as his-tag products (FIG. 161A; 6441=lanes 7-9; 6748=lanes 2-3; 6881=lanes 4-6). The recombinant protein was used to immunise mice, whose sera were used in Western blots (FIGS. 161B, 162 & 163) and for FACS analysis.

These experiments show that cp6441, cp6748 & cp6881 are surface-exposed and immunoaccessible proteins and that they are useful immunogens. These properties are not evident from the sequence alone.

Example 164 and Example 165 Example 166

The following C. pneumoniae protein (PID 4376444) was expressed <SEQ ID 327; cp6444>:

1 MEQPNCVIQD TTTVLYALNS FDPRLSDDTH RLGKQSPLEA ENALGEFIEG
51 LDTNSFPLEE VAIPILPGYH PKFYLSFIDR DDQGVHYEVL DGVFLKTVAA
101 CIIENSFLTD SMSPELLSEV KEALKR*

The cp6444 nucleotide sequence <SEQ ID 328> is:

1 ATGGAGCAAC CCAATTGTGT GATTCAGGAT ACTACAACTG TTTTGTATGC
51 CTTAAATAGC TTTGATCCTA GACTTAGTGA TGACACTCAC AGACTTGGGA
101 AGCAATCACC TCTTGAAGCA GAAAATGCTC TTGGAGAATT TATTGAAGGT
151 TTGGATACAA ATAGCTTTCC TTTAGAGGAA GTTGCCATTC CCATCCTGCC
201 AGGTTATCAC CCTAAGTTTT ATTTATCTTT CATAGATAGG GACGATCAAG
251 GTGTCCACTA TGAAGTTTTA GATGGCGTAT TTTTAAAGAC AGTCGCTGCT
301 TGTATTATAG AGAACTCCTT CTTAACTGAT TCTATGAGCC CGGAGCTTCT
351 CAGCGAAGTT AAGGAAGCTC TGAAACGATG A

The PSORT algorithm predicts cytoplasm (0.2031).

The following C. pneumoniae protein (PID 4376413) was also expressed <SEQ ID 329; cp6413>:

1 MAVQSIKEAV TSAATSVGCV NCSREAIPAF NTEERATSIA RSVIAAIIAV
51 VAISLLGLGL VVLAGCCPLG MAAGAITMLL GVALLAWAIL ITLRLLNIPK
101 AEIPSPGNNG EPNERNSATP PLEGGVAGEA GRGGGSPLTQ LDLNSGAGS*

The cp6413 nucleotide sequence <SEQ ID 330> is:

1 ATGGCTGTTC AATCTATAAA AGAAGCCGTA ACATCAGCCG CAACATCAGT
51 AGGATGTGTA AACTGTTCTA GAGAGGCTAT ACCAGCATTT AATACAGAGG
101 AGAGAGCAAC GAGTATTGCT AGATCTGTTA TAGCAGCTAT CATTGCTGTT
151 GTAGCTATCT CCTTACTCGG ACTAGGTCTT GTAGTTCTTG CTGGTTGCTG
201 TCCTTTAGGA ATGGCTGCGG GTGCTATAAC AATGCTGCTG GGTGTAGCAT
251 TATTAGCTTG GGCAATACTG ATTACTTTGA GACTGCTTAA TATACCTAAG
301 GCTGAAATAC CGAGTCCAGG GAACAACGGT GAGCCTAATG AAAGAAATTC
351 AGCAACTCCT CCTCTAGAGG GTGGTGTTGC AGGAGAAGCC GGTCGCGGCG
401 GGGGGTCACC TTTAACCCAA CTTGATCTCA ATTCAGGGGC GGGAAGTTAG

The PSORT algorithm predicts inner membrane (0.6180).

The following C. pneumoniae protein (PID 4377391) was also expressed <SEQ ID 331; cp7391>:

1 MMLRVIELPL IPIKQALEKA FVQYNSYKAK LTKVEPCFRE SPAYITSEER
51 LQSLDQTLER AYKEYQKRFQ EPSRLESEVS GCREHLREQV KQFETQGLDL
101 IKEFLIFVSD VLFRKMVSCL VSTVHVPFME FYYEYFELHR LRLRAQWMAN
151 AEIYSKVRKA FPEMLKETLE KAKAPREEEY WLLCEERKSK EKRLILNKIE
201 AAQQRVKDLE PPPIKETGKQ KRKKEYSFFI RLKS*

The cp7391 nucleotide sequence <SEQ ID 332> is:

1 ATGATGCTTC GTGTCATAGA GCTTCCACTA CTTCCTATAA AGCAAGCGTT
51 GGAGAAGGCT TTTGTACAAT ATAATAGCTA CAAAGCGAAG TTAACCAAGG
101 TAGAACCTTG CTTTAGAGAG AGCCCTGCCT ATATAACTAG CGAAGAGCGA
151 CTCCAGAGTT TGGATCAGAC TTTAGAACGT GCGTACAAAG AGTACCAGAA
201 GAGATTCCAG GAGCCTTCAC GTTTGGAATC GGAAGTAAGT GGATGTAGAG
251 AGCATCTTAG AGAGCAGGTA AAACAATTTG AAACTCAAGG ACTAGACTTG
301 ATCAAAGAAG AGCTTATTTT TGTTAGTGAT GTGTTATTCC GAAAAATGGT
351 CAGTTGTCTA GTGTCGACAG TGCATGTTCC CTTTATGGAG TTTTATTATG
401 AGTATTTTGA GTTGCATAGA TTGAGGTTGC GGGCCCAATG GATGGCGAAT
451 GCCGAGATTT ATAGCAAAGT TAGAAAAGCA TTCCCAGAGA TGTTGAAGGA
501 GACCTTAGAA AAAGCTAAGG CTCCCAGAGA AGAAGAGTAT TGGTTACTTT
551 GCGAGGAGAG AAAGAGTAAG GAGAAGCGTT TGATTCTCAA CAAGATAGAG
601 GCAGCTCAGC AGCGGGTAAA AGATTTAGAA CCTCCTCCTA TTAAAGAGAC
651 AGGGAAACAG AAACGGAAGA AAGAATATTC GTTTTTCATT CGATTAAAAT
701 CGTGA

The PSORT algorithm predicts inner membrane (0.1489).

The proteins were expressed in E. coli and purified as his-tag and GST-fusion products (FIG. 164A; 6444=lanes 11-12; 7391=lanes 2-3; 6413=lanes 4-6). The recombinant protein was used to immunise mice, whose sera were used in Western blots (FIGS. 164B, 165 & 166) and for FACS analysis.

These experiments show that cp6444, cp6413 & cp7391 are surface-exposed and immunoaccessible proteins and that they are useful immunogens. These properties are not evident from the sequence alone.

Example 167, Example 168, Example 169 and Example 170

The following C. pneumoniae protein (PID 4376463) was expressed <SEQ ID 333; cp6463>:

1 MKKKVTIDEA LKEILRLEGA ATQEELCAKL LAQGFATTQS SVSRWLRKIQ
51 AVKVAGERGA RYSLPSSTEK TTTRHLVLSI RHNASLIVIR TVPGSASWIA
101 ALLDQGLKDE ILGTLAGDDT IFVTPIDEGR LPLLMVSIAN LLQVFLD*

The cp6463 nucleotide sequence <SEQ ID 334> is:

1 ATGAAAAAAA AAGTAACTAT AGATGAGGCT TTAAAAGAAA TTTTACGTCT
51 TGAAGGAGCG GCAACTCAGG AGGAATTATG TGCAAAACTC TTAGCTCAAG
101 GTTTTGCTAC AACCCAGTCG TCTGTATCTC GTTGGCTACG AAAGATTCAG
151 GCTGTAAAGG TTGCTGGAGA GCGTGGTGCT CGTTATTCTT TACCCTCTTC
201 AACAGAGAAG ACCACGACCC GTCATTTGGT GCTCTCTATT CGCCATAACG
251 CCTCTCTTAT TGTAATTCGT ACGGTTCCTG GTTCAGCTTC TTGGATCGCT
301 GCTTTGTTAG ATCAAGGGCT CAAAGATGAA ATTCTTGGAA CTTTGGCAGG
351 AGATGACACG ATTTTTGTCA CTCCTATAGA TGAAGGGAGG CTCCCATTGT
401 TGATGGTTTC GATTGCAAAT TTACTGCAAG TTTTCTTGGA TTAA

The PSORT algorithm predicts inner membrane (0.1510).

The following C. pneumoniae protein (PID 4376540) was also expressed <SEQ ID 335; cp6540>:

1 MSQCQSSSTS TWEWMKSFVP NWKNPTPPLS PIPSEDEFIL AYEPFVLPKT
51 DPENAQANPP GTSTPNVENG IDDLNPLLGQ PENQNNANNP GTSGSNPTSL
101 PAPERLPETE ENSQEEEQGS QNNEDLIG*

The cp6540 nucleotide sequence <SEQ ID 336> is:

1 ATGTCTCAAT GTCAGAGTAG CAGTACATCT ACCTGGGAAT GGATGAAATC
51 TTTTGTGCCA AACTGGAAGA ATCCAACTCC CCCCTTATCT CCTATACCTT
101 CTGAGGACGA ATTTATATTA GCATACGAGC CATTTGTTCT ACCGAAAACA
151 GATCCAGAAA ACGCACAAGC TAATCCTCCA GGCACATCTA CACCGAATGT
201 AGAAAACGGG ATCGATGATC TCAACCCTCT TCTGGGGCAA CCCAACGAAC
251 AAAACAATGC CAACAATCCA GGAACTTCTG GATCTAATCC TACATCTCTA
301 CCCGCCCCCG AACGACTCCC TGAAACTGAA GAGAACAGCC AAGAAGAAGA
351 ACAAGGATCT CAAAATAATG AGGATCTTAT AGGATAA

The PSORT algorithm predicts cytoplasm (0.3086).

The following C. pneumoniae protein (PID 4376743) was also expressed <SEQ ID 337; cp6743>:

1 LREFGSVSFR EYFRAYMCDK IVAQKNFLFT LDAVIKQAGW RSQEKLNLFY
51 VESQALGREI KVSLEEYIQS MVGILGSQRT KKSFKFSVDF TPLEQALQER
101 CSSDDDEDAT ATSTATGATA SPTDMHEDE*

The cp6743 nucleotide sequence <SEQ ID 338> is:

1 TTGAGAGAAG AAGGTAGTGT TTCTTTCAGA GAATATTTCA GAGCCTATAT
51 GTGTGATAAA ATCGTGGCAC AGAAGAACTT CTTATTTACT TTAGACGCTG
101 TAATTAAACA GGCCGGTTGG AGATCACAAG AGAAACTCAA TTTATTTTAT
151 GTTGAAAGTC AGGCTTTAGG AAGAGAAATC AAAGTCAGCT TAGAGGAATA
201 TATTCAGAGT ATGGTCGGGA TTTTGGGATC TCAGAGAACC AAGAAAAGCT
251 TTAAGTTTTC TGTCGACTTT ACCCCTTTAG AGCAGGCTCT ACAAGAAAGA
301 TGCTCTTCTG ATGATGACGA AGATGCAACA GCAACTTCGA CCGCTACAGG
351 GGCAACAGCA TCTCCGACTG ACATGCACGA AGATGAGTAA

The PSORT algorithm predicts cytoplasm (0.2769).

