MORAXELLA CATARRHALIS ANTIGENS

Abstract

The present invention relates to isolated nucleic acid molecules which encode an antigen from a Moraxella catarrhalis (Meat) species, a vector which comprises such nucleic acid molecule, and a host cell comprising such vector. Furthermore, the invention provides antigens from a Moraxella catarrhalis species, as well as fragments and variants thereof, a process for producing such antigens, and a process for producing a cell which expresses such antigen. More specifically, such antigens are produced by or associated with bacterial infections caused by Moraxella catarrhalis. Moreover, the present invention provides antibodies binding to such antigen, a hybridoma cell producing such antibodies, methods for producing such antibodies, a pharmaceutical composition comprising such nucleic acid molecule, antigen, vector or antibody, the use of such nucleic acid molecule, antigen, vector or antibody for the preparation of a pharmaceutical composition, methods for identifying an antagonist capable of binding such antigen or of reducing or inhibiting the interaction activity of such antigen, methods for diagnosing an infection with Moraxella catarrhalis and methods for the treatment or prevention of an infection with Moraxella catarrhalis.

Description

The present invention relates to isolated nucleic acid molecules which encode an antigen from a Moraxella catarrhalis (Mcat) species, a vector which comprises such nucleic acid molecule, and a host cell comprising such vector. Furthermore, the invention provides antigens from a Moraxella catarrhalis species, as well as fragments and variants thereof, a process for producing such antigens, and a process for producing a cell which expresses such antigen. More specifically, such antigens are produced by or associated with bacterial infections caused by Moraxella catarrhalis. Moreover, the present invention provides antibodies binding to such antigen, a hybridoma cell producing such antibodies, methods for producing such antibodies, a pharmaceutical composition comprising such nucleic acid molecule, antigen, vector or antibody, the use of such nucleic acid molecule, antigen, vector or antibody for the preparation of a pharmaceutical composition, methods for identifying an antagonist capable of binding such antigen or of reducing or inhibiting the interaction activity of such antigen, methods for diagnosing an infection with Moraxella catarrhalis and methods for the treatment or prevention of an infection with Moraxella catarrhalis.

Moraxella catarrhalis (Mcat) is a Gram-negative bacterium. Microscopically, the organism appears as a diplococcus, and grows aerobically. Previously used names for Mcat include Branhamella catarrhalis, Neisseria catarrhalis, Micrococcus catarrhalis and Mikrokokkus catarrhalis (Mawas et al., 2009; Karalus et al., 2000; Catlin 1990). Mcat has a genome of approximately 1.9 Mbp, comprising approximately 1,800 open reading frames (Ruckdeschel et al., 2008). Different types of Mcat can differentiated by molecular methods such as multilocus sequencing, sequencing ribosomal RNA, pulsed field gel electrophoresis, and mass spectrometric analysis of membrane proteins (Schaller et al., 2006; Pingault et al., 2007; Pingault et al., 2008; Verduin et al., 2002). The lipooligosaccharide of Mcat is thought too conserved for practical serotyping, but nevertheless three different lipooligosaccharide serotypes have been described (Verduin et al., 2002).

Mcat is identified based on a combination of microbiological culture criteria, such as oxidase production, absence of acid production from carbohydrates such as glucose, maltose, sucrose, lactose and fructose, DNase production, nitrate/nitrite reduction, and conversion of specific chromogenic enzyme substrates (Doern et al., 1980; Janda et al., 1987; Catlin 1990; Verduin et al., 2002). Also, Mcat can be diagnosed using PCR (Verduin et al., 2002).

Pathogenesis of Mcat Infection

Mcat is an obligate commensal of humans and possibly nonhuman primates (McMichael 2001; VandeWoude et al., 1991). Mcat is the third-most frequently isolated bacterium from human respiratory secretions, with Streptococcus pneumoniae and nontypeable Haemophilus influenzae being first and second, respectively (Karalus et al., 2000; Mawas et al., 2009; McMichael 2001; Verduin et al., 2002).

Mcat was originally thought to be an innocuous commensal of the human respiratory epithelium. However, over the last approximately 20 years it has been realized that Mcat is an important respiratory pathogen. Most children experience Mcat colonization by the age of 1 year (Murphy 2005; Tan et al., 2007a). In children, Mcat in the third-most frequent causative agent of otitis media after Streptococcus pneumoniae and nontypeable Haemophilus influenzae, being responsible for 15-20% of otitis media cases (Murphy 2005; Verduin et al., 2002; Tan et al., 2007a). Mcat also is a frequent cause of sinusitis in children (Murphy 2005; Verduin et al., 2002). More rarely, Mcat can cause bacteriaemia and meningitis in children (Karalus et al., 2000). In adults with chronic obstructive pulmonary disease (COPD), Mcat causes lower respiratory tract infections and exacerbation of the COPD (Verduin et al., 2002). Finally, Mcat can cause severe, systemic infections in immunocompromised patients, and is also a nosocomial pathogen (Verduin et al., 2002).

Before 1970, Mcat isolates were generally sensitive to beta lactam antibiotics, but resistance has developed quickly after the first detection of beta lactamase-producing Mcat in 1976-1977 (Verduin et al., 2002; Karalus et al., 2000). Currently, up to 90% of Meat isolates are beta lactamase positive and thus resistant to beta lactam antibiotics such as penicillin or amoxicillin (Budhani et al., 1998; Verduin et al., 2002; Tan et al., 2007a). Mcat has 2 species of beta lactamase enzymes termed BRO-1 and BRO-2. The Mcat beta lactamases are different from those found in other pathogenic bacteria, and have been suggested to have origin in lipoproteins of Gram-positive bacteria (Karalus et al., 2000; Verduin et al., 2002).

Beta lactamase secreted from Mcat can protect co-infecting bacteria such as Streptococcus pneumoniae or nontypeable Haemophilus influenzae against beta lactam antibiotics (Verduin et al., 2002; Mawas et al., 2009; Budhani et al., 1998). Also, Mcat can protect co-infecting bacteria such as nontypeable Haemophilus influenzae from complement-mediated lysis (Tan et al., 2007b).

Finally, in a mouse model of otitis media, coinfection with Mcat facilitated Streptococcus pneumoniae infection (Krishnamurthy et al., 2009). Thus, Mcat is thought to have a double importance for otitis media development: Not only does Mcat act as a primary pathogen, but Mcat potentially also facilitates clinical establishment of other pathogens such as Streptococcus pneumoniae or nontypeable Haemophilus influenzae.

Key to Mcat pathogenesis is the ability to bind to and replicate on the human respiratory tract epithelial layer. Interactions with epithelial cells occur through bacterial membrane adhesin proteins such as ubiquitous surface proteins A1 and A2 (UspA1 and UspA2), MID/Hag OMP CD and McaP (Tan et al., 2007a). Bacterial lipooligosaccharides also contribute to adhesion (Tan et al., 2007a). In some Mcat isolates, pili may contribute to adhesion (Verduin et al., 2002). Finally, Mcat expresses surface lactoferrin and transferrin receptors, to ensure an adequate iron supply, and allow replication in its human host (Verduin et al., 2002).

In vitro studies have shown that in addition to adhering to epithelial surfaces, Mcat can also invade bronchial and lung epithelial cells, as well as phagocyte-like type II lung alveolar cells (Slevogt et al., 2007). Studies on clinical biopsy specimens have confirmed that in vivo, Mcat is able to penetrate epithelial layers, and to persist at sub-epithelial and intracellular locations (Heiniger et al., 2007).

Following colonization, resistance to lysis by human complement is thought to help Mcat maintain its presence on epithelia despite the host immune response (Verduin et al., 2002). While horizontal transfer of the beta lactamase BRO1 and BRO2 genes occurs frequently between Mcat strains, complement resistance appears to be a multigene phenomenon, that is stably inherited and not easily exchanged between Mcat strains (Verduin et al., 2000; Bootsma et al., 2000).

Serum bactericidal antibodies are more frequently found in convalescent sera than in acute phase sera of adult patients with lower respiratory tract Mcat infection (Verduin et al., 2002). Likewise, Mcat-induced otitis media in children is associated with increases in serum anti-Mcat antibodies measured by ELISA (Verduin et al., 2002). In children, antibodies of the IgG3 subclass have been suggested to be involved in protection (Verduin et al., 2002). Finally, it is known that otitis media fluid contains locally produced IgG and IgA antibodies, i.e., mucosal immunity is thought to play a role in otitis media (Verduin et al., 2002). Nevertheless, the protective immune mechanisms in Mcat infections are as yet poorly explored, and while the presence of bactericidal antibodies are sometimes used for this purpose, there are as yet no firmly established in vitro correlates of protection (Murphy 2005).

Otitis Media

Otitis media (OM) is a disease of children. OM is very prevalent, affecting 75% of children before the age of 3 years (Cripps and Otczyk 2006). Clinically, acute OM is defined by an abrupt onset of middle ear effusion and inflammation, diagnosed by various methods to visualize bulging of the tympanic membrane, and presence of effusion in the middle ear cavity (Marcy 2004). Typically, acute OM is preceded by an upper respiratory tract infection 3-5 days prior, and acute OM is often associated with ear pain (Marcy 2004). In some children, OM becomes recurrent and persistent, causing problems with hearing, and speech and language development (Cripps and Otczyk 2006). Treatment of OM is initially symptomatic, with more severe cases requiring antibiotic treatment or tympanostomy tube placement and adenoidectomy (Marcy 2004; Cripps and Otczyk 2006). Due to the high prevalence, OM constitutes a major burden to children and the health system. OM is the main presentation in young children visiting medical practitioners, and OM is the reason for over half of the antibiotics prescribed to children below 3 years in the US (Cripps and Otczyk 2006). Finally, the prevalence of OM has been reported to have increased over the last decades in several industrialized countries such as Finland, USA and Australia (Cripps and Otczyk 2006).

While environmental or host factors compromising the integrity of the nasopharyngeal epithelium predispose to OM, the ultimate cause of OM is infectious, comprising bacteria (nontypeable Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis) as well as viruses (respiratory syncytial virus, rhinovirus, influenza virus, parainfluenza virus, metapneumovirus and coronavirus). Bacteria are isolated from the majority (˜80%) of OM cases. Mcat infection is currently thought to be responsible for up to 20% of acute otitis media cases (Karalus et al., 2000; Verduin et al., 2002; Tan et al., 2007a; Murphy 2005).

Vaccination is known to be protective against OM. For example, influenza virus vaccination can reduce acute OM incidence in children during the influenza season (Marcy 2004). Vaccination with a Streptococcus pneumoniae conjugate vaccine, containing polysaccharide antigens of 7 serotypes conjugated to a nontoxic diphtheria toxin variant, has been shown to reduce acute OM incidence (Marcy 2004; Cripps and Otczyk 2006). An 11-valent conjugate vaccine, containing polysaccharide antigens of 11 serotypes of Streptococcus pneumoniae conjugated to the protein D of nontypeable Haemophilus influenzae showed protection against acute otitis media caused by Streptococcus pneumoniae and nontypeable Haemophilus influenzae (Prymula et al., 2006). However, use of the 7-valent pneumococcal vaccine has been shown to cause “pathogen replacement”, i.e., increased incidence of acute OM cases caused by pneumococcal serotypes not covered by the vaccine, or caused by other pathogens such as nontypeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes and Staphylococcus aureus (Cripps and Otczyk 2006; Tan et al., 2007a). The phenomenon of “pathogen replacement” is a potential concern also for the 11-valent pneumococcal vaccine (Tan et al., 2007a). Pathogen replacement is also thought to provide selective pressure for the spread of penicillin resistance to nonvaccine pneumococcal serotypes (Cripps and Otczyk 2006).

Vaccination Against Otitis Media

Otitis media can be caused by Streptococcus pneumoniae, nontypeable Haemophilus influenzae and Moraxella catarrhalis. While Moraxella catarrhalis causes approximately 20% of otitis media cases, Mcat also has an indirect effect due to secretion of beta lactamase, and protection of nontypeable Haemophilus influenzae against complement lysis (Tan et al., 2007b). Finally, Mcat may increase in importance as an otitis media pathogen due to “pathogen replacement” phenomena associated with vaccines against other otitis media pathogens (Tan et al., 2007a).

Thus, a vaccine against Mcat would be valuable for prevention of otitis media, as well as the other clinical manifestations of Mcat infection such as lower respiratory infections in adults with COPD.

Based on the experience that pneumococcal polysaccharide conjugate vaccine promotes pathogen replacement (Cripps and Otczyk 2006; Tan et al., 2007a), it can be hypothesized that an ideal Mcat vaccine would comprise Mcat proteins (as opposed to carbohydrates), that are highly conserved across clinical strains.

A number of candidate Mcat vaccine antigens have been identified, comprising mainly proteins involved in adhesion (UspA1, Hag, OMP CD, McaP), proteins involved in serum resistance (UspA2), proteins involved in iron acquisition (TbpA, TbpB, CopB, LbpA, LbpB), and lipooligosaccharide (Murphy 2005; Mawas et al., 2009; Tan et al., 2007a).

Nevertheless, at most, 15 candidate antigens have been described, and they comprise essentially some of the most abundant Mcat surface proteins (Murphy 2005; Mawas et al., 2009; Tan et al., 2007a).

Thus, it appears very likely that additional vaccine candidate antigens based on Mcat proteins can be found by appropriately designed screening methods.

Based on current knowledge of Mcat pathogenesis outlined above, Mcat protein vaccine antigens should be sought amongst Mcat membrane proteins that are (i) expressed and surface exposed during colonization of human epithelia, (ii) induce antibody responses in humans, (iii) with antibodies preventing adhesion to human nasopharyngeal cells, and/or exhibiting bactericidal activity in vitro.

Preclinical in vivo confirmation of vaccine antigens could be done in animal models of otitis media, of which several are available, e.g. chinchilla, rats and mice (Murphy 2005; Mawas et al., 2009; Tan et al., 2007a).

A vaccine can contain a whole variety of different antigens. Examples of antigens are whole-killed or attenuated organisms, subfractions of these organisms/tissues, proteins, or, in their most simple form, peptides. Antigens can also be recognized by the immune system in form of glycosylated proteins or peptides and may also be or contain polysaccharides or lipids. Short peptides can be used since for example cytotoxic T-cells (CTL) recognize antigens in the form of short, usually 8-11 amino acids long, peptides in conjunction with major histocompatibility complex (MHC). B-cells can recognize linear epitopes as short as 4-5 amino acids, as well as three-dimensional structures (conformational epitopes).

In order to stimulate sustained antigen-specific immune responses, adjuvants need to trigger immune cascades that involve all cells of the immune system. Primarily, adjuvants act, but are not restricted in their mode of action, on so-called antigen presenting cells (APCs). These cells usually first encounter the antigen(s) followed by presentation of processed or unmodified antigen to immune effector cells. Intermediate cell types may also be involved. Only effector cells with the appropriate specificity are activated in a productive immune response. The adjuvant may also locally retain antigens and other factors that may be co-injected. In addition, the adjuvant may act as a chemoattractant for other immune cells or may act locally and/or systemically as a stimulating agent for the immune system.

Antibodies naturally produced against Mcat by the human immune system and present in human sera are indicative of the in vivo expression of the antigenic proteins and their immunogenicity. The recognition of linear epitopes recognized by serum antibodies can be based on sequences as short as 4-5 amino acids. Of course it does not necessarily mean that these short peptides are capable of inducing the given antibody in vivo. For that reason the defined epitopes, polypeptides and proteins are further to be tested in animals (mainly in mice) for their capacity to induce T cells and antibodies against the selected proteins in vivo.

In addition to the T cell-mediated immune response, antibodies against cell wall proteins induced by B cell epitopes may aid the T cell-mediated immune response and serve multiple purposes: they may inhibit adhesion, interfere with nutrient acquisition, inhibit immune evasion and promote phagocytosis (Hornef, M. et al., 2002). Antibodies against secreted proteins are potentially beneficial in neutralization of their function as toxins or virulence components. It is also known that bacteria communicate with each other through secreted proteins. Neutralizing antibodies against these secreted proteins can interrupt growth-promoting cross-talk between or within the bacteria. The described experimental approach is based on the use of Mcat-induced antibodies which are purified from human sera. The antigens identified by the genomic screens, therefore, are shown to be both expressed in vivo in the host and to be capable of inducing an antibody response. Since it has been shown for many proteins that B cell and T cell epitopes reside in the same protein, the most promising candidates identified by the genomic screens can be further evaluated for the induction of a potent T cell response in vivo. As a first step, bioinformatic analyses have been used to identify potential T cell epitopes in silico, which can then be tested in an appropriate murine model of infection. Thus the present invention combines the experimental identification of immunogenic proteins with the bioinformatic prediction of T cell epitopes in order to provide candidates for an efficient vaccine to treat or prevent Mcat infections.

The problem underlying the present invention was to provide means for the development of pharmaceutical compositions such as vaccines against infections caused by Mcat. More particularly, the problem was to provide an efficient, relevant and comprehensive set of nucleic acid molecules or antigens, or fragments or variants thereof, from Mcat that can be used for the preparation of said pharmaceutical compositions. A still further problem was to provide methods and means for producing an antigen, a fragment or variant thereof. Yet another problem was to provide pharmaceutical compositions comprising said nucleic acids or said antigens. A still further problem of the invention was to provide antibodies, pharmaceutical compositions comprising said antibodies, methods for the production of said antibodies and the use of said antibodies for the preparation of a pharmaceutical preparation. Furthermore, the object of the present invention was to provide methods for identifying an antagonist capable of binding an antigen, or a fragment or variant thereof, as well as to provide methods for identifying an antagonist capable of reducing or inhibiting the interaction activity of such an antigen to its interaction partner. A further problem of the present invention was to provide methods for diagnosing an infection with an Mcat organism. Still another problem underlying the invention was to provide methods for treating Mcat infections, and to provide methods for immunizing an animal or human.

The problem underlying the present invention is solved in one aspect by an isolated nucleic acid molecule encoding an antigen or a fragment thereof, comprising a nucleic acid sequence, which is selected from the group consisting of:

• • a) a nucleic acid molecule having at least 70% sequence identity to a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NOs 860 to 886 and 1 to 214,
  • b) a nucleic acid molecule which is complementary to the nucleic acid molecule of a),
  • c) a nucleic acid molecule comprising at least 15 sequential bases of the nucleic acid molecule of a) or b),
  • d) a nucleic acid molecule which anneals under stringent hybridisation conditions to the nucleic acid molecule of a), b), or c), and
  • e) a nucleic acid molecule which, but for the degeneracy of the genetic code, would hybridise to the nucleic acid molecule defined in a), b), c), or d).

In an embodiment of the invention the sequence identity to SEQ ID NOs 860 to 886 or 1 to 214 is at least 80%, more preferably at least 90%, still more preferably at least 95%, 96%, 97%, 98%, or 99%, or most preferably 100%.

In an embodiment the nucleic acid is DNA.

In an alternative embodiment the nucleic acid is RNA.

In still another embodiment the nucleic acid molecule is isolated from a genomic DNA, preferably from an Mcat species.

In an embodiment of the invention the encoded antigen fragment is an active fragment or an active variant thereof.

In an embodiment the nucleic acid encodes an antigen or fragment thereof, which comprises or consists of a polypeptide or peptide fragment from Mcat.

The problem underlying the present invention is further solved by a vector comprising a nucleic acid molecule as described above.

In an embodiment the vector is adapted for recombinant expression of the antigen, or fragment thereof, encoded by the nucleic acid molecule as defined above.

The present invention also relates to a host cell comprising the vector as defined above.

The problem underlying the present invention is solved in a further aspect by an antigen that is immunologically reactive with sera from a human having an Mcat infection, or an uninfected healthy human who was previously infected with Mcat, wherein the antigen comprises an isolated polypeptide or an active fragment or an active variant thereof from Mcat.

The term “uninfected healthy human” as used herein comprises those individuals who have or had multiple encounters with the pathogen, which may result in colonization, but which either do not result in any symptoms, or which result in mild diseases. Said term and the rationale of selecting sera of uninfected healthy humans for antigen identification is further defined in Nagy, E. et al., (2003).

Another aspect of the present invention relates to an antigen, comprising or consisting of an isolated polypeptide selected from the group consisting of SEQ ID NOs 887 to 913 and 215 to 428, or an active fragment or an active variant thereof.

In an embodiment of the invention said polypeptide is encoded by a nucleic acid molecule as defined above.

In another embodiment the active fragment of the antigen consists of at least 50%, especially at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, 96%, 97% or 98%, most preferably 99% of said polypeptide, especially of a polypeptide as defined by any of the SEQ ID NOs 887 to 913 or 215 to 428.

In an embodiment the active variant of the antigen has at least 50%, especially at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, 96%, 97% or 98%, most preferably 99% sequence identity to said polypeptide, especially to a polypeptide as defined by any of the SEQ ID NOs 887 to 913 or 215 to 428.

The additional amino acid residue(s) may be homologous to the antigen as defined above. Homologous refers to any amino acid residue(s) which is/are identical or similar to the amino acid sequence of the Mcat antigen from which the fragment is derived.

Alternatively or additionally, the polypeptide may comprise or consist of the antigen, optionally the additional sequence as defined above and at least one amino acid residue heterologous to the antigen.

In an embodiment of the invention, the antigen further comprises or consists of at least one amino acid residue heterologous to the antigen, preferably an amino acid sequence of a marker protein.

The additional sequence or amino acid residue(s) as defined above consist(s) of (an) amino acid residue(s), which may be any amino acid, which may be either an L- and/or a D-amino acid, naturally occurring and otherwise. Preferably the amino acid is any naturally occurring amino acid such as alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine.

However, the amino acid may also be a modified or unusual amino acid. Examples of those are 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine or ornithine. Additionally, the amino acid may be subject to modifications such as posttranslational modifications. Examples of modifications include acetylation, amidation, blocking, formylation, gamma-carboxyglutamic acid hydroxylation, glycosylation, methylation, phosphorylation and sulfatation. If more than one additional or heterologous amino acid residue is present in the peptide, the amino acid residues may be the same or different from one another.

The feature “heterologous amino acid” or “amino acid heterologous to the antigen” refers to any amino acid which is different from that amino acid located adjacent to the antigen in any naturally occurring protein of Mcat. Therefore, the protein of the invention encompassing at least one heterologous amino acid refers to a protein which is different from any naturally occurring protein of Mcat or fragments thereof.

In one embodiment, the additional amino acid residue(s) is/are flanking the antigen N-terminally, C-terminally or N- and C-terminally.

In another embodiment, the antigen further comprises or consists of either a leader or a secretory sequence, a sequence employed for purification, or a proprotein sequence.

The problem underlying the present invention is solved in another aspect by an antigen, whereby the antigen comprises a core amino acid sequence as indicated in Table 4, whereby more preferably the core amino acid sequence is selected from the group consisting of:

amino acids 4-10, 12-40, 46-52, 61-76, 78-94, 96-103, 107-114, 126-133, 135-150, 153-162, 167-174, 182-195, 199-208, 210-218, 226-244, 247-255, 262-276, 285-292, 9-99, 108-128, 132-169, 179-286, and 17-72 of SEQ ID NO 215; amino acids 7-17, 25-32, 36-53, 66-76, 83-91, 100-113, 124-131, 151-158, 168-179, 184-198, 213-225, 227-237, 247-256, 261-270, 275-281, 284-293, 295-305, 315-329, 345-358, 388-415, 436-443, 449-456, 471-483, 1-168, 180-204, 212-253, 261-313, 326-467, 472-486, 10-60, 92-130, and 207-280 of SEQ ID NO 216; amino acids 45-53, 58-65, 70-78, 81-87, 94-100, 163-179, 184-192, 201-207, 212-222, 224-230, 254-260, 263-268, 18-158, 163-232, and 211-260 of SEQ ID NO 217; amino acids 4-12, 25-33, 40-50, 62-69, 74-85, 97-109, 127-138, 141-149, 153-161, 197-203, 206-215, 217-223, 258-265, 272-286, 308-316, 325-335, 343-358, 366-371, 373-381, 400-406, 417-426, 444-458, 466-486, 498-514, 522-539, 544-555, 561-572, 603-619, 621-638, 658-672, 687-695, 710-723, 731-744, 747-752, 764-769, 789-805, 808-823, 830-840, 873-885, 888-900, 902-920, 930-943, 964-970, 992-1003, 1005-1016, 1033-1038, 1045-1057, 1059-1068, 1089-1096, 1104-1110, 1115-1142, 1150-1156, 1158-1169, 1188-1198, 1200-1205, 1-80, 88-153, 167-222, 231-249, 256-289, 304-337, 340-488, 491-679, 681-780, 803-878, 880-942, 951-986, 988-1014, 1030-1106, 1109-1133, 1136-1179, 1183-1203, 128-158, 161-232, 277-314, 590-627, 712-848, 957-963, 1007-1033, and 1074-1093 of SEQ ID NO 218; amino acids 4-29, 45-56, 68-78, 84-97, 100-109, 127-141, 145-151, 173-179, 189-197, 202-209, 214-220, 238-263, 266-279, 288-297, 307-315, 330-336, 343-351, 358-367, 373-382, 388-403, 412-421, 423-429, 435-441, 449-459, 462-468, 488-501, 503-522, 536-542, 559-572, 603-611, 623-630, 675-681, 684-690, 705-719, 1-33, 45-204, 210-414, 431-480, 486-532, 536-582, 597-691, 704-724, 496-512, and 542-618 of SEQ ID NO 219; amino acids 14-24, 50-70, 76-92, 100-128, 130-143, 150-155, 178-187, 215-221, 233-251, 261-267, 275-288, 295-301, 319-324, 339-364, 368-384, 404-453, 461-472, 493-510, 522-532, 534-550, 570-577, 598-633, 641-655, 666-672, 676-682, 762-772, 793-808, 819-826, 836-858, 860-867, 872-896, 900-907, 909-918, 929-948, 961-974, 990-1003, 1005-1015, 1018-1025, 1066-1076, 1081-1090, 1100-1113, 1124-1131, 1146-1152, 1191-1197, 1202-1208, 1214-1231, 1237-1254, 1282-1310, 1313-1325, 1333-1353, 1365-1378, 6-26, 35-55, 58-163, 166-184, 190-252, 258-300, 305-493, 495-559, 567-587, 591-654, 672-750, 753-813, 827-866, 870-889, 898-983, 993-1011, 1038-1107, 1139-1162, 1168-1191, 1199-1256, 1261-1355, 1365-1387, 199-293, 420-438, 643-694, 719-771, 823-881, 1050-1144, and 1267-1339 of SEQ ID NO 220; amino acids 14-42, 50-65, 69-81, 96-109, 122-129, 146-161, 170-184, 194-200, 206-215, 221-234, 241-248, 264-270, 273-281, 288-299, 303-316, 320-338, 345-352, 365-372, 426-443, 475-484, 499-515, 518-524, 537-546, 555-563, 565-572, 582-588, 601-608, 612-629, 642-647, 662-683, 698-703, 706-711, 718-731, 739-746, 758-766, 776-782, 801-807, 825-835, 840-852, 878-886, 889-897, 908-915, 920-933, 935-946, 972-979, 982-992, 1003-1008, 1013-1023, 1030-1042, 1065-1081, 1095-1103, 1116-1126, 1132-1138, 1155-1173, 1176-1184, 1186-1199, 1203-1209, 1216-1224, 1242-1262, 1270-1282, 1295-1306, 1308-1316, 1342-1348, 1352-1357, 1-74, 84-228, 233-258, 264-383, 391-449, 451-490, 496-547, 559-610, 616-635, 640-731, 752-778, 799-862, 874-990, 1064-1110, 1114-1138, 1144-1167, 1183-1203, 1209-1230, 1241-1281, 1289-1363, 443-561, 644-713, 767-806, and 990-996 of SEQ ID NO 221; amino acids 4-19, 25-33, 36-51, 53-67, 105-110, 1-72, 74-108, and 55-108 of SEQ ID NO 222; amino acids 15-36, 42-96, 101-112, 3-115, and 4-23 of SEQ ID NO 223; amino acids 4-10, 12-22, 25-32, 35-48, 54-59, 65-70, 100-111, 115-126, 132-149, 156-164, 166-188, 197-207, 220-235, 243-265, 272-278, 309-318, 322-329, 340-353, 355-370, 378-388, 393-398, 410-417, 441-447, 449-455, 1-227, 248-378, 395-427, 445-459, and 310-399 of SEQ ID NO 224; amino acids 5-39, 1-40, and 3-52 of SEQ ID NO 225; amino acids 10-16, 20-40, 58-80, 83-89, 98-119, 126-132, 138-149, 173-179, 193-210, 229-235, 242-248, 266-272, 293-301, 303-315, 335-350, 355-362, 368-377, 379-390, 402-408, 420-431, 438-443, 451-464, 471-476, 486-501, 504-525, 547-568, 606-612, 637-644, 649-656, 685-701, 728-740, 774-780, 784-791, 793-800, 806-816, 818-834, 853-860, 878-888, 890-895, 926-937, 969-976, 983-997, 1-48, 54-82, 101-122, 126-184, 186-235, 237-325, 350-443, 448-512, 523-545, 553-583, 595-623, 634-657, 660-729, 732-786, 804-841, 845-975, 980-1000, 23-130, 227-257, 269-346, 680-775, 817-894, and 922-983 of SEQ ID NO 226; amino acids 4-31, 34-55, 98-105, 127-137, 157-175, 187-194, 228-238, 255-262, 268-273, 281-296, 302-307, 314-327, 329-339, 346-352, 394-401, 407-416, 452-458, 466-477, 534-550, 561-571, 573-580, 597-605, 610-622, 624-630, 639-644, 650-656, 666-672, 689-696, 706-712, 728-735, 743-748, 785-790, 803-812, 820-825, 827-833, 843-848, 861-868, 4-51, 95-116, 125-207, 217-244, 254-363, 375-426, 440-469, 474-506, 527-574, 576-641, 653-682, 750-800, 803-835, 29-69, 204-337, 400-502, and 578-765 of SEQ ID NO 227; amino acids 8-16, 19-52, 61-68, 77-86, 88-98, 106-116, 126-131, 143-153, 163-178, 183-191, 211-217, 6-56, 85-99, 106-127, 140-222, and 101-185 of SEQ ID NO 228; amino acids 15-26, 46-53, 59-68, 75-86, 93-104, 116-122, 126-140, 154-165, 175-181, 206-216, 230-243, 251-257, 294-301, 305-311, 324-336, 364-372, 374-381, 394-400, 419-427, 435-447, 451-459, 1-108, 114-192, 208-285, 310-348, 375-483, 32-98, 122-144, 157-323, 336-351, 369-389, and 433-440 of SEQ ID NO 229; amino acids 4-10, 12-23, 27-40, 42-49, 59-67, 94-99, 1-41, 47-100, and 7-58 of SEQ ID NO 230; amino acids 4-22, 38-52, 110-117, 122-132, 144-154, 167-175, 1-29, 33-61, 71-128, 138-189, 24-38, 46-67, and 128-177 of SEQ ID NO 231; amino acids 4-9, 13-29, 39-45, 50-64, 67-77, 85-94, 103-120, 127-146, 148-156, 163-178, 180-192, 204-211, 213-238, 241-256, 266-295, 1-21, 42-140, 145-261, 263-315, and 135-227 of SEQ ID NO 232; amino acids 4-15, 33-58, 67-120, 129-139, 144-171, 1-178, and 7-61 of SEQ ID NO 233; amino acids 18-24, 26-34, 40-47, 53-70, 74-88, 103-125, 128-137, 150-159, 164-174, 181-188, 201-209, 220-230, 232-238, 249-272, 278-287, 290-303, 318-332, 346-353, 379-387, 394-419, 421-430, 433-441, 443-450, 470-488, 498-507, 509-524, 532-539, 543-550, 557-579, 602-608, 611-617, 624-632, 641-657, 671-676, 694-701, 720-730, 753-762, 764-785, 792-797, 801-807, 817-840, 853-859, 868-874, 878-894, 899-913, 915-921, 929-935, 1-43, 49-70, 100-164, 173-192, 198-258, 265-306, 315-371, 373-433, 438-461, 465-523, 525-741, 761-785, 787-808, 810-925, 937-953, 288-408, 577-608, 622-764, 826-899, and 923-948 of SEQ ID NO 234; amino acids 10-23, 36-44, 46-63, 70-79, 89-106, 118-128, 140-149, 161-170, 197-207, 233-245, 1-185, 190-234, 240-254, and 182-251 of SEQ ID NO 235; amino acids 6-20, 26-32, 41-47, 50-72, 75-83, 102-112, 114-140, 156-172, 178-187, 197-218, 228-269, 275-297, 307-336, 352-368, 370-377, 387-397, 1-399, and 303-363 of SEQ ID NO 236; amino acids 8-38, 40-46, 53-70, 88-94, 127-136, 140-145, 154-174, 186-191, 198-226, 236-243, 249-256, 260-266, 269-280, 320-333, 342-349, 357-363, 365-381, 385-430, 433-449, 456-465, 467-475, 488-493, 498-503, 510-520, 557-609, 618-624, 631-644, 646-673, 679-685, 690-696, 698-734, 780-797, 802-808, 810-829, 831-870, 879-890, 910-916, 926-942, 944-980, 1-76, 82-103, 105-156, 158-289, 295-474, 506-557, 565-803, 806-857, 867-898, 914-988, and 888-947 of SEQ ID NO 237; amino acids 16-23, 25-33, 41-53, 56-63, 70-79, 87-98, 100-111, 120-128, 131-149, 160-171, 214-230, 232-242, 247-255, 259-266, 273-282, 284-292, 308-347, 355-364, 370-376, 379-386, 554-563, 10-182, 212-232, 236-358, 364-409, 461-475, 478-498, 542-577, 140-160, 232-300, 321-394, and 514-522 of SEQ ID NO 238; amino acids 9-35, 39-51, 54-67, 71-78, 93-102, 105-116, 122-129, 138-147, 155-162, 168-192, 194-231, 253-261, 264-276, 281-293, 8-241, 244-265, 269-302, and 152-279 of SEQ ID NO 239; amino acids 35-56, 61-70, 74-101, 120-129, 152-165, 171-183, 192-224, 241-271, 278-285, 291-301, 303-319, 334-340, 367-391, 401-426, 437-442, 446-469, 475-488, 495-520, 523-529, 532-541, 543-550, 554-564, 570-598, 624-630, 633-638, 643-655, 663-685, 687-694, 698-725, 735-751, 758-765, 778-795, 1-22, 36-70, 72-128, 141-223, 231-267, 270-334, 339-605, 613-652, 655-778, 115-232, 312-332, 539-555, and 558-620 of SEQ ID NO 240; amino acids 15-39, 54-61, 65-71, 84-89, 98-111, 126-144, 154-160, 189-196, 199-206, 218-224, 259-275, 1-170, 175-230, 244-278, and 162-217 of SEQ ID NO 241; amino acids 22-37, 45-57, 59-65, 82-93, 102-112, 160-172, 200-207, 229-236, 244-268, 275-322, 327-344, 346-369, 376-414, 428-451, 459-490, 495-527, 531-555, 561-582, 600-612, 616-622, 625-635, 638-669, 683-709, 726-732, 735-763, 11-57, 72-88, 99-122, 139-221, 236-274, 279-328, 332-766, 60-145, and 187-223 of SEQ ID NO 242; amino acids 4-12, 32-78, 81-89, 91-100, 105-111, 125-132, 170-176, 179-189, 194-204, 223-229, 235-245, 259-276, 282-289, 303-313, 318-326, 334-345, 350-356, 364-370, 387-405, 409-422, 424-430, 442-457, 459-465, 490-516, 521-532, 534-541, 566-571, 1-208, 230-310, 315-438, 440-456, 467-580, 186-238, and 398-572 of SEQ ID NO 243; amino acids 14-20, 28-35, 41-47, 52-61, 73-86, 92-103, 124-141, 143-158, 172-180, 185-191, 193-221, 239-250, 262-273, 280-290, 304-324, 328-357, 367-376, 378-384, 394-401, 420-428, 439-458, 460-469, 474-492, 22-125, 130-150, 157-177, 179-368, 403-495, 59-74, 94-141, 143-203, and 297-362 of SEQ ID NO 244; amino acids 8-17, 25-39, 41-67, 69-85, 93-115, 123-143, 145-157, 166-174, 178-185, 188-199, 204-210, 221-238, 245-252, 257-268, 284-290, 294-306, 312-320, 1-170, 175-247, 261-323, 17-28, 61-99, and 221-240 of SEQ ID NO 245; amino acids 5-14, 16-26, 28-45, 54-60, 72-82, 98-104, 106-114, 121-132, 137-164, 169-175, 188-194, 214-220, 233-239, 283-296, 302-310, 336-347, 357-364, 400-407, 419-425, 433-439, 443-452, 464-470, 492-499, 511-521, 528-534, 536-548, 564-571, 577-595, 597-611, 615-623, 637-649, 652-670, 681-700, 716-724, 726-738, 766-792, 806-833, 839-851, 853-872, 880-899, 905-912, 919-927, 935-956, 958-970, 980-988, 990-995, 1004-1013, 1017-1031, 1033-1039, 1043-1055, 1059-1068, 1087-1093, 1105-1114, 1116-1122, 1128-1134, 1149-1158, 1164-1172, 1197-1204, 1208-1215, 1230-1244, 1284-1290, 8-53, 68-252, 265-283, 287-320, 329-353, 357-383, 389-470, 475-495, 507-527, 534-749, 773-870, 876-898, 906-928, 938-1150, 1153-1255, 1267-1287, 1291-1314, 291-408, 411-425, 500-552, 719-741, 975-1034, 1089-1156, 1203-1234, and 1273-1301 of SEQ ID NO 246; amino acids 9-20, 25-40, 48-61, 70-84, 89-113, 116-145, 161-181, 183-193, 196-212, 217-229, 235-246, 251-276, 284-290, 300-325, 338-349, 365-376, 387-393, 401-421, 429-438, 448-458, 6-382, 385-465, 204-286, and 310-463 of SEQ ID NO 247; amino acids 9-47, 52-61, 63-76, 82-88, 95-106, 112-126, 132-138, 145-154, 162-176, 178-223, 228-234, 236-247, 253-290, 300-307, 311-325, 327-347, 353-361, 379-408, 426-436, 454-460, 490-504, 506-517, 526-532, 539-545, 550-574, 580-591, 598-612, 616-622, 625-634, 652-668, 670-679, 682-688, 1-463, 466-510, 519-641, 647-702, 7-54, 198-314, 333-381, 404-452, and 523-549 of SEQ ID NO 248; amino acids 4-20, 24-38, 40-64, 66-85, 8-88, and 15-72 of SEQ ID NO 249; amino acids 6-15, 28-33, 35-46, 54-65, 74-108, 141-146, 172-178, 204-217, 221-231, 242-248, 251-257, 276-294, 298-304, 316-332, 341-353, 356-363, 369-382, 3-44, 64-172, 175-191, 202-348, 56-110, 143-224, and 252-308 of SEQ ID NO 250; amino acids 4-28, 49-60, 66-71, 158-164, 215-221, 254-275, 288-294, 318-326, 338-344, 366-380, 389-395, 400-421, 423-430, 450-458, 465-470, 474-491, 495-504, 512-517, 543-563, 577-582, 587-594, 617-623, 631-637, 644-652, 677-691, 694-700, 705-733, 748-783, 791-819, 833-840, 847-854, 876-886, 1-39, 46-64, 80-100, 105-130, 142-162, 166-233, 249-342, 352-467, 470-511, 539-591, 596-687, 689-859, and 609-738 of SEQ ID NO 251; amino acids 7-31, 42-54, 56-64, 94-110, 112-122, 128-136, 172-195, 198-224, 256-263, 281-287, 298-310, 340-346, 356-372, 1-147, 155-276, 278-319, 329-387, 391-405, and 87-164 of SEQ ID NO 252; amino acids 4-20, 33-41, 58-65, 78-86, 93-100, 102-108, 114-161, 194-200, 202-212, 218-225, 230-235, 243-250, 269-278, 291-301, 312-317, 329-340, 352-375, 416-422, 427-434, 443-453, 468-476, 481-491, 510-532, 537-554, 560-566, 574-588, 612-623, 653-663, 678-702, 721-728, 742-748, 770-778, 796-805, 807-817, 824-832, 856-864, 871-878, 885-900, 1-20, 27-172, 175-195, 218-270, 276-308, 334-388, 409-449, 451-712, 741-824, 828-913, 16-32, 133-197, 277-385, 556-573, and 798-874 of SEQ ID NO 253; amino acids 7-14, 17-30, 35-40, 62-69, 80-86, 88-99, 109-118, 120-133, 154-179, 203-209, 216-226, 237-245, 253-260, 265-273, 286-291, 315-339, 353-371, 390-398, 412-430, 435-448, 456-464, 476-487, 492-503, 522-533, 537-549, 579-590, 606-622, 648-654, 664-679, 681-692, 700-711, 718-730, 743-752, 763-798, 808-815, 823-838, 846-864, 891-925, 927-933, 935-948, 25-79, 83-126, 151-284, 292-408, 415-511, 532-577, 594-627, 634-735, 740-809, 815-866, 885-916, 53-163, 191-246, 457-515, 520-528, 620-642, 759-800, and 894-943 of SEQ ID NO 254; amino acids 21-31, 48-58, 60-66, 83-89, 102-115, 144-154, 159-168, 175-186, 195-200, 206-218, 244-250, 259-272, 293-299, 301-307, 324-330, 339-348, 358-369, 380-391, 404-421, 429-437, 456-467, 481-486, 495-502, 509-533, 536-550, 553-571, 575-580, 599-620, 1-93, 98-116, 118-149, 174-199, 202-302, 319-393, 410-468, 493-578, 582-623, 251-272, 290-363, and 424-502 of SEQ ID NO 255; amino acids 12-19, 30-48, 55-61, 66-74, 103-115, 117-131, 176-183, 192-197, 200-222, 8-52, 80-171, 173-201, 208-237, 240-258, 15-64, 77-168, and 190-252 of SEQ ID NO 256; amino acids 4-19, 31-58, 61-70, 84-96, 104-112, 131-138, 143-165, 168-178, 197-208, 213-220, 255-268, 273-281, 313-320, 334-354, 359-367, 369-376, 382-399, 406-420, 429-439, 441-461, 463-507, 513-544, 550-557, 569-580, 583-589, 598-618, 640-650, 675-680, 683-711, 713-739, 797-805, 1-55, 59-164, 168-185, 193-238, 244-333, 343-375, 378-517, 521-583, 602-656, 670-761, 788-808, 124-151, 326-341, and 567-720 of SEQ ID NO 257; amino acids 5-12, 22-37, 47-75, 78-94, 102-107, 110-118, 124-130, 140-154, 161-166, 185-193, 205-213, 218-237, 244-257, 264-269, 276-283, 289-315, 317-323, 334-341, 1-105, 121-344, 141-168, and 256-313 of SEQ ID NO 258; amino acids 33-48, 56-63, 65-79, 84-101, 109-122, 127-160, 211-216, 224-229, 239-256, 1-108, 110-265, 4-75, and 108-199 of SEQ ID NO 259; amino acids 6-14, 24-40, 47-60, 79-98, 102-108, 114-120, 122-132, 154-160, 168-179, 187-206, 221-226, 231-253, 255-268, 296-314, 316-337, 341-347, 357-378, 383-407, 418-425, 443-452, 456-462, 485-495, 503-510, 512-523, 533-543, 550-555, 1-101, 115-152, 154-203, 210-314, 321-368, 375-412, 414-434, 438-494, 497-529, 544-558, 35-136, and 348-355 of SEQ ID NO 260; amino acids 4-11, 14-22, 24-29, 33-40, 42-52, 54-70, 87-111, 121-128, 132-143, 149-168, 180-190, 197-215, 223-232, 244-251, 296-319, 328-349, 363-368, 378-399, 414-431, 434-440, 451-457, 1-27, 29-48, 59-199, 206-257, 264-378, 398-459, and 322-374 of SEQ ID NO 261; amino acids 4-20, 28-34, 36-42, 56-72, 80-86, 97-110, 118-127, 132-138, 145-150, 173-179, 181-195, 232-244, 250-263, 270-276, 308-314, 324-331, 336-343, 355-364, 398-403, 1-37, 52-81, 93-152, 173-252, 266-406, 29-90, and 125-197 of SEQ ID NO 262; amino acids 15-23, 31-38, 51-66, 89-105, 123-130, 135-140, 149-154, 198-208, 212-220, 228-239, 249-257, 261-267, 275-281, 300-310, 322-343, 354-365, 372-384, 394-404, 409-420, 422-429, 446-455, 459-468, 1-87, 104-194, 199-392, 404-425, 443-466, 82-164, 236-325, and 392-453 of SEQ ID NO 263; amino acids 4-10, 17-46, 51-80, 87-92, 99-121, 135-150, 170-176, 200-212, 243-252, 277-282, 310-331, 340-349, 1-160, 168-188, 200-263, 276-355, and 126-294 of SEQ ID NO 264; amino acids 4-18, 35-48, 52-67, 73-80, 90-96, 108-147, 156-171, 188-207, 227-234, 239-248, 263-286, 301-330, 347-363, 365-395, 409-419, 421-431, 442-447, 454-468, 471-482, 484-510, 517-525, 545-555, 1-62, 84-178, 182-264, 295-396, 403-452, 461-502, 520-568, 53-62, 112-187, 233-265, and 375-402 of SEQ ID NO 265; amino acids 13-31, 36-48, 54-64, 67-83, 93-104, 106-126, 128-137, 144-154, 161-169, 179-196, 205-221, 232-255, 257-263, 265-305, 307-324, 326-340, 354-367, 372-378, 383-402, 14-409, 23-124, and 134-172 of SEQ ID NO 266; amino acids 5-21, 26-35, 37-44, 57-69, 71-78, 84-93, 95-101, 1-78, 81-112, and 41-103 of SEQ ID NO 267; amino acids 13-19, 21-42, 47-55, 57-66, 68-74, 77-82, 84-90, 92-101, 106-112, 147-169, 171-183, 185-200, 221-230, 240-252, 269-275, 284-296, 299-308, 311-322, 332-342, 344-358, 374-382, 386-391, 403-412, 445-451, 462-477, 484-494, 557-563, 565-574, 1-47, 50-65, 80-108, 121-135, 145-234, 236-322, 326-379, 396-488, 502-522, 564-589, 14-77, 217-269, and 380-542 of SEQ ID NO 268; amino acids 4-34, 36-44, 51-57, 63-68, 70-80, 91-130, 136-173, 186-193, 195-201, 207-213, 226-234, 242-266, 268-284, 290-308, 315-332, 368-378, 386-402, 413-437, 1-87, 89-172, 175-193, 222-244, 247-366, 381-409, 416-437, 191-197, and 350-438 of SEQ ID NO 269; amino acids 11-24, 29-51, 61-91, 98-116, 119-129, 132-142, 10-66, 69-102, 111-145, and 2-98 of SEQ ID NO 270; amino acids 16-49, 55-74, 78-88, 91-112, 119-127, 140-145, 156-165, 167-173, 199-209, 234-249, 251-263, 265-272, 282-290, 299-305, 322-330, 1-333, 32-89, 108-141, and 246-333 of SEQ ID NO 271; amino acids 19-43, 49-55, 78-92, 1-65, 68-113, and 45-108 of SEQ ID NO 272; amino acids 15-23, 52-84, 97-103, 110-133, 149-154, 160-183, 203-227, 233-241, 243-250, 313-330, 338-351, 373-382, 384-396, 401-407, 425-452, 469-474, 476-482, 490-513, 519-528, 533-545, 591-613, 619-649, 23-105, 115-152, 154-220, 226-263, 276-296, 302-368, 378-402, 414-477, 481-536, 539-581, 588-652, 158-222, 327-423, and 427-526 of SEQ ID NO 273; amino acids 4-11, 19-29, 43-52, 82-96, 101-110, 138-144, 157-165, 258-268, 326-334, 336-344, 346-358, 377-383, 386-394, 397-405, 413-427, 429-434, 437-443, 449-458, 486-495, 514-521, 535-546, 554-560, 566-578, 589-596, 603-612, 618-625, 644-659, 662-671, 675-680, 707-720, 722-735, 737-752, 754-771, 788-802, 816-830, 836-844, 851-870, 884-909, 39-105, 132-176, 183-247, 258-292, 300-324, 328-398, 405-506, 512-532, 544-653, 659-838, 840-885, 78-130, 215-292, 655-713, and 818-880 of SEQ ID NO 274; amino acids 5-14, 21-27, 29-37, 46-72, 83-96, 113-121, 129-134, 1-63, 81-141, and 58-141 of SEQ ID NO 275; amino acids 4-9, 20-34, 36-54, 88-94, 124-148, 155-161, 182-202, 204-211, 220-232, 1-53, 75-116, 126-174, 177-235, and 92-234 of SEQ ID NO 276; amino acids 4-22, 31-38, 49-55, 82-91, 1-24, 28-49, and 7-65 of SEQ ID NO 277; amino acids 4-28, 30-83, 85-92, 94-123, 125-157, 163-196, 216-234, 240-258, 260-289, 307-313, 315-323, 328-378, 392-424, 428-440, 458-472, 474-482, 491-506, 508-530, 539-564, 1-298, 301-566, 14-31, and 282-347 of SEQ ID NO 278; amino acids 7-17, 20-28, 30-37, 39-47, 49-61, 77-96, 114-128, 149-154, 166-180, 182-200, 240-248, 256-271, 1-158, 163-275, 60-112, and 149-246 of SEQ ID NO 279; amino acids 6-25, 31-37, 43-57, 63-72, 82-90, 96-107, 109-118, 123-140, 144-149, 155-162, 168-182, 184-190, 213-219, 237-244, 247-256, 262-268, 271-284, 295-301, 307-320, 325-337, 346-364, 1-46, 48-196, 208-231, 234-278, 290-374, and 184-277 of SEQ ID NO 280; amino acids 27-46, 53-77, 103-123, 130-142, 153-159, 171-179, 182-209, 215-222, 224-233, 237-243, 271-276, 2-210, 215-271, and 12-138 of SEQ ID NO 281; amino acids 17-24, 38-45, 49-67, 80-86, 94-112, 119-141, 154-159, 165-171, 183-200, 218-226, 234-252, 263-291, 328-336, 338-349, 357-385, 397-405, 423-440, 445-453, 457-466, 470-477, 487-493, 8-134, 141-167, 175-224, 231-473, 479-496, 284-393, and 462-469 of SEQ ID NO 282; amino acids 4-9, 16-23, 56-64, 70-82, 86-93, 1-20, 39-103, and 11-36 of SEQ ID NO 283; amino acids 5-38, 69-77, 83-99, 143-151, 154-160, 176-186, 198-210, 226-268, 270-288, 293-303, 305-324, 327-342, 344-353, 388-394, 432-438, 1-49, 54-69, 96-110, 130-248, 251-270, 312-368, 382-450, 65-116, 179-231, and 328-395 of SEQ ID NO 284; amino acids 8-23, 58-70, 74-80, 85-91, 95-103, 110-127, 143-150, 153-160, 164-171, 189-201, 218-224, 228-233, 236-242, 248-255, 1-27, 30-46, 53-71, 77-171, 181-264, and 3-91 of SEQ ID NO 285; amino acids 4-19, 30-49, 57-83, 88-100, 111-119, 122-129, 154-159, 168-173, 189-200, 222-229, 237-243, 251-259, 1-22, 33-51, 55-99, 114-139, 163-204, 220-250, and 108-262 of SEQ ID NO 286; amino acids 4-19, 26-51, 53-62, 68-79, 83-96, 99-105, 114-122, 128-135, 138-146, 155-164, 180-199, 201-218, 226-232, 237-249, 257-264, 1-174, 176-277, and 47-117 of SEQ ID NO 287; amino acids 12-17, 23-31, 42-62, 81-88, 96-103, 109-118, 122-128, 136-146, 151-158, 188-203, 210-224, 229-238, 270-287, 296-308, 310-333, 341-354, 361-368, 383-395, 404-420, 