The following C. pneumoniae protein (PID 4377041) was also expressed <SEQ ID 339; cp7041>:

1 MLMMLMMIIG ITGGSGAGKT TLTQNIKEIP GEDVSVICQD NYYKDRSHYT
51 PEERANLIWD HPDAFDNDLL ISDIKRLKNN EIVQAPVFDF VLGNRSKTEI
101 ETIYPSKVIL VEGILVFENQ ELRDLMDIRI FVDTDADERI LRRMVRDVQE
151 QGDSVDCIMS RYLSMVKPMH EKFIEPTRKY ADIIVHGNYR QNVVTNILSQ
201 KIKNHLENAL ESDETYYMVN SK*

The cp7041 nucleotide sequence <SEQ ID 340> is:

1 ATGTTGATGA TGCTTATGAT GATTATTGGA ATTACAGGAG GTTCTGGAGC
51 TGGGAAAACC ACCCTAACCC AAAACATTAA AGAAATTTTC GGTGAGGATG
101 TGAGTGTTAT CTGCCAAGAT AATTATTACA AAGATAGATC TCATTATACT
151 CCTGAAGAAC GTGCCAATTT AATTTGGGAT CATCCGGACG CCTTTGATAA
201 TGACTTATTA ATTTCAGACA TAAAACGTCT AAAAAATAAT GAGATTGTCC
251 AAGCCCCAGT TTTTGATTTT GTTTTAGGTA ATCGATCTAA AACGGAGATA
301 GAAACGATCT ATCCATCTAA AGTTATTCTT GTTGAAGGTA TTCTGGTCTT
351 TGAAAATCAA GAACTTAGAG ATCTTATGGA TATTAGGATC TTTGTAGACA
401 CCGATGCTGA TGAAAGGATA CTACGCCGTA TGGTTCGAGA TGTTCAAGAA
451 CAAGGAGATA GCGTGGACTG CATCATGTCT CGTTATCTTT CTATGGTAAA
501 GCCTATGCAT GAGAAATTTA TAGAGCCGAC TCGGAAATAT GCTGATATCA
551 TTGTACATGG AAATTACCGA CAAAACGTAG TAACAAATAT TTTGTCACAG
601 AAAATTAAAA ATCATTTAGA GAATGCCCTG GAAAGCGATG AGACGTATTA
651 TATGGTCAAC TCTAAGTAA

The PSORT algorithm predicts inner membrane (0.1022).

The proteins were expressed in E. coli and purified as his-tag products (FIG. 167A; 6463=lanes 2-4; 6540=lanes 5-7; 6743=lanes 8-9; 7041=lanes 10-11). The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 167B, 168, 169 & 170) and for FACS analysis.

These experiments show that cp6463, cp6540, cp6743 & cp7041 are surface-exposed and immunoaccessible proteins and that they are useful immunogens. These properties are not evident from the sequence alone.

Example 171 and Example 172 and Example 173

The following C. pneumoniae protein (PID 4376632) was expressed <SEQ ID 341; cp6632>:

1 VQLFQYMNES GWDWICDFDS QGEGFQLSRL VGLLHSSWAL YEAKFQFYLP
51 EVSLLTWEEL IEMQLLSKPT KHGVAKDLCN VFEKHFQRFR QYLGSLDLNQ
101 RFENTFLNYP KYHLDRE*

The cp6632 nucleotide sequence <SEQ ID 342> is:

1 GTGCAATTAT TTCAATATAT GAATGAGTCC GGATGGGATT GGCTTTGTGA
51 TTTTGATTCT CAAGGCGAGG GATTCCAGTT ATCACGTCTG GTTGGGCTGT
101 TACATTCGTC CTGGGCATTA TACGAAGCAA AAGAGCAATT TTACCTTCCT
151 GAGGTTTCTC TATTGACCTG GGAAGAACTG ATAGAAATGC AGTTATTAAG
201 CAAACCAACA AAACACGGGG TTGCAAAAGA TCTTTGTAAT GTATTTGAAA
251 AACACTTTCA AAGGTTTAGA CAGTACCTAG GTTCCTTAGA TCTAAATCAA
301 AGGTTCGAAA ATACCTTCTT GAATTATCCT AAATACCATT TAGATAGGGA
351 GTGA

The PSORT algorithm predicts cytoplasm (0.3627).

The following C. pneumoniae protein (PID 4376648) was also expressed <SEQ ID 343; cp6648>:

1 MPVSSAPLPT SHRPSSGNLG LMEPNSKALK AKHQDKTTKT IKLLVKILVA
51 ILVIEVLGII AAFFIPGTPP ICLIILGGLI LTTVLCVLLL VIKLALVNKT
101 EGTTAEQQIK RKLSSKSIS*

The cp6648 nucleotide sequence <SEQ ID 344> is:

1 ATGCCCGTGT CCTCAGCCCC CCTACCCACA AGCCACCGCC CTTCCTCTGG
51 AAATCTAGGC CTCATGGAAC CAAATTCCAA AGCTCTAAAA GCAAAGCATC
101 AAGATAAAAC GACGAAGACG ATTAAACTTT TAGTTAAAAT CCTTGTTGCC
151 ATTCTAGTAA TAGAAGTTTT AGGAATAATT GCAGCTTTCT TTATTCCTGG
201 GACTCCTCCC ATCTGCTTGA TTATCCTAGG AGGCCTTATT CTTACAACAG
251 TACTCTGTGT GCTTCTTCTT GTTATAAAGC TTGCCCTTGT AAACAAAACC
301 GAAGGAACAA CTGCTGAACA GCAGATAAAA CGTAAACTCT CTTCTAAAAG
351 TATTTCTTAG

The PSORT algorithm predicts inner membrane (0.6074).

The following C. pneumoniae protein (PID 4376497) was also expressed <SEQ ID 345; cp6497>:

1 MKPNSIIFLE NTKHYPDIFR EGFVRDRHGL MEASDWLLST FITIIRSILG
51 AIPILGNILG AGRLYSVWYT SDEDWKKQVV *

The cp6497 nucleotide sequence <SEQ ID 346> is:

1 ATGAAGCCAA ATAGTATTAT TTTTTTAGAA AATACTAAGC ATTATCCCGA
51 CATCTTTCGA GAAGGATTTG TTCGTGATCG TCATGGACTA ATGGAAGCCT
101 CGGATTGGTT ACTTTCTACG GAAATTACGA TCATTCGCTC CATTCTGGGA
151 GCTATCCCTA TTTTAGGAAA TATTCTTGGA GCCGGACGAC TCTATAGCGT
201 TTGGTATACA AGTGACGAAG ATTGGAAAAA ACAAGTGGTT TGA

The PSORT algorithm predicts inner membrane (0.145).

The proteins were expressed in E. coli and purified as his-tag products (FIG. 171A; 6632=lanes 5-7; 6648=lanes 8-10; 6497=lanes 2-4). The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 171B, 172, 173) and for FACS analysis.

These experiments show that cp6632, cp6648 and cp6497 are surface-exposed and immunoaccessible proteins and that they are useful immunogens. These properties are not evident from the sequence alone.

Example 174, Example 175, Example 176, Example 177 and Example 178

The following C. pneumoniae protein (PID 4377200) was expressed <SEQ ID 347; cp7200>:

1 MPVPIDNSSR NLQEVPESLE DLEQHAEESP THQSAESSSL QLSLASSAIS
51 SRVFQLSSLV LGMENSDFSS LRDVPIFSAI YESSTHTPVP TPLVGVGYIN
101 GSQSGYYDTQ RESLHLSQLL GSRRVEVVYN QGNFMEASLL NLCPRRPRRD
151 PSPISLALLE LWEAFFLEHP PGSTFNPIFF W*

The cp7200 nucleotide sequence <SEQ ID 348> is:

1 ATGCCCGTTC CTATAGATAA TTCCTCTCGC AACCTACAAG AAGTTCCAGA
51 AAGCCTAGAA GACCTCGAAC AACACGCAGA AGAATCTCCT ACTCATCAAA
101 GTGCAGAAAG CAGTTCTTTG CAACTGTCTC TAGCCTCCTC AGCAATTTCT
151 AGTAGAGTAG AACAACTATC TTCCCTCGTC TTAGGAATGG AAAATTCAGA
201 TTTCTCCTCT TTAAGAGACG TTCCTATCTT CTCAGCTATC TACGAATCTT
251 CAACACACAC ACCTGTCCCC ACTCCTCTAG TTGGCGTGGG ATATATCAAC
301 GGAAGTCAAT CAGGATACTA CGATACACAA AGAGAATCTC TTCACCTCAG
351 CCAATTGTTA GGAAGCCGAA GAGTTGAAGT TGTCTATAAC CAAGGAAACT
401 TCATGGAGGC CTCTTTGCTA AATCTGTGCC CCAGAAGACC TCGAAGAGAT
451 CCCTCTCCAA TTTCTTTAGC TCTATTAGAG CTCTGGGAAG CATTTTTTTT
501 AGAACACCCC CCAGGTAGCA CTTTTAATCC AATATTTTTT TGGTAA

The PSORT algorithm predicts cytoplasm (0.3672).

The following C. pneumoniae protein (PID 4377235) was also expressed <SEQ ID 349; cp7235>:

1 LNFVSTLTGS DFYAPVLEKL EEAFADTTGQ VILFSSSPDF IVHPIAQQLG
51 ISSWYASCYR DQSAEQTIYK KCLTGDKKAQ ILSYIKKINQ ARSHTFSDHI
101 LDLPFLMLGE EKTVVRPQGR LKKMAKKYYW NIV*

The cp7235 nucleotide sequence <SEQ ID 350> is:

1 TTGAATTTTG TATCGACTCT GACCGGCTCC GATTTTTATG CTCCTGTTTT
51 AGAAAAACTA GAAGAAGCTT TTGCAGATAC CACAGGACAG GTGATCCTTT
101 TTTCTTCTTC TCCAGACTTT ATTGTCCACC CCATAGCGCA GCAACTCGGG
151 ATTAGTTCTT GGTATGCGTC GTGTTATCGC GATCAGTCTG CAGAACAGAC
201 GATCTATAAA AAATGTCTTA CAGGGGATAA AAAAGCGCAA ATTTTGAGTT
251 ATATTAAAAA AATTAATCAA GCAAGAAGCC ATACCTTCTC CGACCATATT
301 TTAGATCTTC CTCTTCTTAT GCTGGGAGAA GAGAAAACCG TCGTTCGCCC
351 TCAGGGACGA CTCAAGAAAA TGGCAAAAAA ATATTACTGG AATATCGTTT
401 AA

The PSORT algorithm predicts cytoplasm (0.3214).

The following C. pneumoniae protein (PID 4377268) was also expressed <SEQ ID 351; cp7268>:

1 MMHRYFIPLL ALLIFSPSLV RAELQPSENR KGGWPTQLSC AEGSQLFCKF
51 EAAYNNAIEE GKPGILVFFS ERPTPEFADL TNGSFSLSTP IAKGFNVVVL
101 CPGLISPLDF FHKMDPVILY MGSFLEMFPE VEAVSGPRLC YILIDEQGGA
151 QCQAVLPLET KN*

The cp7268 nucleotide sequence <SEQ ID 352> is:

1 ATGATGCACC GTTATTTTAT TCCTTTATTA GCACTTCTCA TTTTCTCTCC
51 TTCTTTAGTC AGGGCAGAGC TACAACCAAG TGAAAACAGA AAAGGGGGGT
101 GGCCTACACA ACTTTCCTGT GCAGAAGGTT CGCAACTCTT CTGTAAATTC
151 GAAGCTGCCT ATAATAATGC AATTGAGGAA GGGAAACCTG GGATTTTAGT
201 CTTTTTCTCT GAGCGACCCA CACCAGAATT TGCCGACTTA ACGAATGGTT
251 CATTTTCTCT CTCTACGCCA ATCGCCAAGG GCTTTAATGT CGTTGTGTTA
301 TGCCCCGGGC TTATCAGTCC CTTAGACTTT TTCCACAAAA TGGATCCTGT
351 GATTCTCTAT ATGGGAAGTT TTCTAGAGAT GTTCCCTGAA GTGGAGGCAG
401 TTAGTGGCCC TCGCTTATGT TATATCTTAA TAGATGAACA GGGTGGGGCT
451 CAATGTCAGG CTGTCCTGCC TTTAGAAACA AAGAATTAG

The PSORT algorithm predicts inner membrane (0.1235).