423-435, 443-456, 460-503, 510-517, 521-526, 541-548, 565-570, 575-586, 606-617, 625-637, 648-655, 683-696, 702-713, 722-728, 740-748, 779-786, 789-801, 807-812, 820-829, 849-854, 862-868, 9-61, 75-124, 132-288, 299-547, 592-608, 612-660, 666-695, 733-753, 776-813, 818-854, 858-875, 59-133, 260-272, 382-410, 440-515, and 711-872 of SEQ ID NO 288; amino acids 15-24, 28-56, 63-87, 98-104, 112-121, 125-133, 142-159, 166-176, 189-198, 200-207, 216-223, 225-231, 236-243, 245-252, 262-287, 295-304, 315-323, 326-332, 340-356, 361-368, 370-376, 387-398, 401-410, 412-420, 422-434, 442-455, 469-476, 486-496, 507-514, 519-527, 547-556, 559-564, 569-575, 588-594, 604-609, 611-622, 634-662, 676-686, 712-718, 723-738, 752-758, 779-790, 793-822, 833-850, 866-891, 894-908, 935-950, 1-155, 162-181, 186-321, 346-600, 608-760, 778-897, 910-953, 700-781, and 800-903 of SEQ ID NO 289; amino acids 25-31, 41-54, 79-86, 88-96, 111-125, 135-141, 145-152, 159-182, 188-195, 208-214, 257-267, 275-297, 306-312, 326-333, 342-358, 370-377, 399-414, 420-428, 434-439, 446-492, 499-507, 509-521, 527-533, 2-33, 36-217, 231-251, 263-363, 367-427, 430-527, 2-122, and 192-306 of SEQ ID NO 290; amino acids 6-13, 18-26, 28-46, 49-56, 62-69, 74-84, 88-110, 116-132, 135-149, 156-178, 181-191, 202-223, 247-260, 274-281, 295-303, 336-344, 368-375, 377-390, 395-404, 409-433, 450-458, 470-506, 1-83, 100-280, 295-370, 374-509, 63-151, 220-291, and 321-388 of SEQ ID NO 291; amino acids 4-11, 16-27, 31-41, 62-81, 101-108, 116-144, 169-185, 203-218, 228-234, 236-245, 255-273, 283-294, 298-304, 306-313, 315-340, 342-350, 357-371, 378-390, 402-416, 421-436, 444-456, 458-464, 481-487, 496-508, 510-531, 536-541, 544-551, 556-569, 576-590, 600-614, 627-636, 644-651, 664-670, 675-688, 700-720, 727-770, 775-797, 802-818, 828-835, 864-884, 887-892, 899-907, 918-928, 940-958, 965-980, 7-194, 202-235, 261-383, 401-505, 524-617, 624-727, 735-749, 761-879, 898-973, 975-989, 96-113, 634-656, and 658-734 of SEQ ID NO 292; amino acids 4-12, 14-23, 26-35, 51-78, 83-94, 100-106, 109-119, 121-135, 140-149, 153-177, 179-202, 212-224, 229-239, 241-252, 260-312, 330-336, 342-352, 368-383, 393-403, 1-100, 105-343, 365-406, 76-101, and 295-353 of SEQ ID NO 293; amino acids 4-28, 31-38, 65-81, 1-84, and 9-69 of SEQ ID NO 294; amino acids 4-11, 26-35, 55-74, 78-90, 109-115, 125-141, 164-169, 173-181, 193-203, 212-231, 253-266, 289-295, 297-313, 350-356, 380-388, 410-416, 425-433, 449-462, 466-473, 478-497, 562-584, 586-606, 614-619, 718-724, 727-738, 745-752, 782-789, 812-821, 827-834, 844-854, 864-874, 902-908, 920-927, 952-957, 966-973, 982-988, 1010-1019, 1027-1034, 1050-1057, 1082-1088, 1111-1125, 1129-1135, 1150-1163, 1-104, 108-152, 161-190, 195-323, 337-366, 369-410, 415-449, 453-495, 532-552, 559-624, 677-697, 724-751, 784-802, 804-824, 834-931, 949-977, 979-999, 1060-1149, 378-625, and 795-908 of SEQ ID NO 295; amino acids 7-31, 36-49, 59-66, 78-85, 89-95, 101-120, 127-151, 174-183, 185-194, 207-217, 221-230, 238-247, 258-268, 290-297, 309-315, 1-42, 58-233, 236-279, 290-315, and 181-234 of SEQ ID NO 296; amino acids 17-25, 60-72, 80-90, 97-109, 111-117, 1-72, 74-120, and 22-110 of SEQ ID NO 297; amino acids 4-31, 44-53, 55-70, 76-99, 109-115, 123-130, 136-142, 147-155, 158-191, 199-220, 223-235, 243-257, 264-281, 286-299, 313-323, 350-365, 373-391, 397-441, 455-466, 1-35, 45-218, 225-323, 347-386, 388-403, 405-446, 448-469, 105-193, and 327-355 of SEQ ID NO 298; amino acids 4-19, 47-53, 69-75, 81-87, 109-122, 125-140, 142-156, 191-199, 219-238, 246-271, 279-290, 296-302, 305-313, 321-327, 331-345, 1-32, 75-89, 107-127, 133-171, 188-310, 325-349, 43-142, and 300-314 of SEQ ID NO 299; amino acids 4-43, 49-56, 66-74, 80-94, 97-110, 116-136, 139-145, 151-163, 165-178, 182-210, 213-220, 222-231, 241-252, 256-271, 277-293, 300-314, 330-336, 340-363, 369-375, 381-404, 419-426, 439-445, 447-453, 465-501, 1-38, 43-62, 77-137, 140-173, 179-291, 328-376, 386-400, 406-504, 19-76, 195-221, and 271-292 of SEQ ID NO 300; amino acids 4-19, 25-31, 40-63, 71-80, 89-98, 101-119, 125-151, 159-164, 173-201, 219-227, 247-258, 263-284, 308-319, 321-326, 10-99, 101-185, 200-294, 297-347, and 74-161 of SEQ ID NO 301; amino acids 12-18, 25-31, 37-43, 76-94, 121-135, 146-176, 191-199, 205-212, 259-290, 300-313, 340-347, 364-373, 375-385, 413-420, 424-430, 446-462, 465-473, 475-486, 505-522, 537-544, 557-563, 573-582, 588-602, 604-613, 620-630, 638-675, 677-683, 1-28, 33-134, 137-179, 181-221, 255-323, 333-439, 443-532, 537-692, 25-59, 156-270, 272-335, and 557-565 of SEQ ID NO 302; amino acids 10-22, 24-54, 63-70, 77-86, 94-110, 112-133, 1-60, 88-136, and 17-135 of SEQ ID NO 303; amino acids 7-24, 28-47, 76-86, 92-103, 122-129, 135-141, 152-157, 162-173, 192-205, 210-225, 230-236, 270-286, 289-297, 299-314, 316-328, 334-341, 349-356, 376-382, 384-395, 4-47, 50-248, 261-393, 61-67, 168-195, and 257-320 of SEQ ID NO 304; amino acids 4-9, 20-39, 42-48, 67-101, 129-135, 156-162, 170-176, 178-189, 192-197, 228-235, 260-266, 272-278, 291-301, 330-342, 347-360, 367-373, 383-390, 397-409, 432-443, 466-487, 506-517, 520-545, 551-561, 588-598, 602-608, 622-630, 15-126, 129-166, 171-217, 227-307, 309-361, 363-431, 461-496, 501-637, 172-215, 218-291, 417-440, and 498-574 of SEQ ID NO 305; amino acids 8-28, 34-67, 1-70, and 2-32 of SEQ ID NO 306; amino acids 9-26, 33-40, 79-93, 98-105, 112-121, 128-137, 144-156, 160-167, 183-190, 198-204, 210-224, 250-275, 283-289, 296-302, 314-321, 336-344, 346-353, 355-362, 367-380, 382-391, 393-403, 407-413, 416-423, 434-443, 469-492, 499-509, 550-559, 596-603, 613-633, 635-642, 648-659, 1-30, 33-99, 106-126, 129-331, 340-511, 522-565, 578-599, 608-667, 59-118, and 160-215 of SEQ ID NO 307; amino acids 7-20, 25-33, 36-64, 74-83, 117-124, 126-137, 146-159, 164-173, 181-186, 195-231, 240-255, 258-266, 268-276, 283-290, 5-90, 113-250, 260-293, 9-47, 145-198, and 227-239 of SEQ ID NO 308; amino acids 4-24, 31-41, 43-57, 61-68, 72-78, 83-88, 115-121, 123-135, 190-200, 214-224, 239-248, 291-299, 305-312, 328-333, 351-362, 1-101, 103-140, 202-263, 286-362, and 189-242 of SEQ ID NO 309; amino acids 4-21, 110-126, 128-134, 139-149, 1-29, 31-62, 65-94, 99-149, and 7-80 of SEQ ID NO 310; amino acids 4-9, 23-29, 37-52, 57-73, 80-98, 101-119, 146-163, 175-181, 198-208, 222-228, 243-249, 258-263, 269-278, 283-298, 309-324, 329-339, 342-353, 356-363, 365-374, 389-395, 397-404, 408-415, 424-429, 432-450, 457-490, 503-524, 526-556, 568-575, 584-592, 596-619, 621-646, 653-673, 675-694, 699-706, 1-104, 121-135, 143-176, 182-274, 281-301, 306-385, 393-502, 505-648, 654-709, 162-186, 309-397, 435-463, and 503-565 of SEQ ID NO 311; amino acids 5-21, 24-49, 51-63, 65-76, 111-117, 133-145, 149-156, 189-197, 217-230, 240-245, 288-295, 297-316, 321-335, 339-346, 369-382, 401-421, 1-89, 106-125, 129-207, 210-230, 255-357, 360-453, 166-220, and 369-473 of SEQ ID NO 312; amino acids 4-11, 22-37, 39-54, 62-70, 78-84, 94-100, 106-111, 1-51, 59-102, and 41-100 of SEQ ID NO 313; amino acids 16-26, 29-46, 48-54, 94-101, 103-110, 117-125, 134-148, 156-180, 190-196, 221-239, 1-35, 61-81, 88-116, 139-205, 207-227, and 43-75 of SEQ ID NO 314; amino acids 5-19, 21-27, 30-35, 40-57, 67-75, 104-114, 121-130, 152-161, 167-175, 187-205, 219-227, 244-250, 1-76, 83-155, 158-191, 218-253, and 15-137 of SEQ ID NO 315; amino acids 15-38, 40-49, 57-66, 68-76, 80-88, 91-109, 117-122, 148-154, 161-169, 195-214, 220-230, 233-241, 263-269, 285-293, 301-312, 324-340, 349-367, 372-386, 398-407, 426-434, 436-443, 446-452, 456-476, 478-498, 510-517, 523-550, 558-565, 589-604, 610-619, 634-639, 659-668, 680-688, 698-704, 732-739, 741-754, 758-767, 783-796, 800-812, 816-826, 842-860, 1-83, 85-115, 136-179, 192-223, 257-360, 375-486, 501-527, 530-618, 626-644, 653-867, 598-632, 690-700, and 772-833 of SEQ ID NO 316; amino acids 4-12, 41-49, 61-66, 111-118, 123-128, 139-144, 159-166, 178-185, 206-214, 236-248, 262-268, 275-293, 297-314, 348-360, 368-399, 1-68, 71-150, 193-226, 230-296, 305-406, 37-125, and 272-341 of SEQ ID NO 317; amino acids 8-15, 18-54, 61-80, 109-114, 143-149, 153-160, 172-185, 188-194, 196-207, 221-230, 238-244, 273-279, 291-297, 305-319, 325-333, 342-362, 17-110, 118-139, 149-238, 247-365, 62-107, and 123-182 of SEQ ID NO 318; amino acids 15-28, 34-59, 105-135, 137-144, 155-184, 187-212, 221-233, 240-260, 267-278, 286-291, 299-315, 319-334, 1-32, 39-55, 76-122, 124-279, 282-337, 50-100, 116-176, and 261-288 of SEQ ID NO 319; amino acids 4-22, 43-56, 71-108, 119-125, 127-138, 146-155, 164-171, 173-181, 190-199, 214-234, 1-36, 57-112, 121-181, 190-241, and 4-108 of SEQ ID NO 320; amino acids 11-19, 24-31, 37-42, 61-71, 85-91, 94-100, 105-115, 123-138, 140-148, 153-161, 164-173, 185-192, 194-202, 212-222, 227-233, 244-250, 276-287, 298-304, 309-315, 319-330, 342-353, 367-374, 387-394, 403-425, 439-445, 514-520, 528-543, 545-553, 569-584, 586-595, 603-621, 8-273, 283-299, 309-346, 367-490, 494-537, 558-632, 17-74, 155-189, 220-302, 418-540, and 550-612 of SEQ ID NO 321; amino acids 4-20, 41-47, 49-56, 68-86, 95-101, 108-121, 123-137, 145-150, 158-178, 187-198, 218-226, 231-237, 240-250, 252-262, 269-282, 287-317, 1-62, 70-209, 217-319, 140-149, 174-233, and 243-300 of SEQ ID NO 322; amino acids 16-21, 29-36, 43-61, 68-84, 89-136, 159-197, 206-216, 224-240, 244-251, 270-286, 292-307, 313-323, 327-332, 339-364, 388-395, 402-409, 423-440, 448-461, 464-476, 1-15, 19-260, 292-312, 314-451, 465-483, 242-272, and 435-457 of SEQ ID NO 323; amino acids 8-28, 30-35, 55-64, 69-79, 87-95, 132-143, 161-177, 186-198, 222-227, 247-258, 266-288, 340-352, 1-40, 44-96, 103-154, 158-177, 181-202, 208-268, 272-355, 158-168, and 286-334 of SEQ ID NO 324; amino acids 5-24, 39-46, 50-64, 79-89, 103-109, 116-133, 137-150, 159-167, 169-177, 184-202, 209-229, 245-254, 272-282, 294-301, 340-356, 368-378, 388-395, 429-444, 450-463, 484-490, 536-545, 547-557, 565-601, 615-627, 648-655, 1-33, 35-108, 118-236, 245-260, 264-324, 329-358, 360-397, 448-485, 492-536, 541-599, 607-669, 162-181, 331-422, 481-534, and 591-669 of SEQ ID NO 325; amino acids 26-55, 58-73, 75-81, 87-96, 131-140, 150-158, 6-151, and 15-96 of SEQ ID NO 326; amino acids 4-9, 43-52, 63-71, 97-104, 117-125, 130-136, 143-161, 167-179, 185-191, 194-201, 205-211, 219-225, 240-245, 247-256, 261-279, 286-301, 323-338, 340-349, 359-370, 390-397, 399-414, 439-473, 485-496, 505-521, 534-561, 566-571, 575-588, 598-606, 623-629, 653-665, 675-680, 683-690, 692-700, 730-755, 769-798, 802-815, 818-826, 852-859, 862-875, 27-88, 90-173, 176-283, 303-404, 421-443, 473-487, 507-540, 544-642, 645-673, 677-761, 767-840, 843-878, 24-123, 243-316, 430-472, and 512-571 of SEQ ID NO 327; amino acids 40-55, 59-119, 145-180, 182-210, 215-230, 264-274, 293-305, 308-314, 316-328, 339-346, 348-354, 388-393, 395-414, 416-425, 431-436, 442-451, 467-473, 477-487, 491-498, 505-513, 519-542, 548-565, 1-336, 344-379, 395-438, 440-457, 476-571, 230-331, and 454-460 of SEQ ID NO 328; amino acids 5-24, 32-46, 72-77, 83-98, 100-106, 113-135, 140-150, 154-170, 176-199, 218-240, 259-278, 282-301, 307-323, 379-386, 389-430, 449-457, 465-484, 504-520, 537-561, 568-581, 1-321, 323-344, 353-371, 374-588, 341-374, and 406-416 of SEQ ID NO 329; amino acids 11-20, 27-37, 39-54, 67-73, 79-89, 92-102, 112-120, 125-138, 145-152, 172-178, 181-191, 193-204, 213-220, 222-235, 241-257, 281-288, 298-306, 331-359, 361-403, 420-426, 434-456, 462-470, 477-484, 497-504, 520-527, 535-548, 566-575, 584-592, 606-613, 624-629, 635-651, 654-660, 683-694, 697-703, 714-725, 728-757, 767-779, 787-794, 801-814, 834-843, 858-867, 869-875, 888-899, 912-933, 936-946, 3-31, 36-63, 66-80, 103-208, 231-299, 307-399, 445-517, 528-586, 597-618, 632-652, 661-742, 745-781, 785-852, 855-893, 909-947, 293-326, 691-787, and 896-950 of SEQ ID NO 330; amino acids 4-21, 29-46, 61-77, 81-88, 1-88, and 24-78 of SEQ ID NO 331; amino acids 8-41, 48-53, 73-78, 81-98, 115-140, 143-149, 154-162, 182-196, 207-215, 221-226, 230-239, 257-264, 268-276, 287-303, 1-49, 69-149, 161-227, 230-306, 39-67, and 77-105 of SEQ ID NO 332; amino acids 4-11, 16-23, 31-39, 64-79, 82-89, 98-105, 111-117, 136-146, 162-168, 186-193, 196-205, 219-236, 268-287, 289-300, 308-314, 323-339, 349-365, 368-374, 1-51, 93-245, 259-377, 3-9, 107-183, and 210-318 of SEQ ID NO 333; amino acids 4-28, 35-48, 56-64, 91-125, 135-143, 150-159, 1-26, 54-136, 144-167, and 14-88 of SEQ ID NO 334; amino acids 4-16, 21-39, 53-83, 88-93, 99-133, 152-164, 167-187, 192-209, 211-231, 245-273, 278-284, 289-304, 317-333, 335-341, 346-353, 359-366, 378-389, 394-402, 424-438, 447-460, 473-480, 482-492, 494-500, 506-520, 523-541, 558-574, 577-588, 598-605, 611-619, 622-629, 633-643, 649-669, 685-701, 707-727, 735-749, 768-780, 783-789, 798-810, 1-153, 178-240, 254-282, 284-349, 366-446, 450-784, 794-813, 79-169, 194-251, and 451-509 of SEQ ID NO 335; amino acids 4-30, 38-46, 56-70, 80-90, 101-110, 117-123, 149-157, 162-172, 178-191, 211-218, 230-256, 261-283, 292-310, 335-344, 348-354, 361-369, 382-396, 403-414, 425-431, 452-458, 460-472, 484-504, 508-514, 524-541, 562-580, 597-603, 627-643, 645-657, 688-696, 707-713, 719-736, 746-759, 769-775, 791-797, 802-812, 824-830, 838-847, 856-864, 880-901, 909-916, 1-52, 64-78, 81-202, 221-297, 335-381, 384-454, 464-552, 559-590, 598-620, 622-644, 646-668, 672-821, 833-898, 25-108, and 505-560 of SEQ ID NO 336; amino acids 16-24, 37-46, 60-73, 79-90, 97-116, 130-141, 143-150, 155-161, 179-186, 193-206, 212-225, 230-253, 258-267, 290-296, 306-344, 366-381, 383-390, 398-405, 418-425, 438-449, 465-482, 489-499, 503-509, 530-543, 545-567, 7-44, 51-79, 81-114, 116-263, 285-307, 311-351, 379-416, 427-482, 491-509, 535-570, 126-230, 248-286, 364-508, and 510-517 of SEQ ID NO 337; amino acids 4-10, 13-21, 27-40, 90-95, 121-128, 197-203, 239-246, 253-259, 305-311, 322-336, 349-356, 358-364, 372-379, 393-408, 411-417, 427-434, 457-467, 481-490, 492-497, 504-513, 533-542, 561-571, 592-600, 641-646, 661-667, 671-682, 711-720, 722-734, 763-771, 779-796, 833-838, 855-865, 873-879, 886-896, 901-908, 913-924, 931-939, 1-19, 24-55, 88-108, 118-153, 172-190, 198-281, 299-361, 369-392, 430-578, 661-689, 698-738, 795-850, 861-901, 904-924, 927-946, 37-262, 272-351, 453-474, 499-518, 527-547, 569-643, and 897-954 of SEQ ID NO 338; amino acids 22-28, 39-49, 4-20, 26-44, and 5-65 of SEQ ID NO 339; amino acids 4-39, 52-63, 70-88, 97-112, 1-88, 94-125, and 56-113 of SEQ ID NO 340; amino acids 10-29, 35-40, 46-62, 77-84, 94-129, 146-153, 158-164, 166-174, 196-202, 209-215, 225-240, 250-258, 272-287, 305-313, 318-335, 339-353, 362-381, 384-390, 395-424, 439-452, 454-463, 467-476, 482-496, 504-529, 535-562, 564-573, 576-588, 596-602, 604-619, 625-658, 664-675, 685-696, 714-727, 746-758, 770-777, 780-789, 793-799, 845-861, 863-873, 890-901, 905-910, 921-934, 1-30, 35-106, 117-348, 357-381, 390-551, 553-800, 804-835, 844-882, 893-937, 231-286, 394-429, and 686-758 of SEQ ID NO 341; amino acids 8-31, 35-70, 83-89, 91-102, 108-116, 130-156, 169-191, 203-217, 238-256, 270-292, 298-311, 315-331, 335-345, 364-370, 376-382, 384-401, 405-419, 421-430, 434-441, 445-452, 467-481, 503-511, 516-522, 524-531, 534-539, 547-560, 570-582, 5-104, 122-260, 271-340, 353-564, 566-603, 106-129, 215-224, and 454-522 of SEQ ID NO 342; amino acids 8-27, 29-37, 42-49, 63-69, 74-80, 95-118, 140-148, 156-170, 189-195, 197-209, 218-223, 227-235, 247-255, 268-275, 294-299, 305-313, 316-333, 347-359, 369-376, 381-386, 399-410, 423-432, 436-455, 462-475, 524-537, 578-588, 597-605, 616-622, 624-634, 636-644, 648-655, 657-665, 693-698, 706-723, 726-732, 736-778, 782-805, 820-832, 835-846, 848-870, 882-909, 927-933, 951-957, 962-971, 976-982, 986-1001, 1016-1021, 1042-1050, 1054-1061, 1066-1075, 1-68, 84-100, 152-182, 198-344, 348-413, 416-552, 558-621, 624-788, 800-892, 958-1060, 1064-1078, 155-209, 427-501, 658-688, 809-841, 862-871, and 927-1010 of SEQ ID NO 343; amino acids 15-36, 55-67, 70-76, 81-88, 110-125, 128-134, 136-188, 197-214, 216-223, 244-250, 257-263, 265-280, 300-306, 311-319, 2-36, 52-72, 82-152, 156-188, 191-225, 232-322, 21-78, 90-154, and 168-215 of SEQ ID NO 344; amino acids 4-25, 32-41, 51-58, 71-78, 81-89, 101-123, 130-138, 140-151, 165-171, 176-193, 202-214, 244-259, 266-282, 285-293, 295-301, 316-325, 329-334, 344-350, 381-390, 405-415, 423-431, 1-120, 126-160, 173-257, 266-313, 322-342, 354-434, 8-56, and 181-269 of SEQ ID NO 345; amino acids 4-46, 53-60, 74-86, 97-104, 121-128, 149-160, 206-217, 252-258, 274-280, 287-294, 311-327, 356-370, 378-383, 394-403, 425-432, 434-447, 473-484, 491-502, 543-556, 599-608, 610-623, 641-657, 671-684, 698-707, 719-733, 748-758, 772-779, 798-808, 814-820, 830-843, 854-866, 872-884, 891-899, 901-907, 920-932, 1-91, 114-128, 130-149, 156-190, 207-306, 308-330, 342-429, 436-450, 454-662, 666-937, 103-238, 247-273, 451-470, 687-734, and 753-796 of SEQ ID NO 346; amino acids 35-41, 52-58, 66-74, 78-87, 92-99, 101-109, 120-132, 153-164, 176-182, 186-194, 208-238, 246-262, 272-284, 291-311, 317-322, 328-343, 4-104, 112-131, 150-214, 216-345, 36-58, and 282-336 of SEQ ID NO 347; amino acids 5-27, 29-42, 57-71, 73-80, 92-99, 101-107, 126-136, 142-151, 155-163, 173-179, 214-232, 235-254, 262-274, 297-309, 311-323, 330-358, 362-379, 383-394, 396-416, 432-457, 464-472, 474-485, 487-498, 511-519, 527-536, 1-35, 53-118, 123-280, 283-440, 442-542, and 22-120 of SEQ ID NO 348; amino acids 4-15, 27-61, 64-77, 84-97, 102-122, 144-215, 223-239, 242-252, 257-263, 271-286, 295-302, 312-324, 328-348, 380-400, 403-411, 417-428, 434-440, 444-451, 453-467, 485-496, 522-538, 540-545, 552-560, 566-577, 582-588, 1-149, 151-408, 442-594, and 324-391 of SEQ ID NO 349; amino acids 21-41, 43-123, 125-137, 139-157, 171-190, 193-203, 205-226, 242-256, 264-274, 289-299, 301-311, 323-368, 375-396, 400-408, 418-434, 1-437, 84-149, 163-176, and 260-321 of SEQ ID NO 350; amino acids 4-11, 19-34, 44-65, 70-115, 130-140, 142-168, 170-176, 185-206, 221-227, 1-65, 81-124, 127-206, 215-232, and 157-215 of SEQ ID NO 351; amino acids 4-19, 44-51, 66-104, 122-129, 136-141, 143-150, 154-159, 166-173, 181-205, 207-218, 1-15, 21-119, 123-213, and 77-182 of SEQ ID NO 352; amino acids 4-15, 17-34, 40-51, 61-71, 84-89, 137-146, 153-159, 163-170, 175-191, 203-210, 216-228, 242-250, 259-271, 279-284, 291-329, 333-372, 376-394, 399-404, 406-412, 416-424, 446-451, 1-76, 82-168, 179-209, 227-249, 253-473, and 234-319 of SEQ ID NO 353; amino acids 8-15, 17-26, 30-45, 47-74, 80-92, 94-111, 114-120, 124-133, 148-173, 175-181, 183-196, 198-208, 212-231, 238-244, 248-262, 266-285, 290-302, 308-317, 327-332, 334-341, 346-355, 357-365, 373-380, 1-47, 68-99, 104-174, 178-378, and 135-280 of SEQ ID NO 354; amino acids 12-28, 32-56, 61-72, 74-89, 95-128, 130-144, 150-163, 177-189, 196-214, 218-282, 294-305, 313-382, 384-398, 404-420, 426-454, 456-461, 1-224, 229-283, 296-464, and 350-406 of SEQ ID NO 355; amino acids 6-17, 22-31, 35-63, 67-85, 89-101, 128-147, 149-155, 161-170, 172-179, 187-198, 228-234, 240-247, 284-291, 298-308, 326-337, 344-350, 357-366, 1-28, 30-82, 86-267, 271-307, 310-376, 5-136, and 172-260 of SEQ ID NO 356; amino acids 9-32, 36-52, 72-90, 149-157, 165-177, 188-196, 223-233, 237-245, 264-273, 280-293, 298-307, 309-348, 352-362, 366-374, 381-396, 405-413, 416-421, 428-435, 438-444, 449-457, 464-476, 491-555, 579-597, 602-628, 646-652, 663-670, 674-692, 739-747, 762-771, 779-789, 809-816, 1-40, 49-69, 72-92, 97-265, 271-369, 373-452, 456-476, 496-527, 535-555, 567-637, 652-819, 297-327, 539-627, and 712-776 of SEQ ID NO 357; amino acids 5-16, 21-27, 39-46, 60-77, 92-104, 112-118, 129-136, 141-164, 167-176, 178-214, 220-228, 234-251, 284-301, 303-310, 315-328, 336-353, 362-375, 380-390, 393-409, 411-417, 430-438, 448-459, 461-468, 1-15, 53-86, 89-225, 237-320, 331-398, 406-467, 107-184, and 256-311 of SEQ ID NO 358; amino acids 13-34, 36-42, 49-62, 86-92, 124-130, 132-145, 1-38, 41-140, and 4-89 of SEQ ID NO 359; amino acids 16-22, 45-51, 80-85, 88-108, 115-127, 139-157, 160-176, 184-189, 198-204, 227-234, 249-270, 277-295, 314-325, 331-337, 344-367, 397-424, 432-500, 512-521, 524-530, 555-563, 569-583, 586-617, 622-653, 661-673, 679-696, 710-738, 754-759, 764-786, 793-808, 813-820, 831-837, 844-850, 864-889, 891-899, 912-926, 961-970, 975-982, 1016-1024, 1027-1034, 1051-1059, 1096-1102, 1134-1147, 1158-1177, 1179-1185, 1188-1215, 1235-1250, 1271-1284, 1308-1315, 1-34, 79-113, 129-325, 327-360, 363-383, 385-514, 530-565, 577-701, 704-941, 944-981, 985-1005, 1012-1032, 1049-1088, 1097-1183, 1188-1320, 108-194, 370-410, 411-442, 956-1046, and 1055-1236 of SEQ ID NO 360; amino acids 20-32, 67-73, 84-91, 102-108, 148-163, 198-209, 232-237, 7-94, 98-116, 121-196, 205-224, 240-254, 113-132, and 167-245 of SEQ ID NO 361; amino acids 7-16, 36-43, 69-77, 101-114, 136-150, 155-161, 