The following C. pneumoniae protein (PID 4377375) was also expressed <SEQ ID 353; cp7375>:

1 MQRIIIVGID TGVGKTIVSA ILARALNAEY WKPIQAGNLE NSDSNIVHFL
51 SGAYCHPEAY RLHKPLSPHK AAQIDNVSIE ESHICAPKTT SNLIIETSGG
101 FLSPCTSKRL QGDVFSSWSC SWILVSQAYL GSINHTCLTV FAMRSRNLNI
151 LGMVVNGYPE DEEHWLTQEI KLPIIGTLAK EKEITKTIIS CYAEQWKEVW
201 TSNHQGIQGV SGTPSLNLH*

The cp7375 nucleotide sequence <SEQ ID 354> is:

1 ATGCAACGTA TCATCATTGT AGGAATCGAC ACTGGCGTAG GAAAAACCAT
51 TGTCAGTGCT ATCCTTGCTA GAGCACTTAA CGCAGAATAC TGGAAACCTA
101 TACAAGCAGG GAATCTAGAA AATTCAGATA GCAATATTGT TCATGAGCTA
151 TCGGGAGCCT ACTGTCATCC CGAAGCTTAT CGATTGCATA AGCCCTTGTC
201 TCCACACAAG GCAGCGCAAA TCGATAATGT AAGTATCGAA GAGAGTCATA
251 TTTGTGCGCC AAAAACAACT TCGAATCTGA TTATTGAGAC TTCAGGAGGA
301 TTTTTATCCC CCTGCACATC AAAAAGACTT CAGGGAGATG TGTTTTCTTC
351 TTGGTCATGT TCTTGGATTT TAGTGAGCCA AGCATATCTC GGAAGTATCA
401 ATCACACCTG TTTAACGGTA GAAGCAATGC GCTCACGAAA CCTCAATATC
451 TTAGGTATGG TGGTAAATGG GTATCCAGAG GACGAAGAGC ACTGGCTAAC
501 TCAAGAAATC AAGCTTCCTA TAATCGGGAC TCTTGCCAAG GAAAAAGAAA
551 TCACAAAGAC AATCATAAGC TGTTATGCCG AACAATGGAA GGAAGTATGG
601 ACAAGCAATC ATCAGGGAAT TCAGGGTGTA TCTGGCACCC CTTCACTCAA
651 TCTGCATTAG

The PSORT algorithm predicts cytoplasm (0.0049).

The following C. pneumoniae protein (PID 4377388) was also expressed <SEQ ID 355; cp7388>:

1 MQVLLSPQLP FPPQHSVGSI SSPSKLRVLA ITFLVFGMLL LISGALFLTL
51 GIPGLSAAIS FGLGIGLSAL GGVLMISGLL CLLVKREIPT VRPEFIPEGV
101 SLAPSEEPAL QAAQKTLAQL PKELDQLDTD IQEVFACLRK LKDSKYESRS
151 FLNDAKKELR VFDFVVEDTL SEIFELRQIV AQEGWDLNFL INGGRSLMMT
201 AESFSLDIFH VSKRIGYLPS GDVRGEGLKK SAKEIVARLM SLHCFIHKVA
251 VAFDRNSYAM AEKAFAKALG ALEESVYRSL TQSYRDKFLE SERAKIPWNG
301 HITWLRDDAK SGCAEKKLRD AEERWKKFRK AVFWVEEDGG FDINNLLGDW
351 GTVLDPYRQE RMDEITFHEL YEKTTFLKRL HRKCALAKTT FEKKRSKKNL
401 QAVEEANARR LKYVRDWYDQ EFQKAGERLE KLHALYPEVS VSIRFNKIQE
451 TRSNLEKAYE AIEENYRCCV REQEDYWKEE EKREAEFRER GNKILSPEFL
501 ESSLEQFDHG LKNFSEKLME LEGHILKLQK EATAEVENKI LSDAFSRLFI
551 VFEDVKEMPC RIEEIEKTLR MAELPLLPTK KAFEKACSQY NSCAEMLEKV
601 KPYCKESLAY VTSKERLVSL DEDLRRAYTE CQKRFQGDSG LESEVRACRE
651 QLRERIQEFE TQGLDLVEKE LLCVSSRLRN TECDCVSGVK KEAPPGKKFY
701 AQYYDEIYRV RVQSRWMTMS ERLREGVQAC NKMLKAGLSE EDKVLKEEFY
751 WLYREERKNK EKRLVGTKIV ATQQRVAAFE SIEVPEIPEA PEEKPSLLDK
801 ARSLFTREDH T

The cp7388 nucleotide sequence <SEQ ID 356> is:

1 ATGCAAGTAC TTCTATCTCC GCAGCTACCC CCCCCCCCCC AACACTCTGT
51 AGGGTCGATT TCTTCTCCAT CTAAACTTCG CGTTTTAGCG ATTACTTTTT
101 TAGTTTTTGG TATGCTCTTA CTGATTTCAG GAGCTCTCTT TCTGACGTTA
151 GGGATTCCAG GATTGAGTGC AGCAATTTCT TTTGGATTAG GCATCGGTCT
201 CTCCGCATTA GGAGGAGTGC TGATGATTTC GGGACTACTA TGTCTTTTAG
251 TAAAACGAGA GATTCCGACA GTACGACCAG AAGAAATTCC TGAAGGGGTT
301 TCGCTGGCTC CTTCTGAGGA GCCAGCTCTA CAGGCAGCTC AGAAGACTTT
351 AGCTCAGCTG CCTAAGGAAT TGGATCAGTT AGATACAGAT ATTCAGGAAG
401 TGTTCGCATG TTTAAGAAAG CTGAAAGATT CTAAGTATGA AAGTCGAAGT
451 TTTTTAAACG ATGCTAAGAA GGAGCTTCGA GTTTTTGACT TTGTGGTTGA
501 GGATACCCTC TCGGAGATTT TCGAGTTGCG GCAGATTGTG GCTCAAGAGG
551 GATGGGATTT AAACTTTTTG ATCAATGGGG GACGAAGCCT CATGATGACT
601 GCAGAATCTG AATCGCTTGA TTTGTTTCAT GTATCGAAGC GGCTAGGGTA
651 TTTACCTTCT GGGGATGTTC GAGGGGAGGG GTTAAAGAAA TCTGCGAAGG
701 AGATAGTCGC TCGTTTGATG AGCTTGCATT GCGAGATTCA CAAGGTGGCG
751 GTAGCGTTTG ATAGGAATTC CTATGCGATG GCAGAAAAGG CGTTTGCGAA
801 AGCGTTGGGA GCCTTAGAAG AGAGTGTGTA TCGGAGTCTG ACGCAGAGTT
851 ATAGAGATAA ATTTTTGGAG AGCGAGAGGG CGAAGATCCC ATGGAATGGG
901 CATATAACCT GGTTAAGAGA TGATGCGAAG AGTGGGTGTG CTGAAAAGAA
951 GCTTCGGGAT GCCGAGGAAC GTTGGAAGAA ATTTAGGAAA GCAGTCTTTT
1001 GGGTAGAAGA AGACGGGGGC TTTGACATCA ATAATCTCCT TGGAGACTGG
1051 GGGACAGTGC TTGATCCTTA TAGACAAGAG AGAATGGACG AGATAACGTT
1101 CCATGAGTTG TATGAAAAAA CTACGTTTTT GAAAAGACTG CACAGAAAGT
1151 GTGCGTTAGC GAAAACAACC TTTGAAAAGA AGAGATCTAA AAAGAATTTG
1201 CAGGCAGTCG AGGAGGCGAA TGCACGTAGG TTGAAATATG TAAGGGATTG
1251 GTATGATCAG GAGTTTCAGA AAGCAGGGGA GAGATTAGAG AAACTGCATG
1301 CTTTGTATCC TGAGGTCTCA GTCTCTATAA GAGAGAACAA AATACAAGAG
1351 ACGCGCTCTA ATTTAGAGAA AGCCTATGAG GCTATCGAAG AGAACTATCG
1401 TTGCTGTGTC CGAGAGCAAG AGGACTACTG GAAAGAAGAA GAGAAAAGGG
1451 AAGCGGAGTT TAGGGAGAGG GGAAACAAGA TTCTTTCTCC TGAGGAGCTG
1501 GAAAGTTCTT TGGAGCAATT CGACCATGGT TTGAAAAATT TTTCTGAGAA
1551 ATTAATGGAA TTGGAAGGGC ATATCTTAAA ACTTCAGAAA GAAGCCACAG
1601 CAGAGGTGGA GAATAAAATA CTTTCAGATG CAGAGAGCCG CCTTGAGATT
1651 GTATTTGAAG ATGTCAAGGA GATGCCCTGT CGAATTGAGG AGATAGAGAA
1701 GACGCTGCGT ATGGCGGAGC TGCCCCTACT TCCTACGAAG AAGGCGTTTG
1751 AGAAGGCCTG CTCACAATAT AATAGCTGCG CAGAGATGTT GGAGAAGGTG
1801 AAGCCTTACT GCAAGGAGAG CCTCGCCTAT GTGACTAGCA AAGAGCGTTT
1851 AGTGAGCTTG GATGAAGATT TACGACGAGC CTACACAGAG TGTCAGAAGA
1901 GATTCCAGGG GGATTCGGGT TTGGAGTCGG AAGTAAGAGC CTGTCGAGAG
1951 CAACTGCGAG AGCGGATCCA AGAGTTTGAA ACTCAAGGGC TGGACTTGGT
2001 GGAAAAAGAG TTGCTTTGTG TGAGTAGTAG ATTAAGAAAT ACAGAGTGCG
2051 ATTGTGTATC TGGTGTTAAG AAAGAAGCAC CTCCTGGTAA GAAGTTTTAT
2101 GCCCAGTATT ATGATGAGAT TTATCGAGTT AGAGTTCAAT CCCGATGGAT
2151 GACGATGTCT GAGAGATTGA GAGAGGGAGT TCAAGCATGC AACAAGATGT
2201 TGAAGGCAGG CCTAAGCGAA GAAGATAAGG TTCTTAAAGA AGAAGAGTAT
2251 TGGTTGTATC GAGAGGAGAG AAAGAATAAA GAGAAACGTT TGGTTGGTAC
2301 TAAGATAGTA GCAACGCAGC AGCGAGTTGC AGCATTTGAA TCCATAGAAG
2351 TTCCTGAGAT TCCTGAGGCC CCAGAGGAGA AACCGAGTTT GCTGGATAAA
2401 GCGCGTTCTT TATTTACTCG CGAGGACCAT ACCTAG

The PSORT algorithm predicts inner membrane (0.461).

The proteins were expressed in E. coli and purified as his-tag products (FIG. 174: 7200=lanes 2-3; 7236=lanes 4-5; 7268=lanes 6-8; 7375=lanes 9-10; 7388=lanes 11-12). The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 174, 175, 176, 177 & 178) and for FACS analysis.

These experiments show that cp7200, cp7235, cp7268, cp7375 & cp7388 are surface-exposed and immunoaccessible proteins and that they are useful immunogens. These properties are not evident from the sequence alone.

Example 179

The following C. pneumoniae protein (PID 4376723) was expressed <SEQ ID 357; cp6723>:

1 MATSVAPSPV PESSPLSHAT EVLNLPNAYI TQPEPIPAAP WETFRSKLST
51 KHTLCFALTL LLTLGGTISA GYAGYTGNWI ICGIGLGIIV LTLILALLLA
101 IPLKNKQTGT KLTDEISQDI SSIGSGFVQR YGLMFSTIKS VHLPELTTQN
151 QEKTRILNEI EAKKESIQNL ELKITECQNK LAQKQPKRKS SQKSFMRSIK
201 HLSKNPVILF DC*

The cp6723 nucleotide sequence <SEQ ID 358> is:

1 ATGGCAACTT CCGTAGCCCC ATCACCAGTC CCCGAGAGCA GCCCTCTCTC
51 TCATGCTACA GAAGTTCTCA ATCTTCCTAA TGCTTATATT ACGCAGCCTC
101 ATCCGATTCC AGCGGCTCCT TGGGAGACCT TTCGCTCCAA ACTTTCCACA
151 AAGCATACGC TCTGTTTTGC CTTAACACTA CTGTTAACCT TAGGGGGAAC
201 GATCTCAGCA GGTTACGCAG GATATACTGG AAACTGGATC ATCTGTGGCA
251 TCGGCTTGGG AATTATCGTA CTCACACTGA TTCTTGCTCT TCTTCTAGCA
301 ATCCCTCTTA AAAATAAGCA GACAGGAACA AAACTGATTG ATGAGATATC
351 TCAAGACATT TCCTCTATAG GATCAGGATT TGTTCAGAGA TACGGGTTGA
401 TGTTCTCTAC AATTAAAAGC GTGCATCTTC CAGAGCTGAC AACACAAAAT
451 CAAGAAAAAA CAAGAATTTT AAATGAAATT GAAGCGAAAA AGGAATCGAT
501 CCAAAATCTT GAGCTTAAAA TTACTGAGTG CCAAAACAAG TTAGCACAGA
551 AACAGCCGAA ACGGAAATCA TCTCAGAAAT CATTTATGCG TAGTATTAAG
601 CACCTCTCCA AGAACCCTGT AATTTTGTTC GATTGCTGA

The PSORT algorithm predicts inner membrane (0.6095).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 179A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 179B) and for FACS analysis.

These experiments show that cp6723 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 180

The following C. pneumoniae protein (PID 4376749) was expressed <SEQ ID 359; cp6749>:

1 MSYYFSLWYL KVQQHFQAAF DFTRSLCSRI SNFALGVIAL LPIIGQLYVG
51 LDWLLSRIKK PEFPSDVDQI VRVEHVVGHD HRSRVEDILK RQRLSLEPRD
101 EGKVHGDLPS APFF*

The cp6749 nucleotide sequence <SEQ ID 360> is:

1 ATGAGTTATT ACTTTTCTCT TTGGTATCTG AAGGTGCAAC AGCACTTTCA
51 AGCAGCATTT GATTTTACTC GCTCCCTGTG TTCACGAATT TCTAATTTTG
101 CTTTGGGAGT GATTGCATTG CTTCCTATTA TTGGGCAGTT GTATGTAGGG
151 CTGGACTGGC TCCTCTCTAG GATAAAAAAG CCAGAATTTC CTTCCGATGT
201 GGATCAGATC GTGCGAGTAG AACACGTCGT GGGTCACGAC CATAGAAGTC
251 GAGTTGAAGA TATTCTAAAG AGACAAAGGC TCTCATTAGA GCCTAGAGAC
301 GAGGGGAAGG TTCACGGAGA TCTGCCTTCA GCTCCTTTTT TTTGA

The PSORT algorithm predicts inner membrane (0.2996).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 180A). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 180B) and for FACS analysis.

These experiments show that cp6749 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 181, Example 182, Example 183, Example 184 and Example 185

The following C. pneumoniae protein (PID 4376301) was expressed <SEQ ID 361; cp6301>:

1 LNQDLQNVYQ ECQKATGLES EVSAYRDHLR EQITEFETQG LDVIKEELLF
51 VSSTLKSKLS YDPLIADIPC MKFYEEYYDG IDKARVQSRW LEKSERYRKA
101 KKGFQEMLKE GLFKEDQALK KAEYRLLREK RMNKEKLLIC NKIEAAQQRV
151 QEFGPSDS*

The cp6301 nucleotide sequence <SEQ ID 362> is:

1 TTGAATCAGG ATTTACAAAA TGTATACCAA GAGTGCCAGA AGGCTACAGG
51 TTTAGAATCG GAAGTGAGTG CATATAGAGA TCATCTTAGA GACCAGATCA
101 CAGAGTTTGA AACTCAAGGG CTGGACGTGA TAAAAGAAGA ACTTCTTTTT
151 GTGAGTAGTA CTCTCAAAAG TAAATTGAGC TATGATCCAT TAATAGCAGA
201 CATTCCCTGT ATGAAGTTTT ATGAGGAGTA TTATGATGGC ATTGATAAAG
251 CGAGAGTTCA ATCCCGATGG CTGGAGAAGT CTGAGAGGTA TAGAAAGGCG
301 AAGAAGGGAT TCCAAGAGAT GCTGAAGGAA GGCCTATTCA AAGAAGATCA
351 GGCTTTGAAA AAAGCAGAGT ATAGATTACT TCGAGAGAAG AGAATGAATA
401 AGGAGAAGCT TTTGATTTGC AATAAGATAG AAGCAGCTCA GCAGCGAGTC
451 CAAGAATTTG GACCCTCGGA TTCATAA

The PSORT algorithm predicts cytoplasm (0.4621).

The following C. pneumoniae protein (PID 4376558) was also expressed <SEQ ID 363; cp6558>:

1 MNIPAPQVPV IDEPVVNNTS SYGLSLKSSL RPITYLILAI LAIATLMSVL
51 YFCGIISVGT FVLGMLIPLS VCSVLCVAYL FYQQSSIEKT KVFSITSPSV
101 FFSDEDLNLL LGREEDSVSA IDELLKNFPA DDFRRPKMLP YSNFLDEQGR
151 PNESREEDSH TSKIL*

The cp6558 nucleotide sequence <SEQ ID 364> is:

1 ATGAACATAC CCGCTCCCCA AGTACCAGTC ATAGATGAGC CTGTAGTGAA
51 CAACACAAGT AGCTATGGTC TTTCATTGAA AAGTAGTTTA AGACCGATTA
101 CTTATTTGAT TTTAGCTATC TTAGCTATAG CCACACTGAT GTCTGTTCTC
151 TACTTTTGTG GCATCATTAG TGTTGGGACG TTTGTTTTGG GCATGCTGAT
201 CCCTCTATCG GTCTGCTCTG TTCTTTGCGT TGCCTATTTA TTCTATCAGC
251 AATCTTCTAT AGAAAAGACT AAGGTCTTTT CTATAACCAG TCCTTCAGTA
301 TTTTTCTCTG ATGAGGATCT TAATTTACTC TTAGGTCGAG AAGAAGATTC
351 AGTGTCTGCA ATTGATGAAC TTCTTAAGAA CTTTCCAGCT GATGATTTCC
401 GTAGGCCGAA GATGCTTCCT TATTCAAATT TTCTAGATGA GCAGGGAAGG
451 CCTAATGAGA GTAGGGAAGA AGACTCTCAT ACTTCCAAGA TCTTATAA

The PSORT algorithm predicts inner membrane (0.4630).

The following C. pneumoniae protein (PID 4376630) was also expressed <SEQ ID 365; cp6630>:

1 MSMTIVPHAL FKNHCECHST FPLSSRTIVR IAIASLFCIG ALAALGCLAP
51 PVSYIVGSVL AFIAFVILSL VILALIFGEK KLPPTPRIIP DRFTHVIDEA
101 YGLSISAFVR EQQVTLAEFR QFSTALLCNI SPEEKIKQLP SELRSKVESF
151 GISRLAGDLE KNNWPIFEDL LSQTCPLYWL QKFISAGDPQ VCRDLGVPRE
201 CYGYYWLGPL GYSTAKATIF CKETHHILQQ LTKEDVLLLK NKALQEKWDT
251 DEVKAIVERI YTTYTARGTL KTEAGGLTKE TISKELLLLS LHGYSFDQLQ
301 LITQLPRDAW DWLCFVDNST AYNLQLCALV GALSSQNLLD ESSIDFDVNL
351 GLYVIQDLKE AVQAFSASDE RKKELGKFLL RHLSSVSKRL ESVLRQGLHR
401 IALEHGNARA RVYDVNFVTG ARIHRKTSIF FKD*

The cp6630 nucleotide sequence <SEQ ID 366> is:

1 ATGAGCATGA CGATCGTTCC ACATGCTTTA TTTAAAAATC ATTGCGAGTG
51 TCATTCTACC TTTCCTTTGA GTTCAAGGAC TATTGTAAGA ATAGCCATTG
101 CCAGCCTCTT TTGTATAGGT GCATTAGCAG CTTTAGGCTG TTTGGCTCCT
151 CCCGTTTCTT ATATTGTTGG GAGTGTTTTA GCTTTTATTG CCTTTGTCAT
201 TCTTTCTTTA GTAATTTTAG CTTTGATTTT TGGAGAGAAG AAGCTTCCAC
251 CAACACCAAG AATCATTCCT GATAGATTTA CTCACGTGAT AGATGAAGCT
301 TATGGCCTTT CAATCTCTGC ATTTGTAAGA GAACAGCAGG TAACATTAGC
351 CGAGTTTAGA CAATTTTCTA CTGCCCTGTT GTGTAACATA TCTCCTGAAG
401 AGAAAATCAA ACAATTGCCT TCTGAATTGC GAAGTAAAGT AGAGAGTTTT
451 GGTATTAGCA GGCTCGCAGG TGATTTAGAA AAGAATAATT GGCCAATATT
501 TGAAGATCTT TTAAGCCAAA CCTGCCCGTT ATATTGGCTT CAGAAATTTA
551 TATCAGCAGG AGATCCACAA GTTTGTAGAG ACCTAGGTGT CCCTAGAGAA
601 TGTTATGGGT ACTATTGGCT AGGGCCTTTG GGATACAGTA CAGCTAAGGC
651 TACAATTTTT TGTAAAGAGA CGCATCATAT TCTTCAACAA TTAACGAAAG
701 AGGACGTTCT TTTATTAAAA AACAAGGCTC TTCAAGAGAA ATGGGATACT
751 GATGAAGTCA AAGCAATTGT AGAGCGTATC TACACTACCT ATACGGCACG
801 AGGAACTCTA AAGACCGAAG CAGGGGGACT TACAAAAGAG ACAATCAGTA
851 AGGAATTGCT ATTGTTGAGC TTGCATGGCT ATTCTTTTGA TCAGCTACAG
901 CTGATCACTC AACTTCCTAG AGATGCTTGG GATTGGCTGT GTTTTGTAGA
951 TAACAGTACC GCATACAACC TTCAGCTTTG TGCTCTTGTA GGAGCTTTGT
1001 CATCCCAAAA TCTTCTTGAC GAATCTTCTA TCGATTTTGA TGTAAACCTA
1051 GGCCTGTATG TGATTCAGGA TCTAAAAGAA GCTGTTCAAG CATTTTCTGC
1101 TTCTGATGAG CCAAAGAAAG AACTAGGTAA ATTCTTGTTA AGGCATTTGA
1151 GTTCAGTTTC TAAGCGATTA GAGAGTGTAT TAAGACAGGG TCTTCACAGA
1201 ATAGCTCTAG AGCATGGAAA TGCCAGAGCT AGGGTTTATG ACGTCAATTT
1251 TGTAACAGGA GCTAGAATTC ATAGGAAGAC GAGTATCTTC TTTAAAGACT
1301 AA

The PSORT algorithm predicts inner membrane (0.7092).

The following C. pneumoniae protein (PID 4376633) was also expressed <SEQ ID 367; cp6633>:

1 MVNIQPVYRN TQVNYSQATQ FSVCQPALSL IIVSVVAAVL AIVALVCSQS
51 LLSIELGTAL VLVSIILFAS AMFMIYKMRQ EPKELLIPKK IMELIQEHYP
101 SIVVDFIRDQ EVSIYEIHHL ISILNKTNVF DKAPVYLQEK LLQFGIEKFK
151 DVHPSKLPNF EEILLQHCPL HWLGRLVYPM VSDVTPGTYG YYWCGPLGLY
201 ENAPSLFERR SLLLLKKISF GEFALLEDGL KKNTWSSSEL VQIRQNLFTR
251 YYADKEEVDE AELNADYEQF DSLLHLIFSH KLS*

The cp6633 nucleotide sequence <SEQ ID 368> is:

1 ATGGTTAATA TACAGCCTGT GTATAGGAAT ACCCAAGTCA ACTATAGTCA
51 GGCTACCCAA TTTTCGGTGT GCCAGCCAGC GCTTAGCCTG ATTATCGTTT
101 CTGTTGTTGC TGCTGTACTC GCTATTGTAG CTTTGGTATG CAGTCAATCT
151 CTTTTATCCA TAGAGTTAGG AACTGCTCTT GTTCTAGTTT CTCTTATTCT
201 TTTTGCTTCT GCTATGTTTA TGATTTATAA GATGAGACAA GAACCTAAGG
251 AGTTGCTGAT CCCTAAGAAA ATCATGGAAC TCATCCAAGA ACATTATCCA
301 AGTATTGTTG TTGATTTTAT TAGAGATCAG GAGGTTTCCA TTTATGAGAT
351 ACATCACTTG ATCTCTATTC TTAATAAGAC GAATGTTTTC GACAAAGCAC
401 CAGTATATTT ACAAGAAAAA CTCTTACAGT TTGGCATTGA GAAGTTCAAA
451 GATGTACATC CAAGTAAGCT CCCTAATTTT GAAGAAATTC TTCTACAGCA
501 TTGCCCATTG CATTGGTTGG GACGTCTGGT ATATCCCATG GTATCGGATG
551 TCACTCCAGG AACCTATGGA TACTATTGGT GTGGTCCTTT AGGACTGTAC
601 GAGAACGCTC CCTCTCTTTT TGAACGTCGA TCTCTTCTAT TGTTAAAGAA
651 AATTAGCTTT GGAGAGTTTG CTCTTTTAGA AGATGGTCTC AAGAAAAACA
701 CGTGGAGTTC TTCGGAACTC GTTCAAATCA GACAAAACCT TTTTACAAGA
751 TATTATGCTG ATAAAGAAGA GGTAGATGAA GCAGAGTTAA ACGCTGATTA
801 CGAACAGTTT GATTCCCTCC TTCACCTTAT TTTTTCTCAC AAGCTCTCTT
851 GA

The PSORT algorithm predicts inner membrane (0.7283).

The following C. pneumoniae protein (PID 4376642) was also expressed <SEQ ID 369; cp6642>:

1 MATISPISLT VDHPLVDTKK KSCSNFDKIQ SRILLITAIF AVLVTIGTLL
51 IGLLLNIPVI YFLTGISFIA VVLSNFILYK RATTLLKPRA CGKHKEIKPK
101 RVSTNLQYSS ISIAINRSKE NWEHQPKDLQ NLPAPSALLT DNPYFIWKAK
151 HSLFSLVSLL PGGNPEHLLI SASENLGKTL LIEETSQNAP ISSYVDTTPS
201 PKSLLNEAIQ ETRVEINTEL PAGDSGERLY WQPDFRGRVF LPQIPTTPEA
251 IYQYYYALYV TYIQTAINTN TQIIQIPLYS LREHLYSREL PPQSRMQQSL
301 AMITAVKYMA ELHPEYPLTI ACVERSLAQL PQESIEDLS*

The cp6642 nucleotide sequence <SEQ ID 370> is:

1 ATGGCTACAA TCTCACCCAT ATCTTTAACT GTAGATCATC CCCTAGTAGA
51 CACTAAAAAA AAATCCTGCA GCAACTTTGA TAAGATTCAG TCTCGAATTC
101 TATTGATTAC TGCAATCTTT GCTGTCTTAG TTACTATAGG GACCCTACTT
151 ATTGGTTTGC TTTTAAATAT TCCTGTTATC TATTTCCTCA CAGGAATTTC
201 ATTTATTGCT GTTGTTCTTA GCAACTTTAT CCTTTATAAA CGAGCAACCA
251 CCCTCTTAAA ACCGCGTGCT TGTGGCAAAC ACAAAGAAAT AAAACCAAAA
301 AGGGTCTCCA CCAACCTACA GTATTCTTCT ATCTCTATCG CAATCAATCG
351 TTCTAAAGAA AACTGGGAAC ACCAACCCAA GGACCTACAG AATCTCCCCG
401 CACCCTCTGC ATTACTCACA GATAACCCTT ACGAGATATG GAAAGCTAAA
451 CATTCACTGT TTTCCCTAGT ATCCCTCCTA CCGGGAGGCA ATCCAGAACA
501 TCTCTTAATT TCAGCTTCCG AAAATTTAGG AAAGACTCTG TTAATTGAAG
551 AAACCTCGCA AAATGCGCCT ATATCCTCCT ACGTAGATAC CACTCCCTCC
601 CCAAAATCCT TGCTCAATGA GGCAATTCAG GAAACCAGGG TAGAAATAAA
651 TACAGAACTC CCTGCGGGAG ATTCAGGAGA ACGTTTATAC TGGCAACCCG
701 ATTTCCGAGG CCGCGTCTTC CTCCCACAAA TACCAACAAC TCCTGAAGCC
751 ATCTACCAAT ACTACTATGC ACTCTATGTC ACTTATATCC AGACTGCGAT
801 CAATACGAAC ACCCAAATTA TCCAAATCCC TTTATACAGC TTGAGGGAGC
851 ATCTCTATTC TAGAGAATTG CCCCCGCAAT CAAGAATGCA ACAATCTTTG
901 GCTATGATTA CAGCAGTAAA ATACATGGCC GAGCTGCACC CAGAATATCC
951 GCTAACTATT GCTTGTGTTG AAAGATCCTT AGCCCAACTA CCTCAAGAAA
1001 GTATTGAGGA TCTCTCTTAG

The PSORT algorithm predicts inner membrane (0.5288).

The proteins were expressed in E. coli and purified as GST-fusion products. The recombinant proteins were used to immunise mice, whose sera were used in Western blots (FIGS. 181-185) and for FACS analysis.

These experiments show that cp6301, cp6558, cp6630, cp6633 and cp6642 are surface-exposed and immunoaccessible proteins, and that they are useful immunogens. These properties are not evident from their sequences alone.

Example 186

The following C. pneumoniae protein (PID 4376389) was expressed <SEQ ID 371; cp6389>:

1 MSEVKPLFLK NDSFDLATQR FQNLINMLQE QAEIYNEYEE KNARVQNEIK
51 EQKDFVKRCI EDFEARGLGV LKEELASLTR DFHDKAKAET SMLIECPCIG
101 FYYSIHQEEQ RQRQERLQKM AERYRDCKQV LEAVQVEQKD MISSRVVVDD
151 SYFEEEKEEQ KVDNRKKEQD *

The cp6389 nucleotide sequence <SEQ ID 372> is:

1 ATGTCAGAAG TGAAGCCTTT GTTTTTAAAG AATGACTCTT TTGATTTGGC
51 AACTCAGAGA TTCCAGAATC TAATTAACAT GCTACAAGAG CAAGCCGAGA
101 TATATAACGA GTATGAAGAA AAGAATGCTA GGGTTCAGAA TGAGATTAAG
151 GAGCAAAAGG ACTTTGTGAA AAGATGCATA GAGGACTTTG AAGCCAGAGG
201 ACTGGGGGTG CTAAAAGAAG AGCTTGCATC TTTGACGCGT GATTTCCATG
251 ATAAAGCAAA AGCAGAGACT TCTATGCTCA TTGAATGTCC TTGTATTGGT
301 TTTTATTATA GTATTCATCA GGAGGAACAA AGGCAAAGGC AAGAAAGGCT
351 TCAAAAGATG GCTGAGCGCT ATAGGGACTG TAAACAAGTC TTGGAGGCTG
401 TCCAGGTGGA GCAAAAAGAT ATGATATCTT CTAGAGTCGT TGTCGATGAC
451 AGCTACTTTG AAGAAGAAAA AGAAGAACAA AAGGTGGATA ACAGAAAGAA
501 AGAACAGGAC TAG

The PSORT algorithm predicts cytoplasm (0.3193).

The protein was expressed in E. coli and purified as a GST-fusion product (FIG. 186A) and also in his-tagged form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 186B) and for FACS analysis.

These experiments show that cp6389 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 187

The following C. pneumoniae protein (PID 4376792) was expressed <SEQ ID 373; cp6792>:

1 VLQEHFFLSE DVITLAQQLL GHKLITTHEG LITSGYIVET EAYRGPDDKA
51 CHAYNYRKTQ RNRAMYLKGG SAYLYRCYGM HHLLNVVTGP EDIPHAVLIR
101 AILPDQGKEL MIQRRQWRDK PPHLLTNGPG KVCQALGISL ENNRQRLNTP
151 ALYISKEKIS GTLTATARIG IDYAQEYRDV PWRFLLSPED SGKVLS*

The cp6792 nucleotide sequence <SEQ ID 374> is:

1 GTGCTACAAG AACATTTTTT TCTATCGGAA GATGTAATTA CAGTAGCGCA
51 ACAGCTTTTA GGACATAAAC TCATCACAAC ACATGAGGGT CTGATAACTT
101 CAGGTTACAT TGTAGAAACC GAAGCGTATC GTGGCCCTGA TGACAAAGCA
151 TGCCACGCCT ACAACTACAG AAAAACTCAG AGGAACAGAG CGATGTACCT
201 GAAAGGAGGC TCTGCTTACC TCTACCGTTG CTATGGCATG CATCACCTAT
251 TGAATGTTGT CACTGGACCT GAGGACATTC CCCATGCCGT CCTGATCCGG
301 GCCATCCTTC CTGATCAAGG CAAAGAACTT ATGATCCAAC GCCGCCAATG
351 GAGAGATAAA CCCCCACACC TTCTCACCAA TGGACCCGGA AAAGTGTGCC
401 AAGCTCTAGG AATCTCTTTG GAAAACAATA GGCAACGCCT AAATACCCCA
451 GCTCTCTATA TCAGCAAAGA AAAAATCTCT GGGACTCTAA CAGCAACTGC
501 CCGGATCGGC ATCGATTATG CTCAAGAGTA TCGTGATGTC CCATGGAGAT
551 TTCTCCTATC CCCAGAAGAT TCGGGAAAAG TTTTATCTTA A

The PSORT algorithm predicts cytoplasm (0.180).

The protein was expressed in E. coli and purified as a his-tagged product (FIG. 187A; lanes 2-4). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 187B) and for FACS analysis.

These experiments show that cp6792 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 188

The following C. pneumoniae protein (PID 4376868) was expressed <SEQ ID 375; cp6868>:

1 MVETVLHNFQ RYLSKYLYRV FRFPCRKKTF LSSERVLARP SFPVDYCPGK
51 IYDLQEIYEE LNAQLFQGAL RLQIGWFGRK ATRKGKSVVL GLFHENEQLI
101 RIHRSLDRQE IPRFFMEYLV YHEMVHSVVP REYSLSGRSI FHGKKFKEYE
151 QRFPLYDRAV AWEKANAYLL RGYKKRVGGG YGRA*

The cp6868 nucleotide sequence <SEQ ID 376> is:

1 ATGGTTGAAA CAGTACTTCA TAATTTCCAA CGTTATCTGA GCAAGTATCT
51 CTATAGGGTA TTTCGCTTCC CATGTCGTAA AAAGACGTTC CTATCTTCGC
101 ACAGGGTTCT TGCTCGTCCT TCATTCCCAG TAGACTACTG TCCGGGAAAG
151 ATCTATGATT TGCAGGAGAT CTATGAGGAA TTGAATGCGC AGTTATTTCA
201 AGGTGCACTG CGTTTACAGA TTGGTTGGTT CGGAAGGAAA GCTACCAGAA
251 AAGGCAAGAG TGTTGTCTTG GGATTGTTTC ATGAAAATGA ACAGTTAATT
301 CGAATTCATC GTTCTTTAGA TCGGCAGGAA ATCCCAAGAT TTTTTATGGA
351 ATATCTTGTG TATCATGAAA TGGTTCATAG TGTAGTCCCT AGAGAGTATT
401 CTCTATCGGG GCGTTCGATT TTTCATGGTA AAAAGTTTAA AGAATACGAA
451 CAACGTTTCC CCTTGTATGA TCGTGCTGTT GCTTGGGAAA AGGCAAACGC
501 TTATTTATTG CGAGGGTATA AAAAAAGAGT AGGTGGAGGA TATGGCAGGG
551 CATAG

The PSORT algorithm predicts bacterial cytoplasm (0.325).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 188A; lanes 2-3). The recombinant protein was used to immunise mice, whose sera were used in a Western blot (FIG. 188B) and for FACS analysis.

These experiments show that cp6868 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 189

The following C. pneumoniae protein (PID 4376894) was expressed <SEQ ID 377; cp6894>:

1 MYKRCVLDKI LKGIVAGSLI LLYWSSDLLE RDIKSIKGNV RDIQEDIREI
51 SRVVKQQQTS QAIPAAPGVM LAPKLVRDEA FALLFGDPSY PNLLSLDPYK
101 QQTLPELLGT NFHPHGILRT AHVGKPENLS PFNGFDYVVG FYDLCIPSLA
151 SPHVGKYEEF SPDLAVKIEE HLVEDGSGDK EFHIYLRPNV FWRPIDPKAL
201 PKHVQLDEVF QRPHPVTAHD IKFFYDAVMN PYVATMRAVA LRSCYEDVVS
251 VSVENDLKLV VRWKAHTVIN EEGKEERKVL YSAFSNTLSL QPLPRFVYQY
301 FANGEKIIED ENIDTYRTNS IWAQNFTMHW ANNYIVSCGA YYFAGMDDEK
351 IVFSRNPDFY DPLAALIDKR FVYFKESTDS LFQDFKTGKI DISYLPPNQR
401 DNFYSFMKSS AYNKQVAKGG AVRETVSADR AYTYIGWNCF SLFFQSRQVR
451 CAMNMAIDRE RIIEQCLDGQ GYTISGPFAS SSPSYNKQIE GWHYSPEEAA
501 RLLEEEGWID TDGDGIREKV IDGVIVPFRF RLCYYVKSVT AHTIADYVAT
551 ACKEIGIECS LLGLDMADLS QAFDEKNFDA LLMGWCLGIP PEDPRALWHS
601 EGAMEKGSAN VVGFHNEEAD KIIDRLSYEY DLKERNRLYH RFHEIIHEFA
651 PYAFLFSRHC SLLYKDYVKN IFVPTHRTDL IPEAQDETVN VTMVWLEKKE
701 DPCLSTS*

The cp6894 nucleotide sequence <SEQ ID 378> is:

1 ATGTATAAAA GATGTGTGCT AGATAAAATT TTAAAGGGGA TTGTCGCCGG
51 TTCTTTAATT TTGTTATACT GGTCCTCAGA CCTACTTGAA AGAGACATTA
101 AGTCGATAAA AGGTAACGTA AGAGATATTC AAGAAGACAT TCGTGAAATC
151 TCACGCGTAG TGAAACAACA GCAGACATCA CAAGCTATCC CTGCGGCACC
201 TGGGGTGATG CTCGCTCCTA AGCTCGTCAG AGACGAAGCT TTTGCTCTAC
251 TCTTTGGAGA TCCTAGTTAT CCTAATTTAC TTTCCCTAGA CCCCTATAAA
301 CAGCAGACTC TTCCTGAACT TCTAGGAACA AATTTCCACC CTCATGGTAT
351 CCTACGCACT GCCCATGTCG GAAAACCCGA AAATCTGAGC CCTTTTAATG
401 GCTTTGATTA TGTCGTGGGC TTTTACGATC TCTGTATTCC TAGTTTAGCT
451 TCTCCCCACG TAGGGAAATA CGAAGAATTT TCTCCAGATC TCGCTGTGAA
501 AATAGAAGAA CATCTTGTTG AAGATGGTTC TGGGGATAAA GAGTTTCACA
551 TCTATCTGAG GCCGAATGTT TTTTGGCGTC CTATAGATCC TAAGGCCCTT
601 CCAAAACACG TTCAGTTAGA CGAAGTATTT CAACGTCCTC ATCCTGTGAC
651 AGCTCATGAT ATTAAGTTTT TCTACGACGC TGTTATGAAC CCTTATGTAG
701 CAACCATGCG AGCAGTGGCT CTGCGCTCTT GTTATGAAGA TGTGGTTTCT
751 GTCTCAGTAG AAAACGATTT AAAATTAGTA GTCAGATGGA AAGCACACAC
801 GGTAATCAAT GAAGAAGGAA AGGAAGAGCG CAAAGTGCTC TACTCTGCAT
851 TTTCTAATAC CTTAAGCTTG CAGCCCCTCC CTAGATTTGT ATATCAGTAT
901 TTTGCTAACG GGGAAAAAAT CATTGAAGAT GAGAATATCG ATACCTACCG
951 AACCAATTCC ATTTGGGCGC AAAACTTCAC TATGCATTGG GCAAACAACT
1001 ATATTGTAAG TTGTGGAGCC TACTACTTTG CAGGGATGGA TGATGAGAAA
1051 ATCGTGTTTT CTAGAAATCC TGACTTCTAT GATCCTCTTG CGGCTCTTAT
1101 TGACAAGCGT TTCGTCTATT TTAAGGAAAG CACAGACTCC CTATTCCAAG
1151 ATTTTAAGAC AGGGAAAATA GACATCTCTT ACCTTCCACC CAACCAAAGA
1201 GATAATTTCT ATAGTTTTAT GAAAAGCTCC GCTTATAACA AACAGGTAGC
1251 TAAGGGAGGA GCCGTCCGTG AAACAGTCTC AGCAGATCGA GCATATACGT
1301 ACATAGGATG GAATTGCTTT TCATTATTTT TCCAAAGCCG ACAGGTGCGC
1351 TGTGCTATGA ACATGGCAAT CGATAGAGAG AGGATTATCG AACAGTGCTT
1401 GGATGGCCAA GGCTATACGA TTAGTGGGCC TTTTGCTTCG AGTTCTCCTT
1451 CTTATAATAA ACAGATCGAA GGGTGGCATT ATTCTCCAGA AGAAGCAGCT
1501 CGTCTCCTGG AAGAAGAGGG ATGGATAGAT ACCGATGGCG ATGGAATCCG
1551 AGAAAAAGTT ATCGATGGTG TGATTGTCCC GTTCCGTTTC CGTTTATGCT
1601 ATTATGTAAA GAGTGTCACC GCTCATACCA TTGCAGATTA CGTAGCTACT
1651 GCTTGTAAGG AAATCGGAAT CGAGTGTAGC CTTCTAGGAC TAGATATGGC
1701 CGATCTTTCG CAAGCTTTTG ATGAAAAGAA TTTCGATGCT CTTTTAATGG
1751 GATGGTGTTT AGGAATTCCT CCTGAGGATC CTAGGGCTTT ATGGCATTCT
1801 GAAGGGGCTA TGGAAAAGGG TTCAGCGAAT GTTGTAGGTT TCCATAATGA
1851 AGAAGCTGAT AAAATCATAG ACAGACTCAG CTACGAATAC GATCTGAAAG
1901 AACGTAATCG CCTGTACCAC CGTTTCCATG AAATTATTCA TGAGGAAGCT
1951 CCTTATGCTT TCTTGTTCTC ACGACATTGT TCCTTACTTT ATAAGGATTA
2001 TGTAAAAAAT ATTTTCGTAC CTACACATAG AACAGATTTA ATTCCTGAAG
2051 CTCAGGATGA GACTGTCAAC GTAACTATGG TATGGCTTGA GAAGAAGGAG
2101 GATCCGTGCT TAAGTACATC CTAA

The PSORT algorithm predicts inner membrane (0.162).

The protein was expressed in E. coli and purified as a his-tag product (FIG. 189A) and also in GST/his form. The recombinant proteins were used to immunise mice, whose sera were used in a Western blot (FIG. 189B) and for FACS analysis.

These experiments show that cp6894 is a surface-exposed and immunoaccessible protein, and that it is a useful immunogen. These properties are not evident from the sequence alone.

Example 190

The following C. pneumoniae protein (PID 4377193) was identified in the 2D-PAGE experiment <SEQ ID 379; cp7193>:

1 MKRVIYKTIF CGLTLLTSLS SCSLDPKGYN LETKNSRDLN QESVILKENR
51 ETPSLVKRLS RRSRRLFARR DQTQKDTLQV QANFKTYAEK ISEQDERDLS
101 FVVSSAAEKS SISLALSQGE IKDALYRIRE VHPLALIEAL AENPALIEGM
151 KKMQGRDWIW NLFLTQLSEV FSQAWSQGVI SEEDIAAFAS TLGLDSGTVA
201 SIVQGERWPE LVDIVIT*

A predicted leader peptide is underlined.

The cp7193 nucleotide sequence <SEQ ID 380> is:

1 ATGAAAAGAG TCATTTATAA AACCATATTT TGCGGGTTAA CTTTACTTAC
51 AAGTTTGAGT AGTTGTTCCC TGGATCCTAA AGGATATAAC CTAGAGACAA
101 AAAACTCGAG GGACTTAAAT CAAGAGTCTG TTATACTGAA GGAAAACCGT
151 GAAACACCTT CTCTTGTTAA GAGACTCTCT CGTCGTTCTC GAAGACTCTT
201 CGCTCGACGT GATCAAACTC AGAAGGATAC GCTGCAAGTG CAAGCTAACT
251 TTAAGACCTA CGCAGAAAAG ATTTCAGAGC AGGACGAAAG AGACCTTTCT
301 TTCGTTGTCT CGTCTGCTGC AGAAAAGTCT TCAATTTCGT TAGCTTTGTC
351 TCAGGGTGAA ATTAAGGATG CTTTGTACCG TATCCGAGAA GTCCACCCTC
401 TAGCTTTAAT AGAAGCTCTT GCTGAAAACC CTGCCTTGAT AGAAGGGATG
451 AAAAAGATGC AAGGCCGTGA TTGGATTTGG AATCTTTTCT TAACACAATT
501 AAGTGAAGTA TTTTCTCAAG CTTGGTCTCA AGGGGTTATC TCTGAAGAAG
551 ATATCGCCGC ATTTGCCTCC ACCTTAGGTT TGGACTCCGG GACCGTTGCG
601 TCCATTGTCC AAGGGGAAAG GTGGCCCGAG CTTGTGGATA TAGTGATAAC
651 TTAA

The PSORT algorithm predicts periplasmic (0.925).

This shows that cp7193 is an immunoaccessible protein in the EB and that it is a useful immunogen. These properties are not evident from the protein's sequence alone.

It will be appreciated that the invention has been described by way of example only and that modifications may be made whilst remaining within the spirit and scope of the invention.

TABLE II
sequences of the primers used to amplify Cpn genes.
Orf ID	N-terminus final primer	C-terminus final primer
CP0014P	GCGTC CCG GGTCATATG AAGTCTTCTTTCCCCA	GCGT CTC GAG ATGAAAGAGTTTTTGCG
CP0015P	GCGTCCCGGGTCATATG TCAGCTCTGTTTTCTGA	GCGT CTC GAG GAATTGGTATTTTGCTC
CP0016P	GCGTCCCGGGTCATATG GCCGATCTCACATTAG	GCGT CTC GAG GTCCAAGTTAAGGTAGCA
CP0017P	GCGT CCG GGTCATATG GGTATCAAGGGAACTG	GCGT CTC GAG AAATCCGAATCTTCC
CP0019P	GCGTCCCGGGTCAT ATGCAAGACTCTCAAGACTATAG	GCGT CTC GAG AAATCGGTATTTACCC
CP6260P	GCGTC CCG GGT GCTAGCACTACGATTTCTTTAACCC	GCGT CTC GAG AAAACGAAATTTGCTTC
CP6397P	GCGTC CCG GGTCATATGTTTAAACTGCTAAAAAATCTATT	GCGT CTC GAG ATGAAAGAAGAGTCCTCG
CP6456P	GCGTC CCG GGT CATATG TCATCTCCTGTAAATAACA	GCGT CTC GAG CTGACCATCTCCTGTT
CP6466P	GCGTC CCG GGT CAT ATG TGCAAGGAGTCCAGT	GCGT CTC GAG ATTTTCCTTAGCATAACG
CP6467P	GCGTC CCG GGT CAT ATG TGTTCCCCATCCCAA	GCGT CTC GAG TAGTTTTTCTATAAAACGAAAGTCT
CP6468P	GCGTC CCG GGT CAT ATG TGCTCCTCCTACTCTTC	GCGT CTC GAG GGGGAAATAGGTATATTTGA
CP6469P	GCGTC CCG GGT CAT ATG AGCTGCTCAAAGCAA	GCGT CTC GAG ACTTAAGATATCGATATTTTTGA
CP6552P	GCGTC CCG GGT CAT ATG TGCCATAAGGAAGATG	GCGT CTC GAG ACCATTGTCTTGAGTCAT
CP6567P	GCGTC CCG GGT CAT ATG ACCTCACCGATCCCC	GCGT CTC GAG AGAAGCCGGTAGAGGC
CP6576P	GCGTC CCG GGT CAT ATG ACTGAAAAAGTTAAAGAAGG	GCGT CTC GAG GAA CATGCCCCCTAA
CP6727P	GCGTC CCG GGT CATATGCTACATCCACTAATGGC	GCGT CTC GAG GAAAGAATAACGAGTTCC
CP6729P	GCGTC CCG GGT CAT ATGGCAGATGCTTCTTTATC	GCGT CTC GAG GAATGAGTATCTTAGCC
CP6731P	GCGTC CCG GGT CATATGGCTGTTGTTGAAATCAAT	GCGTC CAT GGC GGC CGC GAACTGGAACTTACCTCC
CP6736P	GCGTC CCG GGT GCT AGCGTAGAAGTTATCATGCCTT	GCGTC CAT GGC GGC CGC AAATCGTAATTTGCTTC
CP6737P	GCGT GGA TCC CAT ATG GAGACTAGACTCGGAGG	GCGT CTC GAG AAATGTGGATTTTAGTCC
CP6751P	GCGTC CCG GGT GCT AGC AATGAAGGTCTCCAACT	GCGT CTC GAG AAATCTCATTCTACTCGC
CP6752P	GCGTGA ATT CAT ATGTTCGGGATGACTCCT	GCGT CTC GAG GAATTTTAAGGTACTTCCTG
CP6753P	GCGTC CCG GGT GCT AGCACTCCCTACTCTCATAGAG	GCGT CTC GAG AAACTTAAAGGTCGTTC
CP6767P	GCGTC CCG GGT CAT ATG ATAAAACAAATAGGCCGT	GCGT CTC GAG TTCGTAAGCAACTTCAGA
CP6829P	GCGTC CCG GGT CAT ATG AAGCAGATGCGTCTTT	GCGTC CAT GGC GGC CGC GAAACTAAGGGAGAGGC
CP6830P	GCGTC CCG GGT CAT ATG GATCCCGCGTCTGTT	GCGTC CAT GGC GGC CGC GAATACAAACCGGATCC
CP6832P	GCGTC CCG GGT CAT ATG CATAAAGTAATAGTTTTCATTT	GCGT CTC GAG TAAACTAGAAAAAGTCGTC
CP6848P	GCGTC CCG GGT CAT ATG TCATCAAATCTACATCCC	GCGT CTC GAG AACGCGAGCTATTTTAC
CP6849P	GCGTC CCG GGT GCT AGC AGCGGGGGTATAGAG	GCGT CTC GAG ATACACGTGGGTATTTTC
CP6850P	GCGTC CCG GGT CAT ATG TGCCGCATTGTAGAT	GCGT CTC GAG CTGTTTGCATCTGCC
CP6854P	GCGTC CCG GGT GCT AGC TCAATAGCTATTGCAAG	GCGT CTC GAG TTATCGAAATGTCTTTG
CP6879P	GCGTC CCG GGT CAT ATG GCAACACCCGCTCAA	GCGTC CAT GGC GGC CGC TCCTTGAAATTGCTCTTGC
CP6894P	GCGTC CCG GGT CAT ATG TATAAAAGATGTGTGCTAGA	GCGT CTC GAG GGATGTACTTAAGCACG
CP6900P	GCGTC CCG GGT CAT ATG AAGATAAAATTTTCTTGGAAG	GCGT AAG CTT GGGAAGACGATACCG
CP6952P	GCGTC CCG GGT CAT ATG CTCTCGGATCAATATATAGG	GCGT CTC GAG TCGAATTTCTTTTTTAGC
CP7034P	GCGTC CCG GGT CAT ATG AAAAAACAGGTATATCAATG	GCGT AAG CTT AAACGCTGAAATTATACC
CP7090P	GCGTC CCG GGT CAT ATG TGTAGCCTTTCCCCT	GCGT CTC GAG GCGTGCATGAATCTTA
CP7091P	GCGTC CCG GGT CAT ATG GAAGAATTAGAAGTTGTTGT	GCGT CTC GAG TAGTGTTCTCTTTATCGGT
CP7170P	GCGTC CCG GGT CAT ATG CTAGGGGCTGGAAACC	GCGT AAG CTT AAACTGCAGACCTGACG
CP7228P	GCGTC CCG GGT CAT ATG ACTGCTGTTCTTATTCTTACA	GCGT CTC GAG ATCTGAAAGCGGAGG
CP7249P	GCGTC CCG GGT CAT ATG ATCCCATCCCCTACC	GCGT CTC GAG ATCAGGTTGCTGAGACTT
CP7250P	GCGTC CCG GGT CAT ATG AATCTTTCAAACAGGTCT	GCGT CTC GAG ATTTTTTCTAGAGAGACTCTC
CP0018P	GTGCGT CATATG GCAACCACTCCACTAA	ACTCGCTA GCGGCCGC TAATGAGGTCCCCAG
CP6270P	GTGCGT CATATG AATTTATTAGGAGCTGCT	ACTCGCTA GCGGCCGC AAATTTGATTTTGCTACC
CP6735P	GTGCGT CATATG GCAGCACAAGTTGTATAT	ACTCGCTA GCGGCCGC TGGCGTAGAAGTGATC
CP6998P	GTGCGT CATATG TTGCCTGTAGGGAAC	ACTCGCTA GCGGCCGC GAATCTGAACTGACCAGA
CP7033P	GTGCGT CATATG GTTAATCCTATTGGTCCA	ACTCGCTA GCGGCCGC TTGGAGATAACCAGAATATA
CP7287P	GTGCGT CATATG TTACACAGCTCAGAACTAGA	ACTCGCTA GCGGCCGC GAAAATAATACGGATACCA
CP0010P	GTGCGT CATATG GCAACTGCTGAAAATATA	GCGT CTCGAG GAATTGGAACTTACCC
CP0468P	GTGCGT GCTAGC ATTTTTTATGACAAACTCTAT	GCGT CTCGAG AAATGTGCAATGACTCT
CP6272P	GTGCGT CATATG TTGACTCATCAAGAGGCT	GCGT CTCGAG GAAGGGAGGTTTTTTAGGT
CP6273P	GTGCGT CATATG ACATATCTGGAAGCTC	ACTCGCTA GCGGCCGC CTCCACAATTTTTATG
CP6362P	GTGCGT CATATG CCCTTTGATATTACTTATTATACA	GCGT CTCGAG TCGTTTCCAAATCCA
CP6372P	GTGCGT CATATG AAACAACACTATTCTCTAAATA	GCGT CTCGAG TTTCTTGTGGTTTTTCT
CP6390P	GTGCGT CATATG CGAGAGGTGCCTAAG	ACTCGCTA GCGGCCGC TCTCCTAGACAGCCTT
CP6402P	GTGCGT CATATG AATGTTGCGGATCTCCTTT	GCGT CTCGAG GAAGGGGTTGGCCGT
CP6446P	GTGCGT CATATG TGTAATCAAAAGCCCTCTT	GCGT CTCGAG GGGCTGAGGAGGAAC
CP6520P	GTGCGT GCTAGC AAACACTACCTATCATTTTCT	GCGT CTCGAG CAGAAAGGCTTTTCTTT
CP6577P	GTGCGT CATATG AATTTAGGCTATGTTAATTTA	GCGT CTCGAG GTTTTGTTTTTTGAAAGA
CP6602P	GTGCGT CATATG GCAGCATCAGGAGGCA	GCGT CTCGAG TGACCAAGGATAGGGTTTAG
CP6607P	GTGCGT CATATG CCTCGTGGTGACACTTT	GCGT CTCGAG CGCTGCTTCTTGCTC
CP6615P	GTGCGT CATATG TGCTCTCAAAAAACGACAA	GCGT CTCGAG TGAAGAGGCGCCATC
CP6624P	GTGCGT CATATG GATGCGAAAATGGGA	GCGT CTCGAG TCTTTGACATTCAAGAGC
CP6672P	GTGCGT CATATG ATTCCTACCATGTTAATG	GCGT CTCGAG GTCATACAATTTCCTTATATA
CP6679P	GTGCGT CATATG TGCACTCACTTAGGCT	GCGT CTCGAG CGAGTAGTTAGCACAAAC
CP6717P	GTGCGT GCTAGC AAGACAATCGTAGCTTCA	ACTCGCTA GCGGCCGC GGCTGGCATATAGGT
CP6784P	GTGCGT GCTAGC AAATCAAGATGTTCTATTGATA	GCGT CTCGAG TCCAAAACAACCCTCT
CP6802P	GTGCGT CATATG TGCGTAAGTTATATTAATTCCTT	GCGT CTCGAG CAGTCGGGCTTGTTG
CP6847P	GTGCGT CATATG TCGGATCTTTTACGAG	GCGT CTCGAG TTTCCTACACTGTTGTAATAAA
CP6884P	GTGCGT CATATG AATCAGCTGCTTTCT	GCGT CTCGAG AGAGAAGGTAATTGTACC
CP6886P	GTGCGT CATATG TGTCTACTTATTATCTATCTCTAC	GCGT CTCGAG TTCAGAAAAATGGCT
CP6890P	GTGCGT CATATG TCCCCACGACGACAA	GCGT CTCGAG TCCTGCAGCATTTAGC
CP6960P	GTGCGT CATATG TGTGACGTACGGTCTA	ACTCGCTA GCGGCCGC TTCACCTTGATTTCCT
CP6968P	GTGCGT CATATG TGCGATGCAAAAC	ACTCGCTA GCGGCCGC GGAAGTATGCTTAGATATT
CP6969P	GTGCGT CATATG TGCTGTGGTTACTCTATT	ACTCGCTA GCGGCCGC AAAAAGGTCATAGTATACCT
CP7005P	GTGCGT CATATG AAAACTGTGATATTGAACA	GCGT CTCGAG CTGAGCTTCTATTTCTATTAT
CP7072P	GTGCGT CATATG CCCATTTATGGGAAA	GCGT CTCGAG GTTGAGCAAAGGTTTG
CP7101P	GTGCGT CATATG TATTCGTGTTACAGCAA	GCGT CTCGAG GAAAAATTCTTTAGGGAG
CP7102P	GTGCGT CATATG GCCGCTAAAGCAAAT	GCGT CTCGAG TGAAAATGAAAGGATGGT
CP7105P	GTGCGT GCTAGC AGTCTATATCAAAAATGGTG	GCGT CTCGAG ATCTTTCATTTGGTTATCT
CP7106P	GTGCGT CATATG AAAGATTTGGGGACTCT	GCGT CTCGAG GAATCCTAAGGCATACCTA
CP7107P	GTGCGT GCTAGC AGTATAGTCAGAAATTCTGCA	GCGT CTCGAG GAAGCTAAGATTATAGCTACTTT
CP7108P	GTGCGT GCTAGC GCGGCCCTTTCCA	ACTCGCTA GCGGCCGC TTTATGTATATGGAACAGATAGG
CP7109P	GTGCGT CATATG GGACATTTTATTGATATTG	ACTCGCTA GCGGCCGC ATCATCAAGGTAGATAAAG
CP7110P	GTGCGT CATATG GGTTATTGCTATGTAATTACA	GCGT CTCGAG TTCTGATTGGACTCCA
CP7127P	GTGCGT CATATG GTGGCTTTAACGATAGC	ACTCGCTA GCGGCCG GCAGCCATCGTATTC
CP7130P	GTGCGT CATATG TTCAATATGCGAGG	GCGT CTCGAG CTTCTTATTTGAACTTTG
CP7140P	GTGCGT CATATG ACAGCCGGAGCAGCT	GCGT CTCGAG AGCACCCTCAATTTCATTG
CP7182P	GTGCGT CATATG GGATATGTTTTCTATGTGATC	GCGT CTCGAG GCTACTAAATCGAATCGA
CP6262P	GTGCGT CATATG ATCCCTGGATTAAGTTCA	ACTCGCTA GCGGCCGC TTCACTGGGAGCTTGA
CP6269P	GTGCGT CATATG TACCAGGAGAATCTAAGAT	ACTCGCTA GCGGCCGC GATTTTCTTCTTCAGCTC
CP6296P	GTGCGT CATATG GAGGAGGTGTCTGAGTAT	ACTCGCTA GCGGCCGC ATGTTTCTTTTTACTCTTTCT
CP6419P	GTGCGT CATATG GCTCCAGTCCGTGTT	GCGT CTCGAG AAGTGTTCGTTGGAAGT
CP6601P	GTGCGT CATATG AATAAGCTACTCAATTTCGT	GCGT CTCGAG GAAAATCTGAATTCTTCCT
CP6639P	GTGCGT CATATG TTAAATTCAAGCAATTCA	GCGT CTCGAG AGGAACTAAAACCTCATCT
CP6664P	GTGCGT GCTAGC GTTTTATTTCATGCTCAA	ACTCGCTA GCGGCCGC CTTAGAAAGACTATTTTCTAAGTA
CP6696P	GTGCGT CATATG TGCGTGATAATGGG	GCGT CTCGAG ATTCATCTTCGTAAAGAAT
CP6757P	GTGCGT CATATG GCAGTTGGTGGCGT	ACTCGCTA GCGGCCGC CTGTCCCTCTGGAGC
CP6790P	GTGCGT GCTAGC AGTGAACACAAAAAATCA	ACTCGCTA GCGGCCGC CTTATCGTCGTTATCAATA
CP6814P	GTGCGT CATATG CATGACGCACTTCTAAG	GCGT CTCGAG TACAGCTGCGCGA
CP6834P	GTGCGT CATATG GTTATGGGAACCTATATCG	GCGT CTCGAG TACATTTGTATTGATTTCAG
CP6878P	GTGCGT CATATG AACGTCCCTGATTCC	GCGT CTCGAG GCTAGCGGCTCTTTC
CP6892P	GTGCGT CATATG CAGAAGCATCCTTCCT	ACTCGCTA GCGGCCGC TCCTCTTTAGGAAATGG
CP6909P	GTGCGT CATATG TCCTCTTTAGGAAATGG	GCGT CTCGAG CAGTGCCAAGTAGGGA
CP7015P	GTGCGT CATATG GCAGTACGATTAATTGTTG	GCGT CTCGAG TTTATTGTAGTCTATTTTATATTTC
CP7035P	GTGCGT GCTAGC AGCAGAAAAGACAATGA	GCGT CTCGAG ATTTTGAGTGTCTTGCA
CP7073P	GTGCGT CATATG ATTACCATAAATCACGTG	GCGT CTCGAG TATCCATCGACTTATAGC
CP7085P	GTGCGT GCTAGC TGTATTTTCCCTTACGTA	ACTCGCTA GCGGCCGC GGATTCTGCATACTCTG
CP7092P	GTGCGT CATATG TCTCCTCTTCCTAAAAAA	GCGT CTCGAG GGATTCATTACTGACCA
CP7093P	GTGCGT CATATG AAATACCGTTCACG	GCGT CTCGAG ATTCTGTAGGGCTACGT
CP7094P	GTGCGT CATATG GTACACTTCTCTCATAACCC	GCGT CTCGAG TAAGTTTGTATTGCGGTAT
CP7132P	GTGCGT CATATG TTGTTATTAGGGACTTTAGGA	GCGT CTCGAG TTTCCCAACCGCA
CP7133P	GTGCGT CATATG GCTGCGAATGCTC	GCGT CTCGAG TAATTCAATACTCTTTGAAGG
CP7177P	GTGCGT CATATG CCTACTCAAGTTAAAACAGA	GCGT CTCGAG AAGTTTATATTTCAGCACTT
CP7184P	GTGCGT GCTAGC CATATAGGATTTTGCCA	GCGT CTCGAG GTACTTAGCAAAGCGAT
CP7206P	GTGCGT GCTAGC AAGAAGCTATATCACCCTA	GCGT CTCGAG CACACCGAGGAAAC
CP7222P	GTGCGT CATATG GTAGTTTCAGAAGAAAAAAGTC	GCGT CTCGAG ACGTATGCGCAACTG
CP7223P	GTGCGT CATATG GAAGTATTAGACCGCTCT	GCGT CTCGAG CGAGAAAAAGCTTCC
CP7224P	GTGCGT CATATG ATGAAGAAAATTCGAAA	ACTCGCTA GCGGCCGC TAAGCATTCACAAATGA
CP7225P	GTGCGT CATATG CATATTTTGCTTGATCGT	GCGT CTCGAG TCTTTTAACTAAATCTTGTTCTT
CP7303P	GTGCGT CATATG CTTGTCTATTGTTTTGATCC	GCGT CTCGAG AAAATATACGGAACTCGC
CP7304P	GTGCGT GCTAGC GAAGTTTATAGTTTTTCCC	GCGT CTCGAG TTTTTGATTCCTTAAGAAG
CP7305P	GTGCGT CATATG GAAGTTTATAGTTTTCACCCT	GCGT CTCGAG ACTCCTTGAGAAGGGAA
CP7307P	GTGCGT CATATG CTTAATCATGCTAAAAAGC	ACTCGCTA GCGGCCGC CTCTTTTATTTTAGGAAGCT
CP7342P	GTGCGT CATATG AAAAAAAAATTTATTTTCTACT	ACTCGCTA GCGGCCGC CACACTCTGTTCTTCTG
CP7347P	GTGCGT CATATG TTTTCTAAGGATTTGACTAA	GCGT CTCGAG CGAAGCAGAAGTCGT
CP7353P	GTGCGT CATATG AATATGCCTGTTCCTTCT	GCGT CTCGAG GGGGCGTAGGTTGTA
CP7193P	GTGCGT CATATG TGTTCCCTGGATCCT	ACTCGCTA GCGGCCGC AGTTATCACTATATCCACAAG
CP7248P	GTGCGT GCTAGC CTTGAACATTCTAAACAAGAT	GCGT CTCGAG ACGTAGTTTAAGAGCAGACT
CP7261P	GTGCGT CATATG TGTCTATCTGCCTACATAG	GCGT CTCGAG TTTTGATGCTTCTTTCA
CP7280P	GTGCGT CATATG GACCAGAAAATTGAAAA	GCGT CTCGAG AGAGGTCTTCTGAGTGC
CP7302P	GTGCGT CATATG AATTTCCATTGTAGTGTAGT	GCGT CGCGAG GAACAGTTCGATTTGTG
CP7306P	GTGCGT CATATG CTTCCTTTATCAGGGCA	ACTCGCTA GCGGCCGC TTCTTCAGGTTTCAGG
CP7367P	GTGCGT GCTAGC CGTTATGCCGAGGTC	GCGT CTCGAG TTCGTGCATTTGGTG
CP7408P	GTGCGT CATATG TTGAAAATCCAGAAAAA	GCGT CTCGAG ATTCATTTTCGGAAGAG
CP7409P	GTGCGT CATATG AGACGTTATCTTTTCATGGT	GCGT CTCGAG CCCTTTGCTCTTTACATAG
CP6733P	GTGCGT ACTAGT TGTCACCTACAGTCACTAG	GCGT CTCGAG GAATCGGAGTTTGGTA
CP6728P	GTGCGT ACTAGT AAGTCCTCTGTCTCTTGG	GCGT CTCGAG GAAACAAAACTTAGAGCCC

TABLE III
Proteins with best results in FACS analysis
	Molecular Weight (kDa)
cp number	Theoretical	Western Blot	Fusion type
6260	97.5	94; 70	GST
6270	87.5	—	GST
6272	78.0	90	GST
6273	58.6	74; 64; 50	GST
6296	31.1	—	GST
6390	88.9	102	GST
6456	42.5	89; 67, 45	GST
6466	57.5	59; 56	His
6467	59.0	67	GST
6552	28.4	50; 27	GST
6576	86.0	79; 70; 62; 45	GST
6577	17.3	12	GST
6602	43.4	53; 42; 34	GST
6664	54.5	104; 45	GST
6696	47.9	95; 53	GST
6727	130.0-142.9	123; 61; 39	His
6729	94.8	multiple bands	GST
6731	95.5	97	GST
6733	97.1	104	His
6736	100.1	98; 93; 66; 60	GST
6737	101.2	multiple bands	GST
6751	100.2	95; 71	GST
6752	102.1	97; 48	His
6767	29.1	28	GST
6784	32.9	35	GST
6790	71.3	multiple bands	His
6802	29.7	—	GST
6814	29.6	28	GST
6830	177.4	174; 91; 13	GST
6849	57.3	multiple bands	GST
6850	7.4-9.4	61; 14; 8	GST
6854	42.2	—	GST
6878	40.4	—	GST
6900	28.0	—	GST
6960	25.6	75; 35	GST
6968	34.6	83; 53; 35	GST
6998	39.3	multiple bands	GST
7033	68.2	multiple bands	GST
7101	113	105	GST
7102	63.4	—	GST
7105	29.2	30	GST
7106	39.5	72; 46	GST
7107	71.4	67; 31	His
7108	35.9	35	GST
7111	46.1	51	GST
7132	17.9	57; 47; 17	His
7140	36.2-29.8	50; 38; 34	GST
7170	34.4	77; 33	GST
7224	39.4	40	GST
7287	167.3	180	GST
7306	50.1	50	GST

TABLE IV
FACS-positive proteins not found in C. trachomatis
cp7105 cp6390
cp7106 cp6784
cp7107 cp6296
cp7108

TABLE V
Proteins identified by MALDI-TOF following 2D electrophoresis
cp6270
cp6552
cp6576
cp6577
cp6602
cp6664
cp6727
cp6728
cp6729
cp6733
cp6736
cp6737
cp6752
cp6767
cp6784
cp6790
cp6830
cp6849
cp6900
cp6960
cp6998
cp7033
cp7108
cp7111
cp7170
cp7287
cp7306

Claims

1. A protein comprising an amino acid sequence selected from the group consisting of SEQ IDs 97, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, & 377.

2. A protein having 50% or greater sequence identity to a protein according to claim 1.

3. A protein comprising a fragment of an amino acid sequence selected from the group consisting of SEQ IDs 97, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, & 377.

4. A nucleic acid molecule which encodes a protein according to any one of claims 1 to 3.

5. A nucleic acid molecule according to claim 4, comprising a nucleotide sequence selected from the group consisting of SEQ IDs 98, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, & 378.

6. A nucleic acid molecule comprising a fragment of a nucleotide sequence selected from the group consisting of SEQ IDs 98, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, & 378.

7. A nucleic acid molecule comprising a nucleotide sequence complementary to a nucleic acid molecule according to any one of claims 4 to 6.

8. A nucleic acid molecule comprising a nucleotide sequences having 50% or greater sequence identity to a nucleic acid molecule according to any one of claims 4 to 7.

9. A nucleic acid molecule which can hybridise to a nucleic acid molecule according to any one of claims 4 to 8 under high stringency conditions.

10. A composition comprising a protein or a nucleic acid molecule according to any preceding claim.

11. A composition according to claim 10 being a vaccine composition.

12. A composition according to claim 10 or claim 11 for use as a pharmaceutical.

13. The use of a composition according to claim 10 in the manufacture of a medicament for the treatment or prevention of infection due to Chlamydia bacteria, particularly Chlamydia pneumoniae.