183-195, 197-206, 210-218, 225-237, 1-77, 97-156, 172-215, 221-240, and 92-164 of SEQ ID NO 362; amino acids 17-23, 52-62, 75-87, 107-112, 120-135, 140-146, 170-176, 188-206, 210-220, 228-255, 273-282, 304-314, 324-331, 344-355, 363-370, 2-35, 81-179, 181-264, 267-288, 292-376, 2-126, and 245-300 of SEQ ID NO 363; amino acids 4-9, 11-17, 24-85, 89-103, 114-119, 126-135, 151-158, 169-175, 191-203, 226-237, 247-256, 261-266, 278-283, 306-316, 321-331, 333-340, 350-358, 361-392, 397-408, 412-419, 1-113, 117-183, 186-275, 294-362, 370-448, 468-485, 63-136, 248-305, and 458-466 of SEQ ID NO 364; amino acids 4-23, 41-47, 52-59, 62-75, 86-92, 98-104, 106-112, 126-143, 152-157, 160-166, 183-192, 196-201, 207-214, 227-235, 238-245, 248-265, 276-285, 304-314, 324-342, 350-356, 360-375, 390-403, 411-435, 437-446, 453-461, 1-39, 43-79, 88-160, 165-236, 241-426, 434-470, 132-199, and 214-286 of SEQ ID NO 365; amino acids 8-31, 35-48, 75-98, 100-106, 128-134, 151-166, 168-177, 202-223, 252-257, 265-277, 288-302, 306-316, 371-377, 385-396, 408-413, 429-445, 472-480, 1-331, 352-386, 404-488, 80-156, and 237-303 of SEQ ID NO 366; amino acids 4-10, 22-32, 39-46, 52-69, 90-96, 104-117, 126-142, 148-161, 163-174, 178-195, 200-210, 216-222, 225-236, 238-246, 248-265, 279-285, 313-322, 324-336, 1-203, 205-339, 31-111, and 205-228 of SEQ ID NO 367; amino acids 6-26, 33-63, 66-74, 125-148, 153-185, 225-230, 234-250, 253-278, 286-299, 312-319, 335-346, 348-361, 383-401, 414-419, 423-430, 432-440, 447-454, 470-478, 488-508, 519-533, 537-554, 16-83, 126-172, 175-210, 245-325, 329-448, 469-557, 81-97, 197-371, and 470-500 of SEQ ID NO 368; amino acids 4-9, 11-32, 44-56, 68-74, 82-98, 104-109, 131-140, 144-164, 176-182, 188-194, 206-212, 219-225, 228-235, 237-244, 254-271, 277-287, 294-302, 312-318, 324-331, 341-350, 352-358, 399-405, 411-419, 438-447, 452-459, 465-470, 475-484, 486-506, 514-523, 525-532, 537-543, 1-73, 112-209, 231-294, 296-314, 326-381, 388-414, 423-457, 462-484, 495-514, 522-544, 547-568, and 495-559 of SEQ ID NO 369; amino acids 4-26, 29-36, 45-52, 57-63, 68-75, 77-88, 93-108, 114-120, 138-143, 148-155, 161-179, 198-211, 214-224, 229-242, 244-253, 268-274, 302-311, 319-331, 335-349, 364-374, 377-384, 388-394, 423-436, 441-446, 452-471, 478-501, 506-514, 522-546, 558-579, 589-604, 623-631, 1-36, 40-114, 125-255, 266-395, 413-511, 530-557, 564-583, 605-640, 54-144, and 543-627 of SEQ ID NO 370; amino acids 5-10, 24-30, 37-53, 62-94, 101-111, 118-125, 145-151, 156-162, 164-186, 191-198, 204-212, 224-233, 244-253, 263-269, 277-297, 304-322, 334-341, 355-372, 379-384, 393-406, 412-425, 427-435, 437-449, 461-471, 483-506, 515-527, 535-543, 546-559, 562-570, 576-584, 588-601, 640-647, 651-673, 681-689, 712-734, 1-63, 73-369, 371-428, 432-454, 457-567, 573-635, 655-698, 707-744, 600-685, and 741-753 of SEQ ID NO 371; amino acids 13-19, 27-38, 78-87, 105-129, 134-142, 151-179, 184-197, 206-214, 225-235, 246-265, 274-314, 322-330, 334-343, 1-47, 49-66, 70-96, 102-285, 287-322, 330-346, 14-57, 76-167, and 191-245 of SEQ ID NO 372; amino acids 15-21, 26-45, 49-60, 62-69, 77-87, 96-104, 108-114, 117-130, 135-144, 149-160, 174-181, 186-193, 203-218, 228-234, 236-242, 257-264, 285-326, 332-342, 344-349, 353-370, 373-425, 428-437, 447-457, 472-483, 490-505, 516-526, 546-554, 556-570, 572-585, 601-609, 613-624, 626-640, 647-663, 665-671, 678-689, 691-697, 707-719, 721-751, 769-778, 1-64, 73-192, 209-327, 329-418, 421-529, 534-586, 599-646, 650-682, 703-793, 86-169, 318-384, 502-520, and 529-602 of SEQ ID NO 373; amino acids 4-10, 19-26, 28-34, 41-60, 62-81, 89-97, 99-115, 143-156, 162-181, 186-193, 197-207, 225-231, 233-240, 254-266, 283-296, 309-323, 325-338, 341-352, 363-373, 388-413, 420-426, 1-37, 41-119, 133-251, 255-277, 291-355, 359-411, 93-182, and 360-391 of SEQ ID NO 374; amino acids 5-37, 48-77, 82-89, 91-102, 119-127, 129-138, 145-179, 186-194, 197-203, 205-212, 219-235, 243-250, 273-289, 294-304, 309-349, 352-363, 377-388, 391-399, 407-415, 418-443, 449-463, 471-477, 479-494, 496-503, 2-44, 85-108, 127-242, 245-259, 271-288, 291-329, 339-433, 443-457, 468-517, 34-88, 104-164, 168-227, 235-259, 279-301, and 345-377 of SEQ ID NO 375; amino acids 4-24, 32-39, 44-51, 60-70, 86-96, 98-105, 108-117, 122-138, 142-156, 167-181, 192-212, 214-223, 231-240, 254-278, 15-131, 139-285, 9-96, and 181-194 of SEQ ID NO 376; amino acids 12-31, 41-47, 50-73, 86-99, 103-109, 141-147, 152-167, 180-195, 201-214, 216-237, 240-246, 251-265, 275-281, 289-294, 301-314, 320-333, 342-349, 351-361, 367-389, 402-408, 426-435, 459-469, 9-27, 31-92, 94-134, 147-199, 202-268, 271-291, 303-333, 336-465, 480-498, and 72-139 of SEQ ID NO 377; amino acids 23-34, 41-52, 74-86, 92-99, 107-121, 123-130, 154-159, 162-169, 193-227, 229-243, 249-255, 260-269, 17-154, 183-272, and 137-270 of SEQ ID NO 378; amino acids 4-29, 81-88, 106-128, 177-200, 207-214, 221-234, 240-250, 255-277, 279-285, 291-324, 326-347, 349-362, 376-392, 400-412, 415-423, 425-440, 444-449, 453-460, 1-52, 60-99, 102-161, 166-189, 203-294, 312-429, 436-468, 487-512, and 419-508 of SEQ ID NO 379; amino acids 4-11, 42-49, 51-68, 91-97, 111-122, 130-142, 156-178, 186-230, 247-252, 278-297, 303-331, 337-344, 355-360, 383-391, 415-421, 439-444, 452-463, 468-481, 516-524, 537-542, 558-570, 576-582, 590-597, 621-633, 655-661, 667-673, 680-688, 728-734, 764-771, 776-789, 803-810, 825-841, 844-853, 877-891, 893-907, 924-933, 947-960, 966-974, 984-989, 998-1011, 1018-1024, 1028-1034, 1044-1053, 1084-1093, 1104-1110, 1114-1121, 1146-1154, 1166-1173, 1189-1196, 1202-1210, 1212-1218, 1220-1228, 1243-1248, 2-25, 30-82, 95-287, 304-370, 444-491, 507-662, 677-698, 704-746, 763-811, 818-920, 926-970, 989-1127, 1130-1198, 1203-1239, 200-260, 562-610, 815-858, 892-903, 961-1060, and 1065-1073 of SEQ ID NO 380; amino acids 25-41, 50-56, 58-70, 101-111, 118-126, 138-147, 161-168, 242-252, 271-291, 320-329, 336-342, 353-364, 369-375, 383-389, 418-432, 473-484, 499-514, 520-532, 537-545, 548-560, 567-575, 588-606, 612-622, 628-634, 647-658, 666-673, 686-693, 700-706, 714-720, 734-744, 754-759, 786-798, 820-827, 852-859, 864-885, 892-899, 908-915, 921-933, 941-946, 950-957, 1-52, 55-157, 189-250, 269-311, 358-576, 582-774, 783-837, 855-924, 930-961, 75-155, 197-242, 257-412, 457-547, 715-788, and 874-936 of SEQ ID NO 381; amino acids 29-36, 49-55, 92-100, 109-116, 127-135, 140-148, 153-158, 169-176, 179-191, 214-221, 227-237, 240-246, 1-43, 55-73, 108-155, 164-249, and 20-87 of SEQ ID NO 382; amino acids 5-19, 30-41, 44-49, 64-70, 72-80, 82-88, 95-100, 115-120, 141-163, 1-39, 57-119, 131-166, and 15-83 of SEQ ID NO 383; amino acids 18-53, 64-75, 91-98, 103-109, 112-119, 154-167, 173-181, 200-208, 226-241, 261-268, 283-289, 294-309, 344-371, 374-382, 388-398, 416-423, 425-439, 442-453, 456-472, 491-497, 502-513, 515-524, 527-542, 547-562, 599-608, 610-620, 623-632, 636-656, 666-675, 683-692, 697-703, 709-716, 747-769, 778-785, 792-805, 829-836, 863-870, 877-905, 913-922, 937-950, 952-985, 991-999, 1-110, 114-160, 169-189, 216-238, 244-364, 378-409, 422-446, 453-475, 487-570, 590-640, 643-713, 722-809, 816-952, 969-1017, 155-173, 239-280, 501-575, 689-727, 766-789, and 826-880 of SEQ ID NO 384; amino acids 29-35, 40-64, 66-75, 115-122, 141-173, 179-187, 199-206, 209-215, 234-240, 244-251, 259-272, 297-303, 319-325, 332-348, 358-368, 372-382, 388-397, 399-405, 431-439, 446-454, 43-130, 136-211, 242-297, 314-333, 338-387, 396-416, 422-459, 19-144, 219-359, and 438-452 of SEQ ID NO 385; amino acids 4-23, 30-35, 77-84, 92-97, 105-113, 115-120, 131-137, 140-151, 200-211, 226-239, 241-247, 255-261, 271-278, 304-309, 395-400, 429-435, 443-453, 478-493, 521-530, 535-553, 563-568, 571-581, 623-628, 660-667, 693-700, 1-28, 74-140, 151-185, 194-218, 225-298, 300-355, 358-443, 445-463, 468-553, 567-595, 597-621, 631-676, 681-699, 16-72, 526-619, and 625-694 of SEQ ID NO 386; amino acids 4-18, 26-42, 58-79, 83-89, 99-108, 110-118, 125-140, 143-156, 168-175, 177-189, 211-227, 262-273, 281-291, 356-363, 383-402, 464-472, 1-49, 59-247, 251-321, 338-404, 410-430, 438-493, and 244-462 of SEQ ID NO 387; amino acids 6-28, 57-65, 72-81, 87-99, 1-43, 46-68, 83-104, and 13-105 of SEQ ID NO 388; amino acids 4-12, 32-55, 67-81, 92-100, 119-126, 143-151, 153-163, 182-188, 201-210, 238-244, 250-256, 278-283, 290-296, 303-308, 312-317, 345-354, 356-367, 378-388, 423-434, 442-449, 458-465, 476-488, 494-501, 510-520, 530-538, 551-563, 570-577, 582-601, 628-635, 700-714, 719-725, 744-752, 761-769, 778-786, 790-796, 821-839, 851-867, 872-878, 920-945, 957-975, 1022-1031, 1038-1047, 1084-1090, 1-91, 102-123, 130-170, 173-290, 307-327, 334-354, 359-396, 416-484, 507-563, 567-590, 600-618, 623-648, 661-686, 692-813, 822-887, 905-984, 994-1076, 1079-1093, 219-278, 280-417, 599-696, and 750-759 of SEQ ID NO 389; amino acids 6-39, 46-61, 67-72, 75-93, 107-116, 129-164, 169-176, 181-189, 226-243, 245-255, 267-274, 277-305, 319-326, 342-363, 375-385, 390-404, 419-428, 439-446, 448-465, 478-488, 507-516, 524-530, 546-555, 1-33, 35-63, 71-209, 217-237, 246-260, 283-313, 317-397, 399-431, 445-476, 479-558, and 151-220 of SEQ ID NO 390; amino acids 5-14, 25-50, 84-91, 99-111, 119-125, 129-144, 188-197, 200-208, 228-234, 243-250, 262-269, 275-282, 294-299, 9-175, 183-302, 104-209, and 229-292 of SEQ ID NO 391; amino acids 8-27, 29-42, 45-62, 1-71, and 8-61 of SEQ ID NO 392; amino acids 13-39, 41-47, 56-75, 78-87, 100-107, 121-127, 146-157, 164-171, 173-183, 201-207, 210-216, 221-231, 244-252, 255-263, 281-289, 291-309, 348-374, 390-401, 424-433, 435-445, 458-474, 509-530, 534-545, 548-565, 582-591, 597-602, 605-620, 622-628, 663-672, 686-691, 699-711, 728-742, 745-758, 773-786, 3-118, 120-171, 183-290, 293-313, 316-343, 345-367, 373-555, 561-651, 655-730, 737-773, 261-330, 337-365, and 587-709 of SEQ ID NO 393; amino acids 4-25, 41-54, 92-98, 104-111, 122-131, 144-151, 154-160, 200-207, 215-233, 243-253, 259-270, 278-297, 299-310, 328-337, 345-352, 373-389, 398-405, 419-425, 438-447, 472-479, 483-489, 492-501, 505-519, 531-546, 550-555, 568-574, 581-589, 595-600, 622-631, 683-689, 714-725, 733-738, 747-759, 761-766, 772-779, 795-801, 814-821, 856-868, 899-907, 923-929, 931-937, 1-30, 37-57, 64-86, 93-191, 193-217, 231-277, 296-361, 365-394, 398-529, 534-735, 742-785, 788-912, 506-517, and 532-590 of SEQ ID NO 394; amino acids 4-10, 16-25, 31-59, 66-75, 90-99, 102-107, 136-149, 151-159, 185-202, 204-215, 224-237, 240-248, 254-262, 272-278, 310-316, 337-349, 390-396, 400-409, 420-429, 478-484, 502-517, 533-539, 570-584, 614-621, 625-634, 645-654, 662-672, 677-686, 719-733, 747-753, 762-772, 782-792, 802-811, 820-826, 832-841, 856-862, 912-918, 931-937, 964-970, 989-994, 1006-1011, 1017-1023, 1036-1043, 1053-1064, 1071-1077, 1082-1090, 1103-1109, 1128-1135, 1137-1143, 1156-1165, 1169-1174, 1197-1204, 1213-1222, 1255-1273, 1305-1311, 1315-1322, 1340-1346, 1352-1359, 1386-1393, 1403-1412, 1441-1447, 1459-1468, 1470-1479, 1493-1500, 1511-1520, 1525-1537, 1544-1557, 1564-1569, 1580-1585, 1591-1596, 1627-1633, 1642-1656, 1690-1697, 1707-1713, 1715-1721, 1751-1761, 1773-1781, 1791-1796, 1804-1810, 1846-1851, 1860-1870, 1876-1882, 1910-1917, 1947-1953, 1962-1970, 2008-2019, 2026-2036, 2043-2051, 2085-2090, 2105-2115, 2132-2142, 1-30, 32-88, 143-330, 337-359, 379-434, 480-583, 604-715, 719-736, 744-761, 768-800, 805-850, 874-889, 898-948, 961-1053, 1058-1110, 1135-1150, 1157-1183, 1189-1230, 1235-1255, 1259-1277, 1308-1377, 1383-1422, 1428-1450, 1453-1472, 1474-1501, 1507-1591, 1596-1660, 1669-1703, 1709-1767, 1788-1848, 1861-1876, 1922-1941, 1944-1979, 1998-2061, 2069-2102, 2115-2136, 60-298, 308-571, 616-842, 971-1120, 1126-1252, 1359-1652, 1688-1980, and 2051-2106 of SEQ ID NO 395; amino acids 17-25, 29-38, 41-60, 97-105, 111-117, 124-159, 172-181, 186-192, 200-207, 212-229, 241-257, 267-298, 305-314, 316-322, 335-341, 345-355, 357-364, 367-375, 377-382, 387-400, 407-413, 417-428, 437-443, 448-454, 456-463, 469-477, 483-489, 495-508, 513-524, 540-551, 585-602, 1-98, 108-194, 205-473, 490-510, 517-557, 559-615, and 16-287 of SEQ ID NO 396; amino acids 10-33, 35-41, 50-55, 60-73, 81-90, 106-114, 121-136, 147-158, 179-191, 212-219, 229-236, 266-275, 285-307, 312-318, 334-345, 351-359, 361-366, 373-390, 398-419, 450-464, 475-483, 495-506, 524-530, 534-546, 555-565, 572-583, 596-603, 623-640, 658-664, 675-685, 690-696, 702-711, 714-723, 742-748, 765-772, 783-790, 811-823, 835-842, 848-854, 874-881, 1-31, 33-74, 85-118, 138-158, 165-184, 204-359, 375-402, 408-478, 491-511, 531-640, 653-745, 756-861, 871-889, 34-89, 124-209, 227-245, 271-338, 402-453, and 496-596 of SEQ ID NO 397; amino acids 19-31, 39-48, 50-61, 70-76, 85-96, 98-106, 114-127, 129-159, 166-184, 195-210, 215-224, 234-240, 247-254, 259-265, 267-284, 286-295, 297-307, 310-318, 320-330, 340-347, 1-32, 34-74, 93-115, 134-190, 192-255, 266-339, 119-185, and 241-349 of SEQ ID NO 398; amino acids 16-31, 45-52, 62-68, 89-149, 154-164, 175-185, 187-193, 195-207, 222-230, 253-279, 287-295, 299-323, 1-62, 85-154, 172-222, 224-248, 252-328, 188-255, and 319-334 of SEQ ID NO 399; amino acids 7-15, 38-65, 67-76, 80-103, 123-139, 146-152, 181-188, 196-206, 223-229, 234-245, 259-267, 285-296, 323-333, 340-358, 387-393, 401-409, 417-427, 431-439, 441-450, 472-477, 483-496, 508-524, 591-598, 602-610, 616-624, 627-641, 655-664, 666-679, 687-694, 696-709, 714-726, 759-766, 768-778, 782-793, 804-810, 1-105, 115-293, 300-342, 344-418, 432-562, 567-598, 613-656, 670-713, 757-803, and 23-98 of SEQ ID NO 400; amino acids 9-18, 35-57, 65-74, 82-87, 92-98, 105-117, 128-136, 147-154, 157-165, 181-187, 189-218, 224-232, 2-174, 176-241, and 26-112 of SEQ ID NO 401; amino acids 4-13, 24-35, 49-56, 71-77, 81-98, 103-109, 134-150, 158-169, 176-186, 188-195, 222-229, 249-262, 273-282, 335-341, 358-366, 375-395, 415-423, 431-448, 468-477, 486-492, 500-517, 526-539, 553-558, 563-571, 573-579, 581-603, 606-641, 649-657, 659-669, 1-40, 46-117, 127-171, 186-201, 203-318, 332-352, 356-390, 411-543, 560-669, 13-84, and 395-470 of SEQ ID NO 402; amino acids 43-56, 95-108, 139-145, 158-178, 186-199, 204-216, 224-231, 234-247, 251-275, 282-290, 303-309, 312-329, 331-341, 349-355, 379-405, 410-416, 429-436, 452-457, 463-474, 476-483, 485-491, 507-531, 546-563, 573-585, 628-635, 663-669, 687-700, 718-725, 731-742, 758-769, 788-795, 800-811, 824-831, 841-852, 859-865, 869-881, 885-891, 896-904, 941-951, 955-972, 1-91, 93-195, 199-284, 324-357, 385-405, 409-499, 506-604, 621-678, 688-749, 753-806, 815-882, 887-927, 933-953, 30-60, 107-144, 235-255, 439-492, 620-673, 767-814, and 864-969 of SEQ ID NO 403; amino acids 6-31, 59-73, 76-87, 98-104, 107-118, 126-145, 156-165, 168-177, 185-193, 206-214, 217-237, 245-252, 256-276, 288-304, 314-331, 333-340, 366-378, 387-406, 409-423, 428-437, 457-463, 480-488, 509-519, 536-542, 550-563, 571-593, 606-612, 6-51, 54-151, 161-194, 203-357, 361-474, 477-497, 501-538, 541-615, and 319-382 of SEQ ID NO 404; amino acids 6-12, 17-31, 33-48, 1-53, and 2-60 of SEQ ID NO 405; amino acids 54-61, 64-86, 91-109, 111-120, 127-133, 135-143, 147-152, 166-176, 179-186, 189-207, 212-220, 226-231, 235-242, 250-267, 271-279, 294-308, 316-336, 347-353, 362-368, 380-389, 391-398, 406-418, 441-463, 471-500, 503-522, 524-560, 562-570, 572-582, 584-590, 634-639, 643-652, 657-663, 666-673, 697-707, 714-721, 727-734, 743-751, 758-776, 796-806, 852-867, 870-896, 898-905, 923-945, 948-955, 957-962, 964-969, 982-988, 1000-1006, 1008-1014, 1030-1042, 1056-1068, 1075-1086, 2-97, 111-196, 209-321, 327-349, 353-367, 377-408, 418-491, 500-664, 691-772, 788-1019, 1024-1049, 1054-1077, 1079-1095, 29-54, 261-267, and 635-691 of SEQ ID NO 406; amino acids 4-16, 18-41, 47-54, 106-113, 120-131, 144-150, 165-171, 192-202, 220-227, 229-239, 245-259, 314-327, 358-365, 377-384, 396-402, 414-430, 436-442, 457-466, 474-480, 498-519, 524-531, 535-541, 564-569, 581-597, 622-628, 653-676, 681-686, 719-727, 1-139, 145-180, 186-261, 271-291, 308-328, 332-365, 372-394, 400-440, 453-530, 533-585, 588-631, 639-687, 695-746, 118-193, 209-355, and 505-657 of SEQ ID NO 407; amino acids 6-27, 50-56, 62-72, 80-86, 98-111, 118-127, 138-168, 176-184, 189-201, 205-245, 256-263, 1-44, 46-115, 132-192, 222-242, 246-264, 161-212, and 218-247 of SEQ ID NO 408; amino acids 6-28, 32-41, 46-66, 74-84, 92-108, 110-117, 137-146, 152-170, 173-200, 226-264, 266-275, 284-301, 308-313, 316-335, 355-364, 374-388, 394-404, 406-416, 424-431, 437-452, 456-477, 1-49, 51-166, 170-203, 208-368, 382-486, 489-507, 5-82, 101-118, and 260-376 of SEQ ID NO 409; amino acids 4-13, 16-26, 28-34, 39-45, 47-63, 68-121, 130-145, 150-162, 165-171, 176-203, 215-240, 242-249, 251-261, 282-290, 293-309, 316-322, 334-352, 360-367, 397-410, 412-422, 430-439, 1-67, 73-274, 288-429, 434-465, 119-189, 200-246, and 399-436 of SEQ ID NO 410; amino acids 4-12, 15-22, 25-34, 36-47, 54-79, 92-99, 106-114, 129-136, 150-160, 168-184, 186-215, 222-229, 239-249, 286-293, 299-314, 322-327, 333-358, 371-378, 387-406, 413-422, 435-443, 448-455, 461-471, 474-480, 489-495, 1-42, 57-82, 87-123, 127-498, and 30-110 of SEQ ID NO 411; amino acids 4-29, 42-68, 70-79, 1-52, 57-80, and 15-86 of SEQ ID NO 412; amino acids 8-51, 78-86, 94-99, 107-113, 140-148, 156-162, 173-181, 189-197, 213-229, 264-270, 274-281, 283-293, 308-321, 347-360, 399-411, 425-436, 442-449, 466-482, 486-498, 516-521, 527-534, 557-565, 586-594, 604-617, 622-630, 652-674, 720-731, 742-754, 773-783, 1-129, 131-247, 263-335, 343-364, 370-639, 652-697, 712-779, 187-241, 319-394, 411-485, and 657-679 of SEQ ID NO 413; amino acids 7-14, 36-46, 48-57, 74-81, 85-108, 113-131, 141-154, 168-179, 16-67, 69-91, 96-134, 137-177, and 48-105 of SEQ ID NO 414; amino acids 4-20, 28-33, 39-55, 57-65, 68-82, 84-92, 117-123, 1-85, 92-148, and 15-142 of SEQ ID NO 415; amino acids 4-9, 17-23, 37-53, 71-89, 104-116, 123-128, 133-138, 145-170, 173-205, 213-232, 234-252, 260-268, 275-280, 287-311, 316-345, 357-364, 1-18, 37-57, 68-278, 306-400, 7-100, and 152-243 of SEQ ID NO 416; amino acids 5-19, 38-45, 77-83, 89-99, 108-119, 125-132, 144-153, 161-171, 180-191, 221-227, 233-243, 274-286, 293-299, 1-27, 44-147, 159-255, 262-280, 288-302, 22-73, and 224-290 of SEQ ID NO 417; amino acids 8-15, 17-39, 48-55, 57-77, 87-95, 99-109, 119-132, 146-152, 171-176, 196-203, 220-229, 231-245, 253-259, 273-285, 293-300, 318-324, 331-344, 367-374, 395-401, 409-414, 434-471, 483-490, 509-517, 519-534, 537-544, 554-574, 584-590, 599-609, 615-625, 632-639, 665-672, 676-686, 688-697, 702-708, 714-720, 730-742, 744-752, 780-786, 790-800, 812-836, 2-17, 28-146, 152-170, 198-214, 235-270, 279-326, 333-368, 381-407, 435-608, 624-650, 656-673, 711-779, 782-799, 809-872, 157-185, and 341-483 of SEQ ID NO 418; amino acids 4-57, 60-67, 80-96, 98-118, 5-114, and 38-112 of SEQ ID NO 419; amino acids 11-34, 43-51, 67-108, 116-138, 151-165, 9-30, 40-168, and 13-78 of SEQ ID NO 420; amino acids 18-24, 40-47, 51-62, 67-74, 87-94, 102-109, 112-121, 141-147, 152-164, 175-198, 200-224, 229-235, 245-260, 262-293, 310-323, 326-349, 390-414, 423-431, 440-447, 456-462, 466-476, 481-493, 497-504, 512-538, 545-550, 560-573, 584-591, 596-602, 607-613, 7-67, 71-131, 134-231, 233-260, 276-340, 342-385, 388-443, 447-475, 509-616, and 504-552 of SEQ ID NO 421; amino acids 11-17, 38-49, 52-58, 67-79, 98-104, 7-106, and 16-107 of SEQ ID NO 422; amino acids 5-17, 32-46, 54-60, 89-102, 112-127, 152-161, 169-178, 186-193, 228-240, 247-256, 259-266, 274-296, 3-38, 55-69, 72-135, 165-301, 22-96, 124-131, 143-172, and 209-275 of SEQ ID NO 423; amino acids 17-26, 32-44, 1-43, and 28-49 of SEQ ID NO 424; amino acids 11-17, 26-32, 72-77, 162-168, 204-211, 218-224, 234-243, 270-276, 287-301, 314-333, 339-345, 360-375, 392-398, 403-409, 419-434, 438-444, 454-468, 473-479, 489-503, 508-514, 524-539, 543-549, 559-574, 578-584, 607-616, 618-628, 664-669, 710-715, 718-730, 734-740, 747-763, 766-772, 774-785, 787-796, 73-125, 133-152, 163-246, 264-330, 337-376, 594-635, 660-680, 693-711, 715-816, 6-428, and 493-608 of SEQ ID NO 425; amino acids 14-29, 32-58, 63-76, 78-86, 89-100, 102-110, 131-140, 148-154, 188-201, 210-219, 230-243, 245-254, 262-281, 298-307, 316-324, 340-352, 373-396, 407-414, 440-453, 456-477, 480-487, 495-508, 515-529, 537-544, 552-560, 564-572, 587-592, 605-619, 625-632, 638-644, 660-674, 684-690, 697-719, 732-738, 1-58, 67-95, 123-168, 176-240, 242-284, 287-317, 325-390, 398-418, 423-499, 510-582, 598-631, 635-683, 692-734, 127-190, 537-604, and 697-727 of SEQ ID NO 426; amino acids 13-27, 38-44, 48-55, 57-69, 72-81, 97-108, 126-141, 152-160, 165-177, 180-186, 197-241, 289-296, 309-328, 334-339, 341-353, 1-106, 125-251, 257-306, 310-356, 42-51, and 97-267 of SEQ ID NO 427; amino acids 4-10, 12-21, 29-35, 44-56, 59-67, 74-86, 91-98, 103-114, 1-124, and 9-86 of SEQ ID NO 428.

In one embodiment the antigen further consists of

• • a) 1 to 50 additional amino acid residue(s), preferably 1 to 40, more preferably 1 to 30, even more preferably at most 1 to 25, still more preferably at most 1 to 10, most preferably 1, 2, 3, 4 or 5 additional amino acid residue(s); and/or
  • b) at least one amino acid residue heterologous to the core amino acid sequence.

Said additional amino acid residue(s) are further defined above.

In another embodiment said amino acid residue(s) is/are flanking the core amino acid sequence N-terminally, C-terminally, or N- and C-terminally.

In an embodiment of the invention the antigen comprises at least 2, at least 3, at least 4, at least 5 or at least 6 core amino acid sequences as defined above.

The problem underlying the present invention is solved in another aspect by a process for producing an antigen, or an active fragment or an active variant thereof, as defined in the present invention, comprising expressing the nucleic acid molecule as defined above.

The present invention further relates to a process for producing a cell which expresses an antigen, or an active fragment or an active variant thereof, as defined above, comprising transforming or transfecting a suitable host cell with the vector as defined above.

In an embodiment, the antigen, or the active fragment or the active variant thereof, is isolated from Mcat.

The problem underlying the present invention is solved in another aspect by a pharmaceutical composition, preferably a vaccine, comprising an antigen, or an active fragment or an active variant thereof, as defined above, or a nucleic acid molecule as defined above, or a vector as defined above.

Another aspect of the present invention provides a pharmaceutical composition, preferably a vaccine, comprising an antigen, or an active fragment or an active variant thereof, as defined above, or a nucleic acid molecule as defined above, or a vector as defined above, for the treatment or prevention of an infection with Mcat.

In a preferred embodiment the pharmaceutical composition of the present invention further comprises an immunostimulatory substance, preferably polycationic polymers, especially polycationic peptides, immuno stimulatory oligo-deoxynucleotides (ODNs), especially Oligo(dIdC)₁₃ (SEQ ID NO 857), peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK (SEQ ID NO 858), neuroactive compounds, especially human growth hormone, alum, Freund's complete or incomplete adjuvants, or combinations thereof.

In a more preferred embodiment of the pharmaceutical composition of the present invention the immunostimulatory substance is a combination of either a polycationic polymer and immunostimulatory deoxynucleotides, or of a peptide containing at least two LysLeuLys motifs and immunostimulatory deoxynucleotides, preferably a combination of KLKLLLLLKLK (SEQ ID NO 858) and Oligo(dIdC)₁₃ (SEQ ID NO 857).

In a still more preferred embodiment of the pharmaceutical composition of the present invention the polycationic polymer is a polycationic peptide, especially polyarginine.

Still another aspect of the present invention provides an antigen, or an active fragment or an active variant thereof, as defined above, or a nucleic acid molecule as defined above, or a vector as defined above for the treatment or prevention of an infection with Mcat.

Another preferred embodiment of the invention relates to the use of an antigen, an active fragment or an active variant thereof as defined above, or a nucleic acid molecule as defined above, or a vector as defined above for the preparation of a pharmaceutical composition, especially for the preparation of a vaccine, for treating or preventing infections with Mcat.

The problem underlying the present invention is solved in a further aspect by an antibody, or at least an effective part thereof, which binds to at least a selective part of an antigen or a fragment thereof, preferably an active fragment thereof, or a variant thereof, preferably an active variant thereof, as defined above.

In a preferred embodiment the antibody is a monoclonal antibody.

In another preferred embodiment said effective part comprises a Fab fragment, a F(ab) fragment, a F(ab) N fragment, a F (ab)₂ fragment or a F_v fragment.

In still another embodiment of the invention the antibody is a chimeric antibody.

In yet another embodiment the antibody is a humanized antibody.

In a further embodiment the antibody is an IgA or IgG antibody.

Another aspect of the invention relates to a hybridoma cell line, which produces an antibody as defined above.

The problem underlying the present invention is furthermore solved by a method for producing an antibody as defined above, characterized by the following steps:

• • a) initiating an immune response in a non-human animal by administering an antigen, or an active fragment or an active variant thereof, as defined above, to said animal,
  • b) removing an antibody containing body fluid from said animal, and
  • c) producing the antibody by subjecting said antibody containing body fluid to further purification steps.

The invention further relates to a method for producing an antibody as defined above, characterized by the following steps:

• • a) initiating an immune response in a non-human animal by administering an antigen or an active fragment or an active variant thereof, as defined above, to said animal,
  • b) removing the spleen or spleen cells from said animal,
  • c) producing hybridoma cells of said spleen or spleen cells,
  • d) selecting and cloning hybridoma cells specific for said antigen or for said active fragment or for said active variant thereof,
  • e) producing the antibody by cultivation of said cloned hybridoma cells, and
  • f) optionally conducting further purification steps.

Another aspect of the present invention is related to a pharmaceutical composition comprising an antibody as specified above.

Still another aspect relates to an antibody as defined above or a pharmaceutical composition comprising an antibody as defined above for the treatment or prevention of an infection with Mcat.

The problem underlying the present invention is solved in another aspect by the use of an antibody as defined above for the preparation of a pharmaceutical composition for treating or preventing infections with Mcat.

According to another aspect the present invention provides an antagonist, which binds or is capable of binding to an antigen, or an active fragment or active variant thereof as disclosed in the present invention. According to a still further aspect the antagonist according to the present invention is an antagonist which is capable of reducing or inhibiting the interaction activity of an antigen, or an active fragment thereof or an active variant thereof, according to the present invention to its interaction partner. Such interaction partner is, in a preferred embodiment, an antibody or a receptor, preferably a physiological receptor, of said antigen, or an active fragment thereof or an active variant thereof.

According to another aspect the present invention provides a method for identifying an antagonist capable of binding to an antigen or an active fragment or an active variant thereof, as defined above, comprising:

• • a) contacting an isolated or immobilized antigen or an active fragment or an active variant thereof, as defined above, with a candidate antagonist under conditions to permit binding of said candidate antagonist to said antigen, or an active fragment or active variant thereof, in the presence of a component capable of providing a detectable signal in response to the binding of the candidate antagonist to said antigen, or an active fragment or an active variant thereof; and
  • b) detecting the presence or absence of a signal generated in response to the binding of the antagonist to said antigen, or an active fragment or active variant thereof.

The problem underlying the present invention is further solved by a method for identifying an antagonist capable of reducing or inhibiting the interaction activity of an antigen or an active fragment or an active variant thereof, as defined above, to its interaction partner comprising:

• • a) providing an antigen, or an active fragment or active variant thereof, as defined above,
  • b) providing an interaction partner to said antigen, or said active fragment or active variant thereof, especially an antibody as defined above,
  • c) allowing interaction of said antigen or said active fragment or active variant thereof, to said interaction partner to form an interaction complex,
  • d) providing a candidate antagonist,
  • e) allowing a competition reaction to occur between the candidate antagonist and the interaction complex,
  • f) determining whether the candidate antagonist inhibits or reduces the interaction activities of the antigen, or the active fragment or the active variant thereof, with the interaction partner.

The present invention further relates to the use of any of the antigens, or an active fragment or an active variant thereof, as defined above, for the isolation and/or purification and/or identification of an interaction partner of said antigen, or said active fragment or active variant thereof.

Another aspect of the present invention relates to a method for diagnosing an infection with an Mcat organism comprising the steps of

• • a) contacting a sample obtained from a subject with an antigen, or an active fragment or active variant thereof, as defined above; and
  • b) detecting the presence of an antibody against said Mcat organism in the sample.

According to an embodiment of said method, the presence of one or more antibodies against said Mcat organism is indicative for the Mcat infection.

In another embodiment of said method the antibody is an IgA or IgG antibody.

In yet another aspect the present invention provides a method for diagnosing an infection with an Mcat organism comprising the steps of:

• • a) contacting a sample obtained from a subject with the antibody as defined above; and
  • b) detecting the presence of an antigen of said Mcat organism in the sample.

In an embodiment of said method the antigen of said Mcat organism is an antigen, or an active fragment or an active variant thereof, as defined above.

According to an embodiment of said method, the presence of one or more antigens of said Mcat organism is indicative for the Mcat infection.

In another embodiment of said method the antibody is an IgA or IgG antibody.

Still another aspect relates to a method for diagnosing an infection with an Mcat organism comprising the steps of:

• • a) contacting a sample obtained from a subject with a primer or a probe specific for a nucleic acid molecule, or a fragment thereof, as defined above; and
  • b) detecting the presence of such nucleic acid molecule or fragment thereof in the sample.

According to an embodiment of said method, the presence of one or more of said nucleic acid molecules or fragments thereof is indicative for the Mcat infection.

The present invention also provides a process for in vitro diagnosing a disease related to expression of an antigen or a fragment thereof according to the present invention comprising determining the presence of a nucleic acid sequence encoding said antigen or fragment thereof according to the present invention or determining the presence of the antigen or fragment thereof according to the present invention.

In an embodiment of any of the above described methods for diagnosing an infection with an Mcat organism, the Mcat organism is a pathogenic Mcat organism.

Moreover, the present invention provides the use of an antigen, or a fragment or a variant thereof, as defined in the present invention for the generation of a peptide binding to said antigen, or a fragment thereof or a variant thereof, wherein the peptide is an anticaline.

Moreover, the present invention provides the use of an antigen, or an active fragment or active variant thereof, as defined above, for the preparation of a functional nucleic acid, wherein the functional nucleic acid is selected from the group consisting of aptamers and spiegelmers.

In another aspect, the present invention provides the use of a nucleic acid molecule as defined above for the preparation of a functional ribonucleic acid, wherein the functional ribonucleic acid is selected from the group consisting of ribozymes, antisense nucleic acids and siRNA.

The problem underlying the present invention is further solved by a method for the treatment of an Mcat infection in an animal or human preferably in need thereof, comprising the step of administering to said animal or human a therapeutically effective amount of an antigen, or an active fragment or an active variant thereof, or a nucleic acid molecule, or a vector, or an antibody or a pharmaceutical composition as defined in any of the preceding aspects.

The problem underlying the present invention is solved in another aspect by a method for immunizing an animal or human against infection with an Mcat organism, comprising the step of administering to said animal or human an effective amount of the antigen, or an active fragment or active variant thereof, as defined above, or the nucleic acid molecule as defined above, or a vector as defined above, or an antibody as defined above, or a pharmaceutical composition as defined above, wherein the effective amount is suitable to elicit an immune response in said animal or human.

The problem underlying the present invention is solved in yet another aspect by a method for stimulating an immune response in an animal or human against an Mcat organism, comprising the step of administering to said animal or human an effective amount of the antigen, or an active fragment or an active variant thereof, as defined above, or of the nucleic acid molecule as defined above or of a vector as defined above, or an antibody as defined above, or a pharmaceutical composition as defined above, wherein the effective amount is suitable to stimulate the immune response in said animal or human.

It is within the present invention that the various methods and uses, respectively, where an antigen as defined in the present invention is used, can also be performed or practiced using a fragment of such antigen, preferably an active fragment thereof, or a variant of such antigen, preferably an active variant thereof, each as preferably described herein. It is also within the present invention that the various kinds of compounds disclosed herein as interacting with or targeting the antigen according to the present invention, can additionally or alternatively interact with or target the active fragment or active variant of said antigen.

It is also within the present invention that each and any method in the practice of which an antibody is used, can, in principle, also be practiced when instead of the antibody the anticalines or the functional nucleic acids as defined herein are used, whereby it is preferred that such functional nucleic acid is selected from the group consisting of aptamers and spiegelmers. This applies equally to the various uses of the present application.

In a preferred embodiment a fragment of an antigen as disclosed herein is a part of such antigen which exhibits at least one feature of such antigen. Preferably such feature is a feature selected from the group consisting of suitability for the treatment of infections, immunization of an animal including human, and/or stimulation of an immune response in an animal including human.

It is also within the present invention that any disclosure made herein in relation to Mcat.

The terms “polypeptide”, “peptide”, “protein” or “antigen” are used interchangeably throughout the present specification and refer in a comprehensive manner to the antigen according to the present invention, including each and any variant, fragment, analogue or derivative thereof, particularly as described herein. Insofar, whenever the term polypeptide, peptide, protein or antigen is used herein, and if not explicitly stated otherwise, the respective disclosure is also made for or in relation to any antigen according to the present invention, including each and any variant, fragment, analogue or derivative thereof, particularly as described herein. Also it is to be understood that any use or aspect described in connection with any of the above mentioned compounds covered by the term polypeptide, peptide, protein or antigen according to the present invention shall be applicable also to each and any other of the above mentioned compounds covered by the term polypeptide, peptide, protein or antigen according to the present invention.

The present invention advantageously provides an efficient, relevant and comprehensive set of isolated nucleic acid molecules and antigens encoded by them, including the active fragments and the active variants thereof, using an antibody preparation from multiple human plasma pools and surface expression libraries derived from the genome of Mcat. Thus, the present invention fulfils a widely felt demand for Mcat antigens, vaccines, diagnostics and products useful in procedures for preparing antibodies and for identifying compounds effective against infections caused by pathogenic Mcat species.

An effective vaccine should be composed of proteins or polypeptides, which are expressed by all strains and are able to induce high affinity, abundant antibodies against cell surface components of said pathogenic Mcat. The antibodies should be IgG1 and/or IgG3 for opsonisation, any IgG subtype, and/or IgA for neutralisation of adherence and toxin action.

Mcat infects the host via the mucosal epithelia of the respiratory tract and mucosal immunity is therefore believed to be important to control infection. IgA is the primary immunoglobulin (Ig) isotype induced at mucosal sites and is thought to mediate defense functions at these sites.

A chemically defined vaccine must be definitely superior compared to a whole cell vaccine (attenuated or killed), since components of said pathogenic Mcat, which cross-react with human tissues or inhibit opsonisation can be eliminated, and the individual polypeptides inducing protective antibodies and/or a protective immune response can be selected.

In a preferred embodiment of the present invention, the nucleic acid molecules exhibit 70% identity over their entire length to a nucleotide sequence set forth in SEQ ID NOs 860 to 886 or 1 to 214. More preferred are nucleic acids that comprise a region that is at least 80% or at least 85% identical over their entire length to a nucleic acid molecule set forth in SEQ ID NOs 860 to 886 or 1 to 214. In this regard, nucleic acid molecules, which are at least 90%, 91%, 92%, 93%, 94%, 95%, or 96% identical over their entire length to the same are particularly preferred. Furthermore, those with at least 97% are highly preferred, those with at least 98% and at least 99% are particularly highly preferred, with at least 99% or 99.5% being the more preferred, with 100% identity being especially preferred. Moreover, preferred embodiments in this respect are nucleic acids, which encode antigens or fragments thereof (polypeptides), which retain substantially the same biological function or activity as the mature polypeptide set forth in the SEQ ID NOs 887 to 913 or 215 to 428. It is also within the present invention that the nucleic acid molecules according to the present invention are coding for a protein which is preferably an antigen. Still further it is within the present invention, that the molecules defined by SEQ ID NOs 887 to 913 or 215 to 428 are proteins, which are preferably antigens.

Identity, as known in the art and used herein, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Identity can be readily calculated. While a number of methods exist to measure identity between two polynucleotide or two polypeptide sequences, the term is well known to skilled artisans (e.g. Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S. et al., 1990).

As a second alternative to the nucleic acid molecules described herein by reference to SEQ ID NOs 860 to 886 or 1 to 214, the description of which is also referred to herein as first alternative, the nucleic acid molecules according to the present invention can also be nucleic acid molecules, which are at least essentially complementary to the nucleic acids described in accordance with the first alternative herein. It will be acknowledged by the ones skilled in the art that an individual nucleic acid molecule is at least essentially complementary to another individual nucleic acid molecule. As used herein complementary means that a nucleic acid strand is base pairing via Watson-Crick base pairing with a second nucleic acid strand. Essentially complementary as used herein means that the base pairing is not occurring for all of the bases of the respective strands but leaves a certain number or percentage of the bases unpaired or wrongly paired. The percentage of correctly pairing bases is preferably at least 70%, more preferably 80%, even more preferably 90% and most preferably any percentage higher than 90%. Such higher percentage includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, whereby such definition is applicable to each aspect of the present application where this kind of terminology is used. It is to be noted that a percentage of 70% matching bases is considered as homology and the hybridisation having this extent of matching base pairs is considered as stringent. Hybridisation conditions for this kind of stringent hybridisation may be taken from Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1987). More particularly, the hybridisation conditions can be as follows:

• • Hybridisation performed e.g. in 5×SSPE, 5×Denhardt's reagent, 0.1% SDS, 100 g/mL sheared DNA at 68° C.

Moderate stringency wash in 0.2×SSC, 0.1% SDS at 42° C.

High stringency wash in 0.1×SSC, 0.1% SDS at 68° C.

Genomic DNA with a GC content of 50% has an approximate T_M of 96° C. For 1% mismatch, the T_M is reduced by approximately 1° C.

In addition, any of the further hybridisation conditions described herein are in principle applicable as well.

Of course, all nucleic acid sequence molecules which encode the same polypeptide molecule as those identified by the present invention are encompassed by any disclosure of a given coding sequence, since the degeneracy of the genetic code is directly applicable to unambiguously determine all possible nucleic acid molecules which encode a given polypeptide molecule, even if the number of such degenerated nucleic acid molecules may be high. This is also applicable for active fragments or active variants of a given antigen, as long as the fragments or variants encode an antigen being suitable to be used such that the same effect can be obtained as if the full-length antigen was used. Preferably, such antigens or active fragments or active variants thereof may be used in a vaccination application, e.g. as an active or passive vaccine.

As a third alternative, the nucleic acid molecule according to the present invention can also be a nucleic acid which comprises a stretch of at least 15 bases of the nucleic acid molecule according to the first or second alternative of the nucleic acid molecules according to the present invention as outlined above. Preferably, the bases form a contiguous stretch of bases. However, it is also within the scope of the present invention that the stretch consists of two or more moieties, which are separated by a number of bases.

The nucleic acid molecules according to the present invention may preferably consist of at least 20, even more preferred at least 30, especially at least 50 contiguous bases from the sequences disclosed herein. The suitable length may easily be optimised due to the intended field of use (e.g. as (PCR) primers, probes, capture molecules (e.g. on a (DNA) chip), etc.). Preferred nucleic acid molecules contain at least a contiguous 15 base portion of one or more of the immunogenic amino acid sequences listed in Table 4. Specifically preferred are nucleic acids containing a contiguous portion of a DNA sequence of any sequence contained in the sequence protocol of the present application which shows 1 or more, preferably more than 2, especially more than 5, non-identical nucleic acid residues compared to the genome sequences of Mcat RH4 (determined by Intercell AG (Vienna, Austria)). Specifically preferred non-identical nucleic acid residues are residues which lead to a non-identical amino acid residue. Preferably, the nucleic acid sequences encode polypeptides, proteins, or antigens having at least 1, preferably at least 2, preferably at least 3 different amino acid residues compared to the published or listed Mcat counterparts mentioned above. Preferably, these kind of polypeptides, proteins, or antigens still have at least one of the characteristics of the molecules disclosed herein having identical amino acid residues. Also preferred are such isolated polypeptides which are fragments of the proteins or of the antigens disclosed herein, e.g. in the Sequence Listing, having at least 6, 7, or 8 amino acid residues and being encoded by the nucleic acids as described herein.

The nucleic acid molecule according to the present invention can, as a fourth alternative, also be a nucleic acid molecule which anneals under stringent hybridisation conditions to any of the nucleic acids of the present invention according to the first, second, or third alternative as disclosed herein. Stringent hybridisation conditions are typically those described herein.

Finally, the nucleic acid molecule according to the present invention can, as a fifth alternative, also be a nucleic acid molecule which, but for the degeneracy of the genetic code, would hybridise to any of the nucleic acid molecules of the present invention according to the first, second, third, and fourth alternative as outlined herein. This kind of nucleic acid molecule refers to the fact that preferably the nucleic acids according to the present invention code for the antigen, or fragments or variants thereof, according to the present invention. This kind of nucleic acid molecule is particularly useful in the detection of a nucleic acid molecule according to the present invention and thus the diagnosis of the respective microorganisms such as Mcat or any pathogenic Mcat species, and any disease or diseased condition where these kinds of microorganisms are involved. Preferably, such microorganism, especially an opportunistic microorganism, is causing such disease directly or indirectly. Preferably, the hybridisation could occur or be performed under stringent conditions as described in connection with the fourth alternative described herein.

Nucleic acid molecule as used herein generally refers to any ribonucleic acid molecule, such as mRNA or cRNA, or deoxyribonucleic acid molecule, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid molecule may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acid molecule as used herein refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded DNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single- and double-stranded regions. In addition, nucleic acid molecule as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may be derived from one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. As used herein, the term nucleic acid molecule includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are nucleic acid molecules as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are nucleic acid molecules as the term is used herein. It will be appreciated that a great variety of modifications can be made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term nucleic acid molecule as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of nucleic acid molecule, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. The term nucleic acid molecule also embraces short nucleic acid molecules often referred to as oligonucleotide(s). “Polynucleotide” and “nucleic acid” or “nucleic acid molecule” are often used interchangeably herein.

Nucleic acid molecules provided in the present invention also encompass numerous unique fragments, both longer and shorter than the nucleic acid molecule sequences set forth in the sequencing listing of the present application, more specifically of the Mcat coding regions, which can be generated by standard cloning methods. To be unique, a fragment must be of sufficient size to distinguish it from other known nucleic acid sequences, most readily determined by comparing any selected Mcat fragment to the nucleotide sequences in biosequence databases such as GenBank. It will be appreciated by the one skilled in the art that what is said herein in any aspect in relation to Mcat applies equally to any other Mcat species, more preferably any pathogenic Mcat species.

Additionally, modifications can be made to the nucleic acid molecules and polypeptides that are encompassed by the present invention. For example, the nucleic acid also includes sequences that are a result of the degeneration of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, nucleotide substitutions can be made which do not affect the polypeptide encoded by the nucleic acid. Accordingly, any nucleic acid molecule which encodes an antigen or fragments thereof is encompassed by the present invention.

Furthermore, any of the nucleic acid molecules encoding antigens or fragments thereof provided by the present invention can be functionally linked, using standard techniques such as standard cloning techniques, to any desired regulatory sequences, whether an Mcat regulatory sequence or a heterologous regulatory sequence, heterologous leader sequence, heterologous marker sequence or a heterologous coding sequence to create a fusion protein.

The present invention further relates to variants of the nucleic acid molecules described herein which encode fragments, analogs and derivatives of the antigens and fragments thereof having a deducted Mcat amino acid sequence set forth in the Sequence Listing. A variant of the nucleic acid molecule may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.

Among variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Preferred are nucleic acid molecules encoding a variant, analog, derivative or fragment, or a variant, analogue or derivative of a fragment, which have an Mcat sequence as set forth in the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to 4, 3, 2, 1 or no amino acid(s) is substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the Mcat polypeptides set forth in the Sequence Listing. Also especially preferred in this regard are conservative substitutions.

The nucleic acid molecules of the present invention may also be used as a hybridisation probe for, e.g., RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the nucleic acid molecules of the present invention. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 20, at least 25 or at least 30 bases, and may have at least 50 bases. Particularly preferred probes will have at least 30 bases, and will have 50 bases or less, such as 30, 35, 40, 45, or 50 bases.

For example, the coding region of a nucleic acid molecule of the present invention may be isolated by screening a relevant library using the known DNA sequence to synthesize an oligonucleotide probe. A labelled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine to which members of the library the probe hybridizes.

The nucleic acid molecules and polypeptides of the present invention may be employed as reagents and materials for the development or preparation of pharmaceutical compositions and/or diagnostics for diseases, particularly human disease, as further discussed herein.

The nucleic acid molecules of the present invention that are oligonucleotides can be used in the processes herein as described, but preferably for PCR, to determine whether or not the Mcat genes identified herein in whole or in part are present and/or transcribed in infected tissue such as skin, synovia or blood. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained. For this and other purposes arrays which are known as such in the art, comprising at least one of the nucleic acids or polypeptides according to the present invention as described herein, may be used.

The nucleic acid molecules according to the present invention may be used for the detection of nucleic acid molecules and organisms or samples containing these nucleic acids. Preferably such detection is for diagnosis, more preferably for the diagnosis of a disease related or linked to the presence or abundance of Mcat.

Eukaryotes (herein also “individual(s)”), particularly mammals, and especially humans, infected with Mcat can be identified by detecting any of the nucleic acid molecules according to the present invention detected at the DNA level by a variety of techniques. Preferred nucleic acid molecule candidates for distinguishing Mcat or other pathogenic Mcat from other organisms can be obtained.

The invention provides a process for diagnosing disease arising from infection with Mcat, comprising determining from a sample isolated or derived from an individual an increased level of expression of a nucleic acid molecule having the sequence of a nucleic acid molecule as disclosed herein and more preferably set forth in the Sequence Listing. Expression of nucleic acid molecules can be measured using any one of the methods well known in the art for the quantification of nucleic acid molecules, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting, other hybridisation methods and the arrays described herein.

Isolated as used herein means separated “by the hand of man” from its natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a naturally occurring nucleic acid molecule or a polypeptide naturally present in a living organism in its natural state is not “isolated”, but the same nucleic acid molecule or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein. As part of or following isolation, such nucleic acid molecules can be joined to other nucleic acid molecules, such as DNAs, for mutagenesis, to form fusion genes, and for propagation or expression in a host, for instance. The isolated nucleic acid molecules, alone or joined to other nucleic acid molecules such as vectors, can be introduced into host cells, in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment. Similarly, the nucleic acid molecules and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of nucleic acid molecules or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated nucleic acid molecules or polypeptides within the meaning of that term as it is employed herein.

The nucleic acids can, for example, be isolated from Mcat by methods known to the one skilled in the art. The same applies to the polypeptides according to the present invention.

Preferably, the nucleic acid molecules of the present invention may be originally formed in vitro, e.g. by chemical synthesis, or in a cell culture and subsequent isolation or purification. In general, the nucleic acids may be obtained by the manipulation of nucleic acids by endonucleases and/or exonucleases and/or polymerases and/or ligases and/or recombinases or other methods known to the skilled practitioner to produce the nucleic acids.

The nucleic acid sequences as defined by SEQ ID NOs 860 to 886 or 1 to 214 start with the first complete codon comprised by the fragment as inserted into the vector and encodes the first amino acid as defined by SEQ ID NOs 887 to 913 or 215 to 428. However, for the recombinant production additional nucleic acids might be useful or necessary to facilitate the cloning and expression.

The present invention also relates to vectors, which comprise a nucleic acid molecule or nucleic acid molecules of the present invention. A vector may additionally include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication, one or more therapeutic genes and/or selectable marker genes and other genetic elements known in the art such as regulatory elements directing transcription, translation and/or secretion of the encoded protein. The vector may be used to transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell. The vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.

The present invention also relates to host cells, which are genetically engineered with vectors of the invention and to the production of the polypeptides according to the present invention by recombinant techniques.

A great variety of expression vectors can be used to express the polypeptides according to the present invention. Generally, any vector suitable to maintain, propagate or express nucleic acids to express a polypeptide in a host may be used for expression in this regard. In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector. Starting plasmids disclosed herein are either commercially available, publicly available, or can be constructed from available plasmids by routine application of well-known, published procedures. Preferred among vectors, in certain respects, are those for expression of nucleic acid molecules and the polypeptides according to the present invention. Nucleic acid constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides according to the present invention can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA construct of the present invention.

Host cells can be genetically engineered to incorporate nucleic acid molecules and express nucleic acid molecules of the present invention. Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, Vero, PER.C6® and Bowes melanoma cells; and plant cells.

The host cells can be transfected, e.g. by conventional means such as electroporation with at least one expression vector containing a nucleic acid of the invention under the control of a transcriptional regulatory sequence.

According to another aspect of the present invention, a comprehensive set of novel polypeptides is provided. Such polypeptides, as mentioned previously herein, are antigens as disclosed herein, and the fragments thereof, preferably the active fragments thereof, and the variants thereof, preferably the active variants thereof. Preferably, the polypeptides according to the present invention are antigens and fragments thereof. In a preferred embodiment of the invention, an antigen comprising an amino acid sequence being preferably encoded by any one of the nucleic acids molecules and fragments thereof as described herein, are provided. In another preferred embodiment of the invention a novel set of proteins and antigens and active fragments as well as active variants thereof is provided which comprise amino acid sequences selected from the group consisting of SEQ ID NOs 887 to 913 and 215 to 428.

The polypeptides according to the present invention, i.e. the antigens, as provided by the present invention preferably include any polypeptide or molecule set forth in the Sequence Listing as well as polypeptides which have at least 70% identity to such polypeptide according to the present invention, preferably at least 80% or 85% identity to such polypeptide according to the present invention, and more preferably at least 90% similarity (more preferably at least 90% identity) to such polypeptide according to the present invention and more preferably as set forth in the Sequence Listing and still more preferably at least 95%, 96%, 97%, 98%, 99% or 99.5% similarity (still more preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to such polypeptide according to the present invention and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 4 amino acids and more preferably at least 8, still more preferably at least 30, still more preferably at most 50 amino acids, such as 4, 8, 10, 20, 30, 35, 40, 45 or 50 amino acids. In a preferred embodiment such portions are active fragments of the polypeptides according to the present invention.

The invention also relates to fragments, analogs, and derivatives of the polypeptides according to the present invention. The terms “fragment”, “derivative” and “analog” when referring to such polypeptide whose amino acid sequence is preferably set forth in the Sequence Listing, means a polypeptide which retains essentially the same or a similar biological activity as such polypeptide. It will be acknowledged by the ones skilled in the art that the meaning of the term “similar biological activity” as used herein preferably depends on the polypeptide under consideration and more specifically its function. The term “biological activity” as used herein is further defined below. More preferably, a similar biological function or activity differs from the function of the non-fragment or the non-derivative in terms of extent of activity, affinity, immunogenicity, stability and/or specificity. In a preferred embodiment the difference is less than 50%, less than 75% or less than 90%.

In an embodiment the fragment, derivative, variant or analog of a polypeptide according to the present invention is 1) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the amino acid residues includes a substituent group, or 3) one in which the polypeptide according to the present invention or a fragment thereof is fused with another compound, such as a compound to increase the half-life of the polypeptide according to the present invention or a fragment thereof such as, for example, polyethylene glycol, or 4) one in which the additional amino acids are fused to the polypeptide according to the present invention or a fragment thereof, such as a leader or secretory sequence or a sequence which is employed for purification of said polypeptide according to the present invention or fragment thereof or a proprotein sequence. Such fragments, derivatives, variants and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The present invention also relates to proteins and antigens of Mcat species, preferably pathogenic Mcat species. Such homologues may easily be isolated based on the nucleic acid and amino acid sequences disclosed herein.

There are multiple serotypes, genotypes or clinical strains distinguished to date for each of the pathogens and the typing is based on serotype specific antisera or molecular approaches. The presence of any antigen can accordingly be determined for every serotype, genotype or strain. The contribution of the various serotypes to the different Mcat infections varies in different age groups and especially geographical regions. It is an important aspect that the most valuable protective antigens need to be conserved among various clinical strains.

Additionally, fusion polypeptides comprising such antigens, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments are also encompassed by the present invention. Such fusion polypeptides and proteins, as well as nucleic acid molecules encoding them, can readily be made using standard techniques, including standard recombinant techniques for producing and expression of a recombinant polynucleic acid encoding a fusion protein.

In another embodiment of the invention the peptide as defined above may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity (as defined above for fragments and variants) as the un-modified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether C-terminal or side chain, may be provided in the form of a salt of a pharmaceutically acceptable cation or esterified to form an ester, or converted to an amide. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be converted to an amide. Hydroxyl groups of the peptide side chains may be converted to alkoxy or to an ester using well recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with alkyl, alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Thiols can be protected with any one of a number of well recognized protecting groups, such as acetamide groups.

Further particularly preferred in this regard are variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments, having the amino acid sequence of any polypeptide according to the present invention as disclosed herein and preferably set forth in the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the peptide of the present invention. Also especially preferred in this regard are conservative substitutions. Most highly preferred are peptides having an amino acid sequence set forth in the Sequence Listing without substitutions.

Variants of any of the antigens in their various embodiments disclosed herein and in particular the antigens and peptides specified herein by SEQ ID NOs 887 to 913 or 215 to 428, can typically also be characterized by means of bioinformatics. Respective tools such as the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul, S. et al., 1990) are available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. For comparisons of amino acid sequences of at least 35 amino acids, the Blast 2 sequences function of NCBI Blast 2.0 was employed using the default BLOSUM62 matrix set to default parameters (gapped blastp; gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 35 amino acids), the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Methods for determining sequence identity over such short windows such as 15 amino acids or less are described at the website that is maintained by the National Center for Biotechnology Information in Bethesda, Md. (http://www.ncbi.nlm.nih.gov/BLAST/).

The active variant of an antigen is obtained by sequence alterations in the antigen, including each and any variant, fragment, analogue or derivative thereof, if not explicitly indicated to the contrary, wherein the polypeptide according to the present invention with the sequence alterations retains a function of the unaltered polypeptide according to the present invention, e.g. having a biological activity similar to that displayed by the complete antigen, including the ability to induce an immune response and/or to show protection against an Mcat organism e.g. in a mouse model of Mcat infection. Suitable animal models are reviewed in T. F. Murphy 2005.

A further example of retaining the function of the unaltered polypeptide according to the present invention is that the active variant of the antigen specifically binds a polypeptide specific antibody that binds an unaltered form of the polypeptide according to the present invention. By “biological function” or “biological activity” is preferably meant a function of the polypeptide in cells or organisms in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells and organisms, respectively. For example, the biological function of a porin is to allow the entry into cell of compounds present in the extracellular medium. The biological function is distinct from the antigenic function. A polypeptide according to the present invention can have more than one biological function.

The sequence alterations of such variants can include, but are not limited to, conservative substitutions, deletions, mutations and insertions. Among preferred variants are those that vary from a reference by conservative amino acid substitutions. Conservative substitutions are those that substitute a given amino acid in a polypeptide according to the present invention by another amino acid of like characteristics, i.e. those substitutions that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains, etc. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe and Tyr.

In one embodiment, one conservative substitution is included in the peptide. In another embodiment, two conservative substitutions or less are included in the peptide. In a further embodiment, three conservative substitutions or less are included in the peptide.

Examples of conservative amino acid substitutions include, but are not limited to, those listed below:

Original Residue Conservative Substitutions
Ala              Ser
Arg              Lys
Asn              Gln; His
Asp              Glu
Cys              Ser
Gln              Asn
Glu              Asp
His              Asn; Gln
Ile              Leu; Val
Leu              Ile; Val
Lys              Arg; Gln; Asn
Met              Leu; Ile
Phe              Met; Leu; Tyr
Ser              Thr
Thr              Ser
Trp              Tyr
Tyr              Trp; Phe
Val              Ile; Leu

Preferably, the active variant exhibits reactivity with human sera of patients with Mcat infections, more preferably mediates seroconversion and most preferably shows bactericidal activity. These characteristics of the active variant can be assessed e.g. as detailed in the Examples. In the context of the present invention a variant specifically binds a specific antibody (preferably being polyclonal antibodies raised against recombinant proteins in animals such as mouse, rabbit or monoclonal antibodies generated in mouse), exhibits reactivity with human sera from patients with Mcat infections, mediates seroconversion or shows bactericidal activity, if the activity of the variant amounts to at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, most preferably at least 99% of the activity of the antigen without sequence alterations.

Said active variants include naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As is known in the art, an allelic variant is an alternate form of a (poly)peptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does essentially not alter the biological function of the polypeptide, as it is described above.

Within any species of the living world, allelic variation is the rule. For example, any bacterial species, e.g. Mcat, is usually represented by a variety of strains (characterized by clonal reproduction) that differ from each other by minor allelic variations. Indeed, a polypeptide that fulfils the same biological function in different strains can have an amino acid sequence that is not identical in each of the strains. Such an allelic variation is equally reflected at the nucleotide level.

In a preferred embodiment, the active variant or the active fragment derived from the polypeptide according to the present invention by amino acid exchanges, deletions or insertions may also conserve, or more preferably improve, the activity (reactivity, seroconversion and/or bactericidal activity as defined herein). Furthermore, these polypeptides may also cover epitopes, which trigger the same or preferably an improved T cell response. These epitopes are referred to as “heteroclitic” as further defined herein. They have a similar or preferably greater affinity to MHC/HLA molecules, and the ability to stimulate the T cell receptors (TCR) directed to the original epitope in a similar or preferably stronger manner. Heteroclitic epitopes can be obtained by rational design i.e. taking into account the contribution of individual residues to binding to MHC/HLA as for instance described by Rammensee H. et al., (1999), combined with a systematic exchange of residues potentially interacting with the TCR and testing the resulting sequences with T cells directed against the original epitope. Such a design is possible for a skilled person in the art without undue experimentation.

In a still more preferred embodiment of the invention the active variant of a polypeptide according to the present invention is any of the polypeptides disclosed herein and more specifically any of the polypeptides defined by the SEQ ID NOs 887 to 913 or 215 to 428, having at least 50% sequence identity to the polypeptides of any of said SEQ ID NOs 887 to 913 or 215 to 428, especially at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, 96%, 97%, 98%, most preferably 99% sequence identity to the polypeptides of any of said SEQ ID NOs 887 to 913 or 215 to 428 and/or is derived from said polypeptides of any of the sequences of SEQ ID NOs 887 to 913 or 215 to 428 by conservative substitutions as defined above.

The polypeptides according to the present invention, and fragments and variants thereof, also include or consist of modified epitopes wherein preferably one or two of the amino acids of a given epitope are modified or replaced according to the rules disclosed in, e.g., Tourdot, S. et al., (2000), as well as the nucleic acid sequences encoding such modified epitopes. The epitopes as presented by the polypeptides according to the present invention are also referred to herein as the present epitopes.

It is clear that also epitopes derived from the present epitopes by amino acid exchanges improving, conserving or at least not significantly impeding the T cell activating capability of the epitopes are covered by the epitopes according to the present invention. Therefore the present epitopes also cover epitopes which do not contain the original sequence as derived from Mcat, but trigger the same or preferably an improved T cell response. These epitope are referred to as “heteroclitic”; they need to have a similar or preferably greater affinity to MHC/HLA molecules the ability to stimulate the T cell receptors (TCR) directed to the original epitope in a similar or preferably stronger manner.

Another possibility for identifying epitopes and more specifically heteroclitic epitopes includes the screening of peptide libraries with T cells directed against one or several of the present epitopes. A preferred way is the positional scanning of synthetic peptide libraries. Such approaches have been described in detail for instance by Hemmer, B. et al., (1999) and the references given therein.

As an alternative to epitopes represented by the present derived amino acid sequences or heteroclitic epitopes as disclosed herein, also substances or compounds mimicking these epitopes which are also referred to herein as “peptidemimetica” or “retro-inverse-peptides” can be applied and are thus within the present invention.

Another aspect of the design of improved epitopes is their formulation or modification with substances increasing their capacity to stimulate T cells. These include T helper cell epitopes, lipids or liposomes or preferred modifications as described in WO 01/78767.

Another way to increase the T cell stimulating capacity of epitopes is their formulation with immune stimulating substances, for instance cytokines or chemokines, like interleukin-2, -7, -12, -18, class I and II interferons (IFN), especially IFN-γ, GM-CSF, TNF-alpha, flt3-ligand and others.

The polypeptides according to the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

In another embodiment of the present invention the variant is a fragment. The fragment is characterized by being derived from the antigen as defined above by one or more amino acid deletions. The deletion(s) may be C-terminal, N-terminal and/or internal. Preferably the fragment is obtained by at most 10, 20, 30, 40, 50, 60, 80, 100, 150 or 200, more preferably by at most 10, 20, 30, 40 or 50, even more preferably at most 5, 10 or 15, still more preferably at most 5 or 10, most preferably 1, 2, 3, 4 or 5 deletion(s). The active fragment of the invention is characterized by having a biological activity similar to that displayed by the complete antigen, including the ability to induce an immune response and/or to show protection against Mcat, e.g. in a mouse model of Mcat infection, such as described above. The fragment of an antigen is active in the context of the present invention if the activity of the fragment amounts to at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, most preferably at least 99% of the activity of the antigen without sequence alteration. These fragments may be designed or obtained in any desired length, including as small as about 50 to 80 amino acids in length.

In a further embodiment a fragment, and more preferably an active fragment, of the polypeptide according to the present invention are characterised by structural or functional attributes, i.e. fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha-amphipathic regions, beta-amphipathic regions, flexible regions, surface-forming regions, substrate binding regions, and high antigenic index regions of the polypeptide according to the present invention, and combinations of such fragments. Preferred regions are those that mediate antigenicity and antibody binding activities of the polypeptides according to the present invention. Most highly preferred in this regard are fragments that have a chemical, biological or other activity of the antigen and fragments thereof of the present invention, including those with a similar activity or an improved activity, whereby such improved activities are immunogenicity and stability, or with a decreased undesirable activity, whereby such decreased undesirable activity is enzymatic and/or toxic function and generation of human cross-reactive antibodies. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Mcat, or the ability to cause disease in humans. Further preferred fragments of the polypeptides according to the present invention are those that comprise or contain antigenic or immunogenic determinants in an animal, especially in a human. Such fragments are also referred to as antigenic fragments.

An antigenic fragment is preferably defined as a fragment, which is antigenic by itself or may be made antigenic when provided as a hapten. Therefore, also antigens or antigenic fragments showing one or, particularly for longer fragments, only a few amino acid exchanges are enabled by the present invention, provided that the antigenicity or antigenic capacities of such fragments with amino acid exchanges are not severely deteriorated on the exchange(s), i.e., suited for eliciting an appropriate immune response in an individual vaccinated with this antigen and identified by individual antibody preparations from individual sera.

Preferred examples of such fragments of the polypeptides according to the present invention are the core amino acid sequence as indicated in Table 4.

All these fragments listed in Table 4 individually and each independently form a preferred selected aspect of the present invention.

It will be appreciated that the invention also relates to, among others, nucleic acid molecules encoding the aforementioned fragments, variants, active variants, and active fragments, nucleic acid molecules that hybridise to nucleic acid molecules encoding the fragments, variants, active variants, and active fragments, particularly those that hybridise under stringent conditions, and nucleic acid molecules, such as PCR primers, for amplifying nucleic acid molecules that encode the fragments. In these regards, preferred nucleic acid molecules are those that correspond to the preferred fragments, as discussed above.

The polypeptides according to the present invention may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N- or C-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, regions may be added to the polypeptide to facilitate purification or to enhance expression. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability, to enhance expression or to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize or purify polypeptides. For example, EP 0464533 discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another protein or part thereof. In drug discovery, for example, proteins have been fused with antibody Fc portions for the purpose of high-throughout screening assays to identify antagonists. See for example, (Bennett, D. et al., 1995) and (Johanson, K. et al., 1995). Fusions also may include the polypeptides according to the present invention fused or coupled to moieties other than amino acids, including lipids and carbohydrates. Further, antigens of this invention may be employed in combination with other vaccinal agents described by the prior art, as well as with other species of vaccinal agents derived from other microorganisms. Such proteins are useful in the prevention, treatment and diagnosis of diseases caused by a wide spectrum of Mcat isolates.

In a further embodiment the peptide of the invention is fused to an epitope tag which provides an epitope to which an anti-tag substance can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the peptide but may be incorporated as an internal insertion or substitution as the biological activity permits. The presence of such epitope-tagged forms of a peptide can be detected using a substance such as an antibody against the tagged peptide. Also, provision of the epitope tag enables the peptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his), poly-histidine-glycine (poly-his-gly) tags, the HA tag polypeptide, the c-myc tag, the Strep tag and the FLAG tag.

The polypeptides of the invention may be prepared by any of a number of conventional techniques. For example, they can be produced by chemical synthesis as well as by biotechnological means. The latter comprise the transfection or transformation of a host cell with a vector containing a nucleic acid according to the present invention. In a preferred embodiment the vector is a vector according to the present invention. The biotechnological production of the polypeptides according to the present invention further comprises the cultivation of the transfected or transformed host cell under conditions that allow expression of the protein and which are known to the one skilled in the art. The expressed protein is recovered, isolated, and optionally purified from the cell (or from the culture medium, if expressed extracellularly) by appropriate means known to one of skill in the art. For example, the proteins are isolated in soluble form following cell lysis, or extracted using known techniques, e.g. in guanidine chloride. The molecules comprising the polypeptides and antigens of this invention may be further purified using any of a variety of conventional methods including, but not limited to: ammonium sulfate or ethanol precipitation, acid extraction, liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with inorganic ligands or monoclonal antibodies), size exclusion chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, immobilized metal chelate chromatography, gel electrophoresis, and the like. One of skill in the art may select the most appropriate isolation and purification techniques without departing from the scope of this invention. Such purification provides the antigen in a form substantially free from other proteinaceous and non-proteinaceous materials of the microorganism.

An alternative approach to prepare polypeptides according to the invention involves generating the fragments of known peptides by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes, expressing the digested DNA and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired peptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed as the 5′ and 3′ primers in the PCR. Techniques for making mutations, such as deletions, insertions and substitutions, at predetermined sites in DNA, and therefore in proteins, having a known sequence are well known. One of skill in the art using conventional techniques, such as PCR, may readily use the antigens and peptides provided herein to identify and isolate other similar proteins. Such methods are routine and not considered to require undue experimentation, given the information provided herein. For example, variations can be made using oligonucleotide-mediated site-directed mutagenesis (Carter, P. et al., 1985; Zoller, M. J. et al., 1987), cassette mutagenesis (Wells, J. A. et al., 1985), restriction selection mutagenesis (Wells, J. A. et al., 1986), PCR mutagenesis, or other known techniques can be performed on the cloned DNA to produce the peptide of the invention.

The polypeptide according to the present invention may be used for the detection of the organism or organisms in a sample containing these organisms or proteins or antigens, including fragments thereof. Preferably such detection is for diagnosis, more preferable for the diagnosis of a disease, most preferably for the diagnosis of a disease related or linked to the presence or abundance of Gram-negative bacteria, especially pathogenic Mcat species.

The nucleic acids according to the present invention can also be used for the diagnosis or detection of organisms in a sample, whereby the organisms are preferably the same ones as disclosed in connection with the use of the polypeptides according to the present invention and the antibody according to the present invention, respectively. Basically, it is within the skills of the person of the art to design and practice such diagnosis and detection assays and methods, respectively, in the light of the present disclosure. More preferably such diagnosis or detection uses primers or probes to specifically interact with the nucleic acid molecules according to the present invention. The length and design of such primers and probes, respectively, varies depending on the particular method or diagnosis practiced. Using, in a preferred embodiment, a primer for, e.g., a PCR based detection or diagnosis system, i.e. method or assay, the length of the primer will range from about 10 nucleotides to about 30 nucleotides and more preferably from about 16 to 25 nucleotides. In case of a probe based detection or diagnosis system the length of the probe is preferably about the same as specified for the primer based system. Additionally, in case of a probe based system, the probe will comprise a moiety which allows its detection, either directly or indirectly. Such moiety for direct detection can be a radioactive label or a fluorescence label as known to the ones skilled in the art. Such moiety for indirect detection can be a biotin or any other moiety which mediates interaction with a further compound which in turn is labelled so as to allow its detection.

The present invention also relates to diagnostic assays, such as quantitative diagnostic assays for detecting levels of the polypeptides according to the present invention, and more preferably antigens and fragments thereof of the present invention, in cells and tissues, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of the polypeptides according to the present invention compared to normal control tissue samples may be used to detect the presence of an infection, for example, and to identify the infecting organism. Assay techniques that can be used to determine levels of such polypeptides in a sample derived from a host are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Among these, ELISA and Western Blot analysis frequently are preferred. An ELISA assay initially comprises preparing an antibody specific to one of the polypeptides according to the present invention, preferably a monoclonal antibody. In addition, a reporter antibody generally is prepared which binds to the monoclonal antibody. The reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent, such as horseradish peroxidase enzyme. One or several of the polypeptides according to the present invention and more preferably an antigen and fragment thereof according to the present invention may be immobilised on ELISA plates for detection of reactive antibodies in sera of patients or subjects to be tested.

A Western blot assay initially separates the polypeptides according to the present invention individually or in combination by SDS-polyacrylamide gel electrophoresis and which subsequently are transferred and immobilised onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. Together with a reporter antibody reactive antibodies can be detected. The reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent, such as horseradish peroxidase enzyme.

The polypeptides according to the present invention or the nucleic acid molecules according to the present invention or primers or probes directed thereto as described herein, may also be used for the purpose of or in connection with an array. In case of the nucleic acid molecule according to the present invention and the primers and probes directed there against, the length of the probes and the primer, can also preferably be in the range from about 25 to about 75 nucleotides, more preferably from about 35 to about 50 nucleotides. More particularly, at least one of the polypeptides according to the present invention may be immobilized on a support. Said support typically comprises a variety of the polypeptides according to the present invention and/or antigens and fragments thereof whereby the variety may be created by using one or several of the antigens and fragments thereof according to the present invention and/or antigens and fragments thereof being different. The characterizing feature of such array as well as of any array in general is the fact that at a distinct or predefined region or position on said support or a surface thereof, a distinct polypeptide is immobilized. Because of this, any activity at a distinct position or region of an array can be correlated with a specific polypeptide. The number of different polypeptides and more preferably different antigens and fragments thereof immobilized on a support may range from as little as 10 to several 1,000 different polypeptides and antigens and fragments thereof, respectively. The density of said molecules per cm² is in a preferred embodiment as little as 10 per cm² to at least 400 different of such polypeptides per cm² and more particularly at least 1,000 different of such polypeptides and more preferably different antigens and fragments thereof per cm². What is said herein about the immobilization of the polypeptides according to the present invention and their use, is also applicable to the nucleic acid molecules and the primers and probes, respectively, directed there against, as will be acknowledged by the ones skilled in the art.

The manufacture of such arrays is known to the one skilled in the art and, for example, described in U.S. Pat. No. 5,744,309. The array preferably comprises a planar, porous or non-porous solid support having at least a first surface. The polypeptides according to the present invention are immobilized on said surface. Preferred support materials are, among others, glass or cellulose. It is also within the present invention that the array is used for any of the diagnostic applications described herein. Apart from the polypeptides according to the present invention also the nucleic acid molecules according to the present invention may be used for the generation of an array as described above which, in principle, can be used for any of the purposes disclosed for the array containing polypeptides. This applies as well to an array made of antibodies, preferably monoclonal antibodies as, among others, described herein.

In a further aspect the present invention relates to an antibody directed to any of polypeptides according to the present invention, derivatives, fragments, variants, active fragments and active variants thereof according to the present invention. The present invention includes, for example, monoclonal and polyclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library. It is within the present invention that the antibody may be chimeric, i.e. that different parts thereof stem from different species or at least the respective sequences are taken from different species.

Such antibodies in general and in particular directed against the antigens and fragments thereof corresponding to a sequence of the present invention can be obtained by direct injection of a polypeptide according to the present invention into an animal or by administering said polypeptide to an animal, preferably a non-human. The antibody so obtained will then bind said polypeptide itself. In this manner, even a sequence encoding only a fragment said polypeptide can be used to generate antibodies binding the whole native polypeptides according to the present invention. Such antibodies can then be used to isolate the polypeptide according to the present invention from tissue expressing antigens and fragments thereof. It will be understood by the ones skilled in the art that this procedure is also applicable to the fragments, variants, active fragments and active variants thereof of said polypeptides.

Another aspect of the present invention relates to methods for producing antibodies according to the invention. This includes, for example, monoclonal and polyclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library, which are able to specifically bind to the peptide or composition according to the invention.

In a preferred embodiment the antibody is a monoclonal, polyclonal, chimeric or humanized antibody or functionally active fragment thereof. In another preferred embodiment the functionally active fragment comprises a Fab fragment.

Antibodies generated against the peptide or antigen or composition according to the invention can be obtained by direct injection of the peptide or antigen or composition according to the invention into an animal or administering of the peptide or antigen or composition according to the invention to an animal, preferably a non-human. The antibody so obtained will then bind the peptide or antigen or composition according to the invention. Such antibodies can then be used to isolate reactive antigens, peptide or proteins from a tissue expressing those.

For preparation of monoclonal antibodies, any technique known in the art which provides antibodies produced by continuous cell line cultures, can be used (as described originally in Köhler, G. et al., 1975).

Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic antigens and fragments thereof in their diverse embodiments according to this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies to the polypeptides according to the present invention.

Antibodies may also be produced using a hybridoma cell line.

Still another aspect of the invention relates to a hybridoma cell line which produces the antibody of the invention.

Hybridoma cell lines expressing desirable monoclonal antibodies are generated by well-known conventional techniques. The hybridoma cell can be generated by fusing a normal-activated, antibody-producing B cell with a myeloma cell. In the context of the present invention the hybridoma cell is able to produce an antibody specifically binding to the antigen of the invention.

Similarly, desirable high titre antibodies are generated by applying known recombinant techniques to the monoclonal or polyclonal antibodies developed to these antigens (see, e.g., PCT Patent Application No. PCT/GB85/00392; British Patent Application Publication No. GB2188638A; Amit, A. G. et al., 1986; Queen, C. et al., 1989; PCT Patent Application No. WO 90/07861; Riechmann, L. et al., 1988; Huse, W. D. et al., 1988).

Alternatively, the antibody may be produced employing display libraries. For example, phage display technology or ribosomal display could be utilized to select antibody genes with binding activities towards the polypeptides according to the present invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing respective target antigen binding antibodies or from naïve libraries (McCafferty, J. et al., 1990; Marks, J. et al., 1992). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., 1991).

If two antigen binding domains are present, each domain may be directed against a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides according to the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.

Thus, among others, antibodies against the polypeptides according to the present invention may be employed to inhibit and/or treat infections, particularly bacterial infections and especially infections arising from pathogenic Mcat species.

The polypeptides according to the present invention and more specifically antigens and fragments thereof in their diverse embodiments include antigenically, epitopically or immunologically equivalent derivatives, which form a particular aspect of this invention. The term “antigenically equivalent derivative” as used herein encompasses such polypeptide according to the present invention or its equivalent which will be specifically recognized by certain antibodies which, when raised to said polypeptide, interfere with the interaction between pathogen and mammalian host. The term “immunologically equivalent derivative” as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the interaction between pathogen and mammalian host.

The polypeptides according to the present invention and more specifically the antigens and fragments thereof in their diverse embodiments, such as an antigenically or immunologically equivalent derivative or a fusion protein thereof can be used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide according to the present invention. Such polypeptide may be associated, for example by conjugation, with an immunogenic carrier protein, for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Alternatively, an antigenic peptide comprising multiple copies of the polypeptide according to the present invention and more preferably an antigen and fragments thereof, or an antigenically or immunologically equivalent antigen and fragments thereof, may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.

Preferably the antibody or derivative thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be “humanized”, wherein the complementarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in (Jones, P. et al., 1986) or (Tempest, P. et al., 1991).

The use of a nucleic acid molecule according to the present invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscle, delivery of DNA complexed with specific protein carriers, coprecipitation of DNA with calcium phosphate, encapsulation of DNA in various forms of liposomes, particle bombardment (Tang, D. et al., 1992; Eisenbraun, M. et al., 1993) and in vivo infection using cloned retroviral vectors (Seeger, C. et al., 1984).

In a further aspect the present invention relates to a peptide binding to any of the polypeptides according to the present invention, and a method for the preparation of such peptides whereby the method is characterized by the use of said polypeptide and the basic steps are known to the one skilled in the art.

Such peptides may be generated by using methods according to the state of the art such as phage display or ribosome display. In case of phage display, basically a library of peptides is generated in the form of phages, and this kind of library is contacted with the target molecule, in the present case a polypeptide according to the present invention. Those peptides binding to the target molecule are subsequently removed, preferably as a complex with the target molecule, from the respective reaction. It is known to the one skilled in the art that the binding characteristics, at least to a certain extent, depend on the particularly realized experimental set-up such as the salt concentration and the like. After separating those peptides binding to the target molecule with a higher affinity or a bigger force, from the non-binding members of the library, and optionally also after removal of the target molecule from the complex of target molecule and peptide, the respective peptide(s) may subsequently be characterised. Prior to the characterisation optionally an amplification step is realized such as, e.g. by propagating the peptide encoding phages. The characterisation preferably comprises the sequencing of the target binding peptides. Basically, the peptides are not limited in their lengths, however preferably peptides having a length from about 8 to 20 amino acids are preferably obtained in the respective methods. The size of the libraries may be about 10² to 10¹⁸, preferably 10⁸ to 10¹⁵ different peptides, however, is not limited thereto. In a preferred embodiment such peptides are high-affinity binding peptides. In an even more preferred embodiment the peptides are peptide aptamers.

Peptide aptamers as used herein refer to peptide molecules that bind a specific target molecule. Peptide aptamers are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to that of an antibody (nanomolar range). The variable loop length is typically comprised of 10 to 20 amino acids, and the scaffold may be any protein which has good solubility and stability properties. Currently, the bacterial protein Thioredoxin-A is the most used scaffold protein, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two Cysteines' lateral chains being able to form a disulfide bridge. Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system. Selection of Ligand Regulated Peptide Aptamers (LiRPAs) has been demonstrated. By displaying 7 amino acid peptides from a novel scaffold protein based on the trimeric FKBP-rapamycin-FRB structure, interaction between the randomized peptide and target molecule can be controlled by the small molecule Rapamycin or non-immunosuppressive analogs.

A particular form of target binding peptides as described above, are the so-called “anticalines” which are, among others, described in German patent application DE 19742706. In so far, the present invention is also related to peptides specifically binding to the polypeptides according to the present invention and the use thereof for any of the therapeutic and diagnostic applications described herein, preferably for antibodies.

In a further aspect the present invention relates to functional nucleic acids interacting with any of the polypeptides according to the present invention, and a method for the preparation of such functional nucleic acids whereby the method is characterized by the use of the polypeptides according to the present invention and the basic steps are known to the one skilled in the art. The functional nucleic acids are preferably nucleic acid aptamers and spiegelmers. In so far, the present invention is also related to nucleic acid aptamers and spiegelmers specifically binding to the polypeptides according to the present invention and the use thereof for any of the therapeutic and diagnostic applications described herein, preferably for antibodies.

Nucleic acid aptamers are D-nucleic acids, which are either single stranded or double stranded and which specifically interact with a target molecule. The preparation or selection of aptamers is, e.g. described in European patent EP 0533838. Basically the following steps are realized. First, a mixture of nucleic acids, i.e. potential aptamers, is provided whereby each nucleic acid typically comprises a segment of several, preferably at least eight subsequent randomised nucleotides. This mixture is subsequently contacted with the target molecule whereby the nucleic acid(s) bind to the target molecule, such as based on an increased affinity towards the target or with a bigger force thereto, compared to the candidate mixture. The binding nucleic acid(s) are/is subsequently separated from the remainder of the mixture. Optionally, the thus obtained nucleic acid(s) is amplified using, e.g. polymerase chain reaction. These steps may be repeated several times giving at the end a mixture having an increased ratio of nucleic acids specifically binding to the target from which the final binding nucleic acid is then optionally selected. These specifically binding nucleic acid(s) are referred to as aptamers. It is obvious that at any stage of the method for the generation or identification of the aptamers samples of the mixture of individual nucleic acids may be taken to determine the sequence thereof using standard techniques. It is within the present invention that the aptamers may be stabilized such as, e.g., by introducing defined chemical groups which are known to the one skilled in the art of generating aptamers. Such modification may for example reside in the introduction of an amino group at the 2′-position of the sugar moiety of the nucleotides. Aptamers are currently used as therapeutic agents. However, it is also within the present invention that the thus selected or generated aptamers may be used for target validation and/or as lead substance for the development of pharmaceutical compositions, preferably of pharmaceutical compositions based on small molecules. This is actually done by a competition assay whereby the specific interaction between the target molecule and the aptamer is inhibited by a candidate drug whereby upon replacement of the aptamer from the complex of target and aptamer it may be assumed that the respective drug candidate allows a specific inhibition of the interaction between target and aptamer, and if the interaction is specific, said candidate drug will, at least in principle, be suitable to block the target and thus decrease its biological availability or activity in a respective system comprising such target. The thus obtained small molecule may then be subject to further derivatisation and modification to optimise its physical, chemical, biological and/or medical characteristics such as toxicity, specificity, biodegradability and bioavailability.

Spiegelmers and their generation or preparation is based on a similar principle. The preparation of spiegelmers is described in international patent application WO 98/08856. Spiegelmers are L-nucleic acids, which means that they are composed of L-nucleotides rather than D-nucleotides, as aptamers are. Spiegelmers are characterized by the fact that they have a very high stability in biological systems and, comparable to aptamers, specifically interact with the target molecule against which they are directed. In the process of generating spiegelmers, a heterogeneous population of D-nucleic acids is created and this population is contacted with the optical antipode of the target molecule, in the present case for example with the D-enantiomer of the naturally occurring L-enantiomer of the antigens and fragments thereof according to the present invention. Subsequently, those D-nucleic acids are separated which do not interact with the optical antipode of the target molecule. But those D-nucleic acids interacting with the optical antipode of the target molecule are separated, optionally identified and/or sequenced and subsequently the corresponding L-nucleic acids are synthesized based on the nucleic acid sequence information obtained from the D-nucleic acids. These L-nucleic acids, which are identical in terms of sequence with the aforementioned D-nucleic acids interacting with the optical antipode of the target molecule, will specifically interact with the naturally occurring target molecule rather than with the optical antipode thereof. Similar to the method for the generation of aptamers it is also possible to repeat the various steps several times and thus to enrich those nucleic acids specifically interacting with the optical antipode of the target molecule.

In a further aspect the present invention relates to functional nucleic acids interacting with any of the nucleic acid molecules according to the present invention, and a method for the preparation of such functional nucleic acids whereby the method is characterized by the use of the nucleic acid molecules and their respective sequences according to the present invention and the basic steps are known to the one skilled in the art. The functional nucleic acids are preferably ribozymes, antisense oligonucleotides and siRNA. In so far, the present invention is also related to this kind of functional nucleic acid specifically binding to the polypeptides according to the present invention and the use thereof for any of the therapeutic and diagnostic applications described herein, preferably for antibodies.

Ribozymes are catalytically active nucleic acids, which preferably consist of RNA, which basically comprises two moieties. The first moiety shows a catalytic activity whereas the second moiety is responsible for the specific interaction with the target nucleic acid, in the present case the nucleic acid coding for the polypeptides according to the present invention. Upon interaction between the target nucleic acid and the second moiety of the ribozyme, typically by hybridisation and Watson-Crick base pairing of essentially complementary stretches of bases on the two hybridising strands, the catalytically active moiety may become active which means that it catalyses, either intramolecularly or intermolecularly, the target nucleic acid in case the catalytic activity of the ribozyme is a phosphodiesterase activity. Subsequently, there may be a further degradation of the target nucleic acid, which in the end results in the degradation of the target nucleic acid as well as the protein derived from the said target nucleic acid. Ribozymes, their use and design principles are known to the one skilled in the art, and, for example described in (Doherty, E. et al., 2001) and (Lewin, A. et al., 2001).

The activity and design of antisense oligonucleotides for the preparation of a pharmaceutical composition and as a diagnostic agent, respectively, is based on a similar mode of action. Basically, antisense oligonucleotides hybridise based on base complementarity, with a target RNA, preferably with an mRNA, thereby activating RNase H. RNase H is activated by both phosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupled DNA, however, is rapidly degraded by cellular nucleases with the exception of phosphorothioate-coupled DNA. These resistant, non-naturally occurring DNA derivatives do not inhibit RNase H upon hybridisation with RNA. In other words, antisense polynucleotides are only effective as DNA RNA hybrid complexes. Examples for this kind of antisense oligonucleotides are described, among others, in U.S. Pat. No. 5,849,902 and U.S. Pat. No. 5,989,912. In other words, based on the nucleic acid sequence of the target molecule which in the present case are the nucleic acid molecules for the antigens and fragments thereof according to the present invention, either from the target protein from which a respective nucleic acid sequence may in principle be deduced, or by knowing the nucleic acid sequence as such, particularly the mRNA, suitable antisense oligonucleotides may be designed base on the principle of base complementarity.

Particularly preferred are antisense-oligonucleotides, which have a short stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3 DNA bases is required for activation of bacterial RNase H and a minimum of 5 bases is required for mammalian RNase H activation. In these chimeric oligonucleotides there is a central region that forms a substrate for RNase H that is flanked by hybridising “arms” comprised of modified nucleotides that do not form substrates for RNase H. The hybridising arms of the chimeric oligonucleotides may be modified such as by 2′-O-methyl or 2′-fluoro. Alternative approaches used methylphosphonate or phosphoramidate linkages in said arms. Further embodiments of the antisense oligonucleotide useful in the practice of the present invention are P-methoxyoligonucleotides, partial P-methoxyoligodeoxyribonucleotides or P-methoxyoligodeoxy-ribonucleotides.

Of particular relevance and usefulness for the present invention are those antisense oligonucleotides as more particularly described in the two above mentioned US patents. These oligonucleotides contain no naturally occurring 5′=>3′-linked nucleotides. Rather the oligonucleotides have two types of nucleotides: 2′-deoxyphosphorothioate, which activate RNase H, and 2′-modified nucleotides, which do not. The linkages between the 2′-modified nucleotides can be phosphodiester, phosphorothioate or P-ethoxyphosphodiester. Activation of RNase H is accomplished by a contiguous RNase H-activating region, which contains between 3 and 5 2′-deoxyphosphorothioate nucleotides to activate bacterial RNase H and between 5 and 10 2′-deoxyphosphorothioate nucleotides to activate eukaryotic and, particularly, mammalian RNase H. Protection from degradation is accomplished by making the 5′ and 3′ terminal bases highly nuclease resistant and, optionally, by placing a 3′ terminal blocking group.

More particularly, the antisense oligonucleotide comprises a 5′ terminus and a 3′ terminus; and from position 11 to 59 5′=>3′-linked nucleotides independently selected from the group consisting of 2′-modified phosphodiester nucleotides and 2′-modified P-alkyloxyphosphotriester nucleotides; and wherein the 5′-terminal nucleoside is attached to an RNase H-activating region of between three and ten contiguous phosphorothioate-linked deoxyribonucleotides, and wherein the 3″-terminus of said oligonucleotide is selected from the group consisting of an inverted deoxyribonucleotide, a contiguous stretch of one to three phosphorothioate 2′-modified ribonucleotides, a biotin group and a P-alkyloxyphosphotriester nucleotide.

Also an antisense oligonucleotide may be used wherein not the 5′ terminal nucleoside is attached to an RNase H-activating region but the 3′ terminal nucleoside as specified above. Also, the 5′ terminus is selected from the particular group rather than the 3′ terminus of said oligonucleotide.

The nucleic acids as well as the polypeptides according to the present invention in their diverse embodiments may be used as or for the preparation of pharmaceutical compositions, especially vaccines. Preferably such pharmaceutical composition, preferably vaccine is, for the prevention or treatment of diseases caused by, related to or associated with Mcat species. In so far another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal, which comprises inoculating the individual with the polypeptides according to the present invention in their diverse embodiments, or a fragment or variant thereof, adequate to produce antibodies to protect said individual from infection by the above microorganisms.

Yet another aspect of the invention relates to a method of inducing an immunological response in an individual which comprises, through gene therapy or otherwise, delivering a nucleic acid molecule according to the present invention, preferably functionally encoding antigens and fragments thereof in their diverse embodiments, for expressing the polypeptide according to the present invention in vivo in order to induce an immunological response to produce antibodies or a cell mediated T cell response, either cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not. One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise.

A further aspect of the invention relates to an immunological composition which, when introduced into a host capable of having induced within it an immunological response, induces an immunological response in such host, wherein the composition comprises recombinant DNA which codes for and expresses at least one of the polypeptides according to the present invention in their diverse embodiments. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.

The polypeptides according to the present invention in their diverse embodiments may be fused with a co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. This fused recombinant protein preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilise the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.

Also provided by this invention are methods using the nucleic acid molecule according to the present invention in their diverse embodiments in such genetic immunization experiments in animal models of infection with Mcat. Such molecules will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. This approach can allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of the Mcat infection in mammals, particularly humans.

The polypeptides according to the present invention in their diverse embodiments may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage and thus damaged tissue include wounds in skin or connective tissue and mucosal tissues caused e.g. by viral infection (esp. respiratory, such as the flu), mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina.

The present invention also includes a vaccine formulation, which comprises one or several of polypeptides according to the present invention in their diverse embodiments together with one or more suitable carriers and/or excipients. The pharmaceutically acceptable carriers and/or excipients useful in this invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the (poly)peptides herein disclosed. In general, the nature of the carrier or excipients will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g. powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Since said polypeptides according to the present invention may be broken down in the stomach, they are preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, intradermal, intranasal or transdermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the body fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in-water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

According to another aspect, the present invention relates to a pharmaceutical composition comprising one or several of the polypeptides according to the present invention in their diverse embodiments for Mcat. Such a pharmaceutical composition may comprise one, preferably at least two or more of said polypeptides against Mcat species. Optionally, such polypeptides may also be combined with antigens against even further pathogens in a combination pharmaceutical composition. Preferably, said pharmaceutical composition is a vaccine for preventing or treating an infection caused by an Mcat species, more preferably a pathogenic Mcat species, and/or other pathogens against which the antigens have been included in the vaccine.

According to a further aspect, the present invention relates to a pharmaceutical composition comprising a nucleic acid molecule according to the present invention. Such a pharmaceutical composition may comprise one or more nucleic acid molecules according to the present invention encoding a polypeptide according to the present invention. Optionally, such nucleic acid molecules encoding the polypeptides according to the present invention are combined with nucleic acid molecules encoding antigens against other pathogens in a combination pharmaceutical composition. Preferably, said pharmaceutical composition is a vaccine for preventing or treating an infection caused by Mcat species, more preferably pathogenic Mcat species, and/or other pathogens against which the antigens have been included in the vaccine.

The pharmaceutical composition may contain any suitable auxiliary substances, such as buffer substances, stabilisers or further active ingredients, especially ingredients known in connection of pharmaceutical composition and/or vaccine production.

In a preferred embodiment the pharmaceutical composition further comprises an immunostimulatory substance such as an adjuvant. The adjuvant can be selected based on the method of administration and may include polycationic substances, especially polycationic peptides, immunostimulatory nucleic acids molecules, preferably immunostimulatory oligo-deoxynucleotides (ODNs), especially Oligo(dIdC)₁₃, peptides containing at least two LysLeuLys motifs, especially peptide KLKLLLLLKLK, alum, mineral oil-based adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, neuroactive compounds, especially human growth hormone, or any combination of one or more of the above mentioned adjuvants. Other suitable adjuvants may be selected from the group consisting of Montanide incomplete Seppic adjuvant such as ISA, oil in water emulsion adjuvants such as the Ribi adjuvant system, syntax adjuvant formulation containing muramyl dipeptide, or aluminum salt adjuvants or combinations thereof.

The term “Oligo(dIdC)₁₃” as used in the present invention means a phosphodiester backboned single-stranded DNA molecule containing 13 deoxy (inosine-cytosine) motifs, also defined by the term [oligo-d(IC)₁₃]. The exact sequence is 5′-dIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdC-3′. Oligo(dIdC)₁₃ can also be defined by the terms (oligo-dIC₂₆); oligo-dIC_26-mer; oligo-deoxy IC, 26-mer; or oligo-dIC, 26-mer, as specified for example in WO 01/93903 and WO 01/93905.

It is also within the scope of the present invention that the pharmaceutical composition, especially a vaccine, comprises apart from one or several of the polypeptides according to the present invention in their diverse embodiments, and/or nucleic acid molecules in accordance with the present invention other compounds which are biologically or pharmaceutically active. Preferably, the vaccine composition comprises at least one polycationic peptide. The polycationic compound(s) to be used according to the present invention may be any polycationic compound, which shows the characteristic effects according to the WO 97/30721. Preferred polycationic compounds are selected from basic polypeptides, organic polycations, basic polyamino acids or mixtures thereof. These polyamino acids should have a chain length of at least 4 amino acid residues (WO 97/30721). Especially preferred are substances like polylysine, polyarginine and polypeptides containing more than 20%, especially more than 50% of basic amino acids in a range of more than 8, especially more than 20, amino acid residues or mixtures thereof. Other preferred polycations and their pharmaceutical compositions are described in WO 97/30721 (e.g. polyethyleneimine) and WO 99/38528. Preferably these polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 residues.

These polycationic compounds may be produced chemically or recombinantly or may be derived from natural sources.

Cationic (poly)peptides may also be anti-microbial with properties as reviewed in (Ganz, T., 1999). These (poly)peptides may be of prokaryotic or animal or plant origin or may be produced chemically or recombinantly (WO 02/13857). Peptides may also belong to the class of defensins (WO 02/13857). Sequences of such peptides can be, for example, found in the Antimicrobial Sequences Database under the following internet address: http://www.bbcm.univ.trieste.it/˜tossi/pag2.html.

Such host defence peptides or defensives are also a preferred form of the polycationic polymer according to the present invention. Generally, a compound allowing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer.

Especially preferred for use as polycationic substances in the present invention are cathelicidin derived antimicrobial peptides or derivatives thereof (International patent application WO 02/13857, incorporated herein by reference), especially antimicrobial peptides derived from mammalian cathelicidin, preferably from human, bovine or mouse.

Polycationic compounds derived from natural sources include HIV-REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides derived from these peptides or proteins by biochemical or recombinant production. Other preferred polycationic compounds are cathelin or related or derived substances from cathelin. For example, mouse cathelin is a peptide, which has the amino acid sequence NH₂-RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE-COOH (SEQ ID NO 859). Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues. Derivations may include the substitution or modification of the natural amino acids by amino acids which are not among the 20 standard amino acids. Moreover, further cationic residues may be introduced into such cathelin molecules. These cathelin molecules are preferred to be combined with the antigen. These cathelin molecules surprisingly have turned out to be also effective as an adjuvant for an antigen without the addition of further adjuvants. It is therefore possible to use such cathelin molecules as efficient adjuvants in vaccine formulations with or without further immunactivating substances.

Another preferred polycationic substance to be used in accordance with the present invention is a synthetic peptide containing at least 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids (International patent application WO 02/32451, incorporated herein by reference).

The pharmaceutical composition of the present invention may further comprise immunostimulatory nucleic acid(s). Immunostimulatory nucleic acids are e.g. neutral or artificial CpG containing nucleic acids, short stretches of nucleic acids derived from non-vertebrates or in the form of short oligonucleotides (ODNs) containing non-methylated cytosine-guanine di-nucleotides (CpG) in a certain base context (e.g. described in WO 96/02555). Alternatively, also nucleic acids based on inosine and cytidine as e.g. described in the WO 01/93903, or deoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues (described in WO 01/93905 and WO 02/095027, incorporated herein by reference) may preferably be used as immunostimulatory nucleic acids in connection with the present invention. Preferably, the mixtures of different immunostimulatory nucleic acids may be used according to the present invention.

It is also within the present invention that any of the aforementioned polycationic compounds is combined with any of the immunostimulatory nucleic acids as aforementioned. Preferably, such combinations are according to the ones as described in WO 01/93905, WO 02/32451, WO 01/54720, WO 01/93903, WO 02/13857, WO 02/095027 and WO 03/047602, incorporated herein by reference.

In addition or alternatively such vaccine composition may comprise, apart from the polypeptides according to the present invention and the nucleic acid molecules according to the present invention, preferably the coding nucleic acid molecules according to the present invention, a neuroactive compound. Preferably, the neuroactive compound is human growth factor as, e.g. described in WO 01/24822. Also preferably, the neuroactive compound is combined with any of the polycationic compounds and/or immunostimulatory nucleic acids as aforementioned.

Also, the pharmaceutical composition in accordance with the present invention is a pharmaceutical composition which comprises at least any of the following compounds or combinations thereof: the nucleic acid molecules according to the present invention, the polypeptides according to the present invention in their diverse embodiments, the vector according to the present invention, the cells according to the present invention, the antibody according to the present invention, the functional nucleic acids according to the present invention and the binding peptides such as the anticalines and high-affinity binding peptides and peptide aptamers, respectively, according to the present invention, any agonists and antagonists according to the present invention, preferably screened as described herein. In connection therewith any of these compounds may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of an antigen and fragments thereof of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.

The composition may be used e.g. for immunization or treatment of a subject. The pharmaceutical composition encompasses at least one peptide of the invention; however, it may also contain a cocktail (i.e., a simple mixture) containing different peptides (including fragments and other variants) of the invention, optionally mixed with different antigenic proteins or peptides of other pathogens. Such mixtures of these peptides, polypeptides, proteins or fragments or variants thereof are useful e.g. in the generation of desired antibodies to a wide spectrum of Mcat isolates. The peptide(s) of the present invention may also be used in the form of a pharmaceutically acceptable salt. Suitable acids and bases which are capable of forming salts with the peptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.

Still another aspect of the present invention is a pharmaceutical composition containing a nucleic acid selected from the group consisting of

(i) a nucleic acid of the invention and/or a nucleic acid complementary thereto, and(ii) optionally a pharmaceutically acceptable carrier or excipient.

The nucleic acid sequences, alone or in combination with other nucleic acid sequences encoding antigens or antibodies or directed to other pathogenic microorganisms, may further be used as components of a pharmaceutical composition. The composition may be used for immunizing or treating humans and/or animals with the disease caused by infection with Mcat. The pharmaceutically acceptable carrier or excipient may be as defined above.

In another embodiment, the nucleic acid sequences of this invention, alone or in combination with nucleic acid sequences encoding other antigens or antibodies from other pathogenic microorganisms, may further be used in compositions directed to actively induce a protective immune response to the pathogen in a subject. These components of the present invention are useful in methods for inducing a protective immune response in humans and/or animals against infection with Mcat.

For use in the preparation of the therapeutic or vaccine compositions, nucleic acid delivery compositions and methods are useful, which are known to those of skill in the art. The nucleic acid of the invention may be employed in the methods of this invention or in the compositions described herein as DNA sequences, either administered as naked DNA, or associated with a pharmaceutically acceptable carrier and provide for in vivo expression of the antigen, peptide or polypeptide. So-called “naked DNA” may be used to express the antigen, peptide or polypeptide of the invention in vivo in a patient. (See, e.g., Cohen, J., 1993, which describes similar uses of “naked DNA”). For example, “naked DNA” associated with regulatory sequences may be administered therapeutically or as part of the vaccine composition e.g., by injection.

Alternatively, a nucleic acid encoding the antigens or peptides of the invention or a nucleic acid complementary thereto may be used within a pharmaceutical composition, e.g. in order to express the antigens or peptides or polypeptides of the invention in vivo, e.g., to induce antibodies.

A preferred embodiment of the invention relates to a pharmaceutical composition, wherein the nucleic acid according to the invention is comprised in a vector and/or a cell. Vectors and cells suitable in the context of the present invention are described above. Vectors are particularly employed for a DNA vaccine. An appropriate vector for delivery may be readily selected by one of skill in the art. Exemplary vectors for in vivo gene delivery are readily available from a variety of academic and commercial sources, and include, e.g., adeno-associated virus (International patent application No. PCT/US91/03440), adenovirus vectors (Kay, M. et al., 1994; Ishibashi, S. et al., 1993), or other viral vectors, e.g., various poxviruses, vaccinia, etc. Recombinant viral vectors, such as retroviruses or adenoviruses, are preferred for integrating the exogenous DNA into the chromosome of the cell.

The pharmaceutical compositions of the present invention may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intratracheal or intradermal routes among others.

In therapy or as a prophylactic, the active agent of the pharmaceutical composition of the present invention may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, which is preferably isotonic.

Preferably, the pharmaceutical composition of the present invention may be administered intranasally, e.g. as an aerosol formulation.

In general, intranasal vaccination represents an attractive non-invasive alternative to needle-based injection and provides superior protection at mucosal surfaces. Furthermore, mucosal as well as systemic immunity can be induced after intranasal immunizations.

Therefore, intranasal application is particularly suited for Mcat vaccines according to the present invention.

Alternatively the composition, preferably the pharmaceutical composition may be formulated for topical application, for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.

In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.

In a preferred embodiment the pharmaceutical composition is a vaccine composition. Preferably, such vaccine composition is conveniently in injectable form or in an aerosol formulation for intranasal delivery. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination with a protein antigen is for adults between 0.02 to 3 μg antigen/per kg of body weight and for children between 0.2 to 10 μg antigen/per kg body weight, and such dose is preferably administered 1-3 times and with an interval of 2 to 24 weeks.

An “effective amount” or “therapeutically effective amount” of an antigen, nucleic acid, vector, an antibody or a pharmaceutical composition of the invention may be calculated as that amount capable of exhibiting an in vivo effect, e.g. preventing or ameliorating a sign or symptom of infection with Mcat. Such amounts may be determined by one of skill in the art.

With the indicated dose range, no adverse toxicological effects are expected with the compounds of the invention, which would preclude their administration to suitable individuals.

In a further embodiment the present invention relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. The ingredient(s) can be present in a useful amount, dosage, formulation or combination. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the preparation, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the preparation, use or sale of the product for human administration.

In connection with the present invention any disease related use as disclosed herein such as, e.g., use of the pharmaceutical composition or vaccine, is particularly a disease or diseased condition which is caused by, linked or associated with Mcat. A disease related, caused, or associated with the bacterial infection to be prevented and/or treated according to the present invention includes chronic infections. Common sites include the upper and lower respiratory tract. The spectrum of clinical syndromes includes recurrent or chronic otitis media, pneumonia, upper respiratory tract disease, bronchitis, sinusitis, asthmatic bronchitis, adult-onset asthma, and chronic obstructive pulmonary disease.

It is within the present invention that each and any of the symptoms, diseases, disorders or syndromes described herein which are either directly or indirectly linked to or arise from a contact of an organism such as any animal or human with an Mcat species, preferably a pathogenic Mcat species are separately and independently indications, diseases or disorders in the meaning of the present invention. Accordingly and just by means of illustration, a disease in the sense of the present application is pneumonia as well as bronchitis and chronic obstructive pulmonary disease.

It is within the present invention that the disease for which the various compounds described herein can be used are also those diseases where the polypeptide according to the present invention is expressed or any disease where the compounds described herein such as the polypeptides according to the present invention, the vaccine, the antibody, and any aptamer and spiegelmer, respectively, are suitable for the treatment and/or diagnosis thereof. Such potential use can arise from cross-reactivity and homology, respectively. It understood by the one skilled in the art that any disease described in connection with the pharmaceutical composition according to the present invention can be subject to the use of the pharmaceutical composition described herein, and vice versa.

Treatment in the context of the present invention refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

In a still further embodiment the present invention is related to a screening method using any of the polypeptides according to the present invention or any of the nucleic acids according to the present invention. Screening methods as such are known to the one skilled in the art and can be designed such that an agonist or an antagonist is screened. In connection with such screening method preferably an antagonist is screened which in the present case inhibits or prevents the binding of any antigen and fragment thereof according to the present invention to an interaction partner. Such interaction partner can be a naturally occurring interaction partner or a non-naturally occurring interaction partner.

The invention also provides a method for screening compounds to identify those which enhance (agonist) or block (antagonist) the function of the polypeptides according to the present invention or of the nucleic acid molecules of the present invention, such as its interaction with a binding molecule. The method of screening may involve high-throughput.

For example, to screen for agonists or antagonists, the interaction partner of the polypeptide according to the present invention, maybe a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, may be prepared from a cell that expresses a molecule that binds to the polypeptides according to the present invention. The preparation is incubated with labelled forms of such polypeptides in the absence or the presence of a candidate molecule, which may be an agonist or antagonist. The ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labelled ligand. Molecules which bind gratuitously, i.e., without inducing the functional effects of said polypeptides, are most likely to be good antagonists. Molecules that bind well and elicit functional effects that are the same as or closely related to the polypeptides according to the present invention are good agonists.

The functional effects of potential agonists and antagonists may be measured, for instance, by determining the activity of a reporter system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of polypeptides according to the present invention or molecules that elicit the same effects as said polypeptides. Reporter systems that may be useful in this regard include but are not limited to colorimetric labelled substrate converted into product, a reporter gene that is responsive to changes in the functional activity of the polypeptides according to the present invention, and binding assays known in the art.

Another example of an assay for antagonists is a competitive assay that combines the polypeptides according to the present invention and a potential antagonist with membrane-bound binding molecules, recombinant binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. The polypeptides according to the present invention can be labelled such as by radioactivity or a colorimetric compound, such that the molecule number of polypeptides according to the present invention bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to polypeptides according to the present invention and thereby inhibit or extinguish its activity. Potential antagonists may also be small organic molecules, peptides, polypeptides such as a closely related proteins or antibodies that bind to the same sites on a binding molecule without inducing functional activity of the polypeptides according to the present invention.

Potential antagonists include a small molecule, which binds to and occupies the binding site of the polypeptides according to the present invention thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see Okano, H. et al., 1991; OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION; CRC Press, Boca Raton, Fla. (1988), for a description of these molecules).

Preferred potential antagonists include derivatives of the antigens and fragments thereof of the invention.

As used herein the activity of a polypeptide according to the present invention is its capability to bind to any of its interaction partner or the extent of such capability to bind to its or any interaction partner.

In a particular aspect, the invention provides the use of the polypeptides according to the present invention antigens and fragments thereof, nucleic acid molecules or inhibitors of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection. In particular the molecules of the invention may be used: i) in the prevention of adhesion of the Mcat species as disclosed herein, and more preferably the pathogenic species thereof to mammalian extracellular matrix proteins; ii) to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial proteins which mediate tissue reaction; iii) or lead to evasion of immune defence; iv) to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques, e.g. through inhibiting nutrient acquisition.

Each of the DNA coding sequences provided herein may be used in the discovery, development and/or preparation of antibacterial compounds. The encoded protein upon expression can be used as a target for the screening of antibacterial drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.

The antagonists and agonists may be employed, for instance, to inhibit diseases arising from infection with Mcat.

In a still further aspect the present invention is related to an affinity device. Such affinity device comprises as least a support material and any of the polypeptides according to the present invention, which is attached to the support material. Because of the specificity of said polypeptides for their target cells or target molecules or their interaction partners, said polypeptides allow a selective removal of their interaction partner(s) from any kind of sample applied to the support material provided that the conditions for binding are met. The sample may be a biological or medical sample, including but not limited to, fermentation broth, cell debris, cell preparation, tissue preparation, organ preparation, blood, urine, lymph liquid, liquor and the like.

The polypeptides according to the present invention may be attached to the matrix in a covalent or non-covalent manner. Suitable support material is known to the one skilled in the art and can be selected from the group consisting of cellulose, silicon, glass, aluminum, paramagnetic beads, starch and dextrane.

The present invention is further illustrated by the following Figures, Tables, Examples and the Sequence Listing, from which further features, embodiments and advantages may be taken. It is to be understood that the present examples are given by way of illustration only and not by way of limitation of the disclosure.

In connection with the present invention

FIG. 1 shows the characterization of human sera as sources of pathogen specific antibodies.

FIG. 2 shows the characterization of the libraries.

FIG. 3 shows the PCR analysis to determine the conservation of identified open reading frames encoding antigenic Mcat proteins.

FIG. 4 shows median bacterial colony numbers (cfu) recovered in broncho-alveolar lavage (BAL) and lung tissue of animals immunized with the candidate antigens or controls (OmpCD or whole killed cells) by systemic (subcutaneous) immunization in the presence of incomplete Freund's adjuvant (IFA); the recovered CFU is expressed as percentage of CFU recovered from animals immunized with the adjuvant vehicle (IFA).

FIG. 5 shows median bacterial colony numbers (cfu) recovered from lung tissue of animals immunized with the candidate antigens, adjuvant without the proteins or the buffer alone, via mucosal (intranasal) route in the presence of Intercell's IC31® adjuvant.

FIG. 6 shows bacterial colony numbers (cfu) recovered in nasopharyngeal lavage (NAL) from animals immunized with the candidate antigens or controls (adjuvant and buffer) via mucosal (intranasal) route in the presence of Intercell's IC31® adjuvant.

Table 1 shows the results of the screenings of the 250 and 50/2 Moraxella catarrhalis libraries with the 4 pools of biotinylated human IgGs (P36, P37, P39 and IC20).

Table 2 shows the reactivity of randomly selected bacterial clones from screenings with biotinylated human IgG in Western blotting.

Table 3 shows an example of validating antigenic Mcat proteins identified by screening bacterial display libraries using peptide ELISA.

Table 4 shows the 214 immunogenic Mcat ORFs identified by screening bacterial display libraries of the fragmented genome of Meat RH4 with human immunoglobulin.

Table 5 shows a list of the 47 Mcat isolates used to examine the conservation of the 214 immunogenic Mcat ORFs identified by screening bacterial display libraries of the fragmented genome of Meat RH4 with human immunoglobulin.

Table 6 shows the conservation in 47 Mcat isolates of the 214 immunogenic Mcat ORFs identified by screening bacterial display libraries of the fragmented genome of Mcat RH4 with human immunoglobulin.

Table 7 shows the PCR primer sequences used for the analysis of the conservation in 47 Mcat isolates of the 214 immunogenic Mcat ORFs identified by screening bacterial display libraries of the fragmented genome of Meat RH4 with human immunoglobulin.

Table 8 shows the list of genes from M. catarrhalis strain RH4 selected for expression and further animal studies.

Table 9 shows the list of selected M. catarrhalis strain RH4 antigens and their corresponding immunization doses.

Table 10 shows the summary of sterile cultures identified per each animal experimental group.

The Figures and Tables to which it might be referred to in the specification are described in the following in more details.

FIG. 1. Characterization of Human Sera for Anti-Mcat Antibodies by Immunoblotting.

Lysates of Moraxella catarrhalis RH4 were separated on 4-20% gradient SDS-PAGE gels, transferred to nitrocellulose membranes and immunoblotted with human sera diluted 1:1,000. Molecular weight markers are indicated on the left.

Horizontal black bars indicate paired serum samples obtained from otitis media patients at the onset of disease (left under horizontal bar), and in the convalescent phase approximately 2 weeks later (right under horizontal bar). The figure indicates the reactivity of 10 such paired samples, i.e. 20 individual serum samples (left panel).

In addition, the figure shows the reactivity of 17 single serum samples, comprising 5 sera from asthma patients (P3918-P3965), 8 sera from healthy individuals (IC58B-IC54A) and 4 sera from the patients with respiratory allergies (P3845-P3867).

FIG. 2. Characterization of the Bacterial Display Libraries of the Fragmented Genome of Moraxella catarrhalis (Mcat) RH4.

A,B, Mcat 50/2 Library (LamB Display Scaffold)

A, The DNA sequences of the displayed fragments were determined for 226 bacterial library clones. Following trimming for vector sequence, the displayed sequences exhibited a size distribution from approximately 25 to 150 bp, with an average size of 87 bp.

B, The 226 bacterial library clone sequences were matched to the complete genome sequence of the library Mcat strain RH4 (sequenced by Intercell AG (Vienna, Austria)). The displayed fragments were distributed equally along the complete Mcat genome, which is 1,863,286 bp. The numbering in the diagram refers to the project-internal numbering with by 1 corresponding to by 315261 in the reverse complemented and slightly corrected and repositioned sequence published with the GenBank accession number CP002005.1 (gi:295919923).

The 50/2 library comprised 860,000 colony-forming units, giving a calculated coverage of the Mcat genome of 40-fold.

C,D, Mcat 250 Library (FhuA Display Scaffold)

C, The DNA sequences of the displayed fragments were determined for 320 bacterial library clones. Following trimming for vector sequence, the displayed sequences exhibited a size distribution from approximately 50 to 350 bp, with an average size of 199 bp.

D, The 320 bacterial library clone sequences were matched to the complete genome sequence of the library Mcat strain RH4 (sequenced by Intercell AG (Vienna, Austria)). The displayed fragments were distributed equally along the complete Mcat genome, which is 1,863,286 bp. The numbering in the diagram refers to the project-internal numbering with by 1 corresponding to by 315261 in the reverse complemented and slightly corrected and repositioned sequence published with the GenBank accession number CP002005.1 (gi:295919923).

The 250 library comprised 913,500 colony-forming units, giving a calculated coverage of the Mcat genome of 98-fold.

FIG. 3. Conservation of Identified Open Reading Frames Encoding Antigenic Moraxella catarrhalis (Mcat) Proteins.

A, an example of the PCR analysis of gene conservation for two Mcat open reading frames encoding antigenic proteins.

Genomic Mcat DNA was used as template for the PCR, at a dilution of 1:50. Negative control consisted of PCR on water. Two microliters of each PCR reaction was loaded on a 1% agarose gel. Top panel, gene MCRH3709. Bottom panel, gene MCRH3844. Expected position of PCR products is indicated by arrowheads (MCRH3709, 1,534 bp; MCRH3844, 974 bp). Numbers in lanes indicate Mcat strains (Table 5, internal Intercell reference number A). “−” indicates PCR on water. “+” indicates PCR on the Mcat library strain RH4.

B, C, summary of the PCR analysis of the conservation in 47 Mcat isolates of all 214 identified ORFs encoding antigenic proteins. For each ORF encoding antigenic Mcat proteins, the level of conservation was calculated as 100× (number of PCR positive Mcat isolates/47). B, The number of Mcat ORFs is expressed as a function of the level of conservation. C, The cumulative percent of Mcat ORFs is expressed as a function of the level of conservation.

FIG. 4. Lung Elimination of the Bacteria in the Mice Immunized by a Systemic Route (Intraperitoneal) with Control or Moraxella catarrhalis Vaccine Candidate Antigens after Challenge with Moraxella catarrhalis Strain RH4.

A, Bacterial clearance from the bronchoalveolar space expressed as a percentage of recovered bacteria (median CFU) from antigen-immunized mice compared to the median bacterial CFU recovered from the mice immunized with adjuvant vehicle control (IFA).

B, Percentage of recovered bacteria from the lung tissue homogenate expressed in reference to the adjuvant control (IFA).

C, Percentage of recovered bacteria from both, bronchoalveolar space and lung tissue homogenate combined. Abbreviations: WKC, whole killed cells; IFA, adjuvanted phosphate buffered saline without proteins added; OmpCD, outer membrane protein CD (used as a benchmark).

FIG. 5. Bacteria Recovered in the Lungs of the Mice Immunized Via Mucosal Route (Intranasal) with Control or M. catarrhalis Vaccine Candidate Antigens after Challenge with M. catarrhalis Strain RH4.

The data represents the mean values of the combined, normalized results from 2-5 independent experiments. Error bars represent the standard error of the mean (for N see Table 10). Figure showing data with (A) and without (B) sterile lung cultures.

Abbreviations: IC31®, mucosal adjuvant without proteins added; PBS, phosphate buffered saline without adjuvant added.

FIG. 6. Bacteria Recovered in Nasopharyngeal Lavage (NAL) of the Mice Immunized Via Mucosal Route (Intranasal) with Control or M. catarrhalis Vaccine Candidate Antigens after Challenge with M. catarrhalis Strain RH4.

The data represents the mean values of the combined, normalized results from 2-5 independent experiments. Error bars represent the standard error of the mean (for N see Table 10). Figure showing data with (A) and without (B) sterile NAL cultures.

Abbreviations: IC31®, mucosal adjuvant without proteins added; PBS, phosphate buffered saline without adjuvant added.

TABLE 1
Screening of Moraxella catarrhalis 250 and 50/2 libraries with the 4 pools of
biotinylated human IgGs (IC20, P36, P37, IC20 and P39). As negative control, no IgG
was added to the libraries before magnetic sorting (MACS). Number of library E. coli used
as input and eluted from the MACS columns are shown for all screens.
	Cfu (Input)	Cfu (Eluate)
	bMcat		Without		Without	20 μg	cSpecific
aScreen	library	IgG pool	IgG	20 μg IgG	IgG	IgG	enrichment
MCF-IC20	250	IC20	1,800,000	1,100,000	240	4,440	30
MCF-P36	250	P36	2,100,000	800,000	40	4,920	323
MCF-P37	250	P37	4,500,000	4,300,000	6,800	40,000	6
MCF-IC20a	250	IC20*	300,000	200,000	400	6,720	25
MCF-P39	250	P39**	400,000	1,300,000	1,200	4,280	1.1
MCL2-IC20a	50/2	IC20*	17,400,000	16,800,000	572	6,280	11
MCL2-P36	50/2	P36	17,400,000	8,700,000	572	10,920	38
MCL2-P37	50/2	P37	17,600,000	25,800,000	1,424	4,904	2
MCL2-P39	50/2	P39**	17,600,000	16,600,000	1,424	3,640	3
*IgG pool preadsorbed against 4 Hag clones and 3 UspA2H clones
**IgG pool preadsorbed against 6 Hag clones and 3 UspA2H clones
aScreen identifier.
b250, FhuA display library. 50/2, LamB library.
cCalculated as: (cfu eluate 20 μg IgG/cfu input 20 μg IgG)/(cfu eluate without IgG/cfu input without IgG). Specific enrichment of above 1 indicates that screening with human IgG bound more library clones than negative-control screening without human IgG, i.e., that the clones bound with human IgG were enriched for display of antigenic Mcat sequences.

TABLE 2
Table 2. Reactivity of randomly selected bacterial library clones
from screenings with biotinylated human IgG in Western blotting.
	Number of	Number of	Percent of the
	bacterial library	bacterial	bacterial
	clones tested	clones positive	clones positive
aScreen	by Western	by Western	by Western
MCF-IC20	182	146	80
MCF-P36	142	80	56
MCF-P37	149	101	68
MCF-IC20a	130	74	57
MCF-P39	79	53	67
MCL2-IC20a	48	23	48
MCL2-P36	72	30	42
MCL2-P37	96	55	57
MCL2-P39	68	18	26
The same serum pools were used for MACS and Western.
aScreening identifier; a total of 9 screens were done, corresponding to Table 1.

TABLE 3
Reactivity of overlapping peptides representing 3 antigenic Mcat proteins in ELISA.
Overlapping peptide sets were synthesized for identified antigenic Mcat ORFs, and human sera
were tested for anti-Mcat peptide reactivity by ELISA.
	Seroconversion (OM patients)	Asthmatic patients
Peptide	P4060	P4070	P4072	P4101	P4115	P4120	P3918	P3923	P3941	P3943	P3965
MCRH1546.01	4.59	0.79	1.62	0.57	1.00	0.69	0	0	0	0	0
MCRH1546.02	6.53	0.85	1.32	1.00	1.00	1.00	0	0	0	228	0
MCRH1565.01	1.00	1.00	1.00	1.00	1.00	1.00	0	0	0	0	0
MCRH1565.02	0.58	0.90	1.00	0.83	1.00	0.81	238	298	207	167	244
MCRH1565.03	1.00	1.00	1.00	0.94	0.67	0.60	147	212	122	141	0
MCRH1565.04	1.00	1.00	1.00	0.87	1.00	1.00	0	0	0	0	0
MCRH1586.01	1.00	1.00	1.00	1.00	1.00	1.00	0	0	0	0	0
MCRH1586.02	0.70	1.00	1.00	1.12	1.00	1.00	149	0	221	0	0
MCRH1586.03	0.85	1.05	0.92	0.94	0.75	0.75	391	428	424	381	427
MCRH1586.04	0.87	1.00	1.00	3.25	1.08	1.00	0	0	0	748	0
MCRH1586.05	0.92	1.00	0.78	1.09	0.78	0.68	428	426	391	399	109
	Healthy individuals	FINAL
	Peptide	IC58B	IC85B	IC86B	IC89B	IC54A	SCORE
	MCRH1546.01	0	0	0	0	0	6
	MCRH1546.02	0	312	0	0	0	8
	MCRH1565.01	0	0	0	0	0	0
	MCRH1565.02	0	0	0	0	399	4
	MCRH1565.03	0	0	0	0	186	1
	MCRH1565.04	0	0	0	0	0	0
	MCRH1586.01	0	0	0	0	0	0
	MCRH1586.02	0	100	0	0	136	1
	MCRH1586.03	253	311	0	356	552	10
	MCRH1586.04	0	0	0	0	0	8
	MCRH1586.05	195	416	0	208	560	8
	“Seroconversion (OM patients)”: paired serum samples were examined from the same otitis media patient, at the onset of disease, and in the convalescent phase, approximately 2 weeks later. The seroconversion values were calculated as the ratio between the ELISA reactivity of convalescent to acute serum samples. High seroconversion values indicate high anti-Mcat antibody levels in the convalescent sera.
	“Asthmatic patients”: asthma patients, single serum samples, including recurrent otitis media patient P3965 single serum sample.
	“Healthy individuals”: healthy individuals, single serum samples. For the “Asthmatic patients/recurrent OM” and “Healthy individuals” serum samples, ELISA reactivity values were calculated as 1,000 × [(OD405 wells with serum added) − (OD405 wells with secondary antibody alone)]. ELISA reactivity values below 100 were considered negative. High ELISA reactivity values indicate high anti-Mcat antibody levels in the sera.
	“Final score”: The immunoreactivity of individual synthetic peptides representing selected epitopes with human sera. 22 sera were tested in all, comprising 6 serum pairs, and 10 unpaired sera, as shown in the table.
	Seroconversion (6 serum pairs) was assigned the following score values: >3 fold, 3 points. From 1.9 to 3-fold, 2 points. From 1.5 to 1.9-fold 1 point. ELISA reactivity (recurrent, asthma and healthy individual sera) was assigned the following score values: >500 ELISA units, 3 points. 250-499 ELISA units, 2 points. 150-249 ELISA units, 1 points. For peptides where seroconversion was not seen with any of the 6 paired serum samples, the ELISA reactivities were reduced by 50%. For each peptide, the final ranking score was calculated as the sum of seroconversion and ELISA reactivity scores for all 22 human sera.

TABLE 4
Immunogenic Mcat proteins identified by bacterial surface display.
					eLocation
			cPredicted	dNo of	of
a Moraxella			classII-	selected	identified
catarrhalis	aPutative	bPredicted	restricted T	clones	immunogenic
RH4 antigenic	function (by	immunogenic	cell epitope	per	region	fSEQ ID NO	fSEQ ID NO
protein	homology)	aa	regions	ORF	(aa)	(DNA)	(Protein)
MCRH0005	Putative	4-10, 12-40,	9-99, 108-128,	1	17-72	1	215
	uncharacterized	46-52, 61-76,	132-169,
	protein -	78-94,	179-286
	Psychrobacters	96-103,
	(strain	107-114,
	PRwf-1)	126-133,
		135-150,
		153-162,
		167-174,
		182-195,
		199-208,
		210-218,
		226-244,
		247-255,
		262-276,
		285-292
MCRH0052	S. UDP-N-	7-17, 25-32,	1-168, 180-204,	4	10-60, 92-130,	2	216
	acetylmuramate--	36-53, 66-76,	212-253,		207-280
	L-alanine	83-91,	261-313,
	ligase (EC	100-113,	326-467,
	6.3.2.8)	124-131,	472-486
	(UDP-N	151-158,
	acetylmuramoyl-	168-179,
	L-alanine	184-198,
	synthetase) -	213-225,
	Psychrobacter	227-237,
	arcticum	247-256,
		261-270,
		275-281,
		284-293,
		295-305,
		315-329,
		345-358,
		388-415,
		436-443,
		449-456,
		471-483
MCRH0079	SSU	45-53, 58-65,	18-158,	2	211-260	3	217
	ribosomal	70-78,	163-232
	protein S2P -	81-87, 94-100,
	Psychrobacter	163-179,
	cryohalolentis	184-192,
	(strain K5)	201-207,
		212-222,
		224-230,
		254-260,
		263-268
MCRH0082	SMC protein	4-12, 25-33,	1-80, 88-153,	9	128-158,	4	218
	(Fragment) -	40-50, 62-69,	167-222,		161-232,
	Terfezia	74-85,	231-249,		277-314,
	boudieri	97-109,	256-289,		590-627,
		127-138,	304-337,		712-848,
		141-149,	340-488,		957-963,
		153-161,	491-679,		1007-1033,
		197-203,	681-780,		1074-1093
		206-215,	803-878,
		217-223,	880-942,
		258-265,	951-986,
		272-286,	988-1014,
		308-316,	1030-1106,
		325-335,	1109-1133,
		343-358,	1136-1179,
		366-371,	1183-1203
		373-381,
		400-406,
		417-426,
		444-458,
		466-486,
		498-514,
		522-539,
		544-555,
		561-572,
		603-619,
		621-638,
		658-672,
		687-695,
		710-723,
		731-744,
		747-752,
		764-769,
		789-805,
		808-823,
		830-840,
		873-885,
		888-900,
		902-920,
		930-943,
		964-970,
		992-1003,
		1005-1016,
		1033-1038,
		1045-1057,
		1059-1068,
		1089-1096,
		1104-1110,
		1115-1142,
		1150-1156,
		1158-1169,
		1188-1198,
		1200-1205
MCRH0103	C-terminal	4-29, 45-56,	1-33, 45-204,	2	496-512,	5	219
	processing	68-78, 84-97,	210-414,		542-618
	peptidase-1.	100-109,	431-480,
	Serine	127-141,	486-532,
	peptidase.	145-151,	536-582,
	MEROPS	173-179,	597-691,
	famil S41A -	189-197,	704-724
	Psychrobacter	202-209,
	arcticum	214-220,
		238-263,
		266-279,
		288-297,
		307-315,
		330-336,
		343-351,
		358-367,
		373-382,
		388-403,
		412-421,
		423-429,
		435-441,
		449-459,
		462-468,
		488-501,
		503-522,
		536-542,
		559-572,
		603-611,
		623-630,
		675-681,
		684-690,
		705-719
MCRH0125	S. DNA-	14-24, 50-70,	6-26, 35-55,	42	199-293,	6	220
	directed RNA	76-92,	58-163,		420-438,
	polymerase	100-128,	166-184,		643-694,
	subunit beta′	130-143,	190-252,		719-771,
	(EC 2.7.7.6)	150-155,	258-300,		823-881,
	(RNAP	178-187,	305-493,		1050-1144,
	subunit beta′)	215-221,	495-559,		1267-1339
	(Transcriptase	233-251,	567-587,
	subunit	261-267,	591-654,
	beta′) (RNA	275-288,	672-750,
	polymerase	295-301,	753-813,
	subunit beta′) -	319-324,	827-866,
	Psychrobacters	339-364,	870-889,
	(strain	368-384,	898-983,
	PRwf-1)	404-453,	993-1011,
		461-472,	1038-1107,
		493-510,	1139-1162,
		522-532,	1168-1191,
		534-550,	1199-1256,
		570-577,	1261-1355,
		598-633,	1365-1387
		641-655,
		666-672,
		676-682,
		762-772,
		793-808,
		819-826,
		836-858,
		860-867,
		872-896,
		900-907,
		909-918,
		929-948,
		961-974,
		990-1003,
		1005-1015,
		1018-1025,
		1066-1076,
		1081-1090,
		1100-1113,
		1124-1131,
		1146-1152,
		1191-1197,
		1202-1208,
		1214-1231,
		1237-1254,
		1282-1310,
		1313-1325,
		1333-1353,
		1365-1378
MCRH0139	S. DNA-	14-42, 50-65,	1-74, 84-228,	6	443-561,	7	221
	directed RNA	69-81,	233-258,		644-713,
	polymerase	96-109,	264-383,		767-806,
	subunit beta	122-129,	391-449,		990-996
	(EC 2.7.7.6)	146-161,	451-490,
	(RNAP	170-184,	496-547,
	subunit beta)	194-200,	559-610,
	(Transcriptase	206-215,	616-635,
	subunit	221-234,	640-731,
	beta) (RNA	241-248,	752-778,
	polymerase	264-270,	799-862,
	subunit beta) -	273-281,	874-990,
	Psychrobacter	288-299,	1064-1110,
	arcticum	303-316,	1114-1138,
		320-338,	1144-1167,
		345-352,	1183-1203,
		365-372,	1209-1230,
		426-443,	1241-1281,
		475-484,	1289-1363
		499-515,
		518-524,
		537-546,
		555-563,
		565-572,
		582-588,
		601-608,
		612-629,
		642-647,
		662-683,
		698-703,
		706-711,
		718-731,
		739-746,
		758-766,
		776-782,
		801-807,
		825-835,
		840-852,
		878-886,
		889-897,
		908-915,
		920-933,
		935-946,
		972-979,
		982-992,
		1003-1008,
		1013-1023,
		1030-1042,
		1065-1081,
		1095-1103,
		1116-1126,
		1132-1138,
		1155-1173,
		1176-1184,
		1186-1199,
		1203-1209,
		1216-1224,
		1242-1262,
		1270-1282,
		1295-1306,
		1308-1316,
		1342-1348,
		1352-1357
MCRH0176	—	4-19, 25-33,	1-72, 74-108	1	55-108	8	222
		36-51, 53-67,
		105-110
MCRH0211	S. Shikimate	15-36, 42-96,	3-115	1	4-23	9	223
	dehydrogenase	101-112
	(EC
	1.1.1.25) -
	Psychrobacter
	cryohalolentis
	(strain K5)
MCRH0249	Anthranilate	4-10, 12-22,	1-227, 248-378,	4	310-399	10	224
	synthase,	25-32, 35-48,	395-427,
	component I	54-59,	445-459
	(EC 4.1.3.27) -	65-70, 100-111,
	Psychrobacter	115-126,
	cryohalolentis	132-149,
	(strain K5)	156-164,
		166-188,
		197-207,
		220-235,
		243-265,
		272-278,
		309-318,
		322-329,
		340-353,
		355-370,
		378-388,
		393-398,
		410-417,
		441-447,
		449-455
MCRH0267	Putative	5-39	1-40	17	3-52	11	225
	uncharacterized
	protein -
	Moraxella
	catarrhalis
MCRH0268	Lactoferrin	10-16, 20-40,	1-48, 54-82,	13	23-130,	12	226
	binding	58-80,	101-122,		227-257,
	protein A -	83-89, 98-119,	126-184,		269-346,
	Moraxella	126-132,	186-235,		680-775,
	catarrhalis	138-149,	237-325,		817-894,
		173-179,	350-443,		922-983
		193-210,	448-512,
		229-235,	523-545,
		242-248,	553-583,
		266-272,	595-623,
		293-301,	634-657,
		303-315,	660-729,
		335-350,	732-786,
		355-362,	804-841,
		368-377,	845-975,
		379-390,	980-1000
		402-408,
		420-431,
		438-443,
		451-464,
		471-476,
		486-501,
		504-525,
		547-568,
		606-612,
		637-644,
		649-656,
		685-701,
		728-740,
		774-780,
		784-791,
		793-800,
		806-816,
		818-834,
		853-860,
		878-888,
		890-895,
		926-937,
		969-976,
		983-997
MCRH0278	Lactoferrin	4-31, 34-55,	4-51, 95-116,	37	29-69, 204-337,	13	227
	binding	98-105,	125-207,		400-502,
	protein B -	127-137,	217-244,		578-765
	Moraxella	157-175,	254-363,
	catarrhalis	187-194,	375-426,
		228-238,	440-469,
		255-262,	474-506,
		268-273,	527-574,
		281-296,	576-641,
		302-307,	653-682,
		314-327,	750-800,
		329-339,	803-835
		346-352,
		394-401,
		407-416,
		452-458,
		466-477,
		534-550,
		561-571,
		573-580,
		597-605,
		610-622,
		624-630,
		639-644,
		650-656,
		666-672,
		689-696,
		706-712,
		728-735,
		743-748,
		785-790,
		803-812,
		820-825,
		827-833,
		843-848,
		861-868
MCRH0342	Putative	8-16, 19-52,	6-56, 85-99,	3	101-185	14	228
	uncharacterized	61-68, 77-86,	106-127,
	protein -	88-98,	140-222
	Psychrobacter	106-116,
	arcticum	126-131,
		143-153,
		163-178,
		183-191,
		211-217
MCRH0357	Lytic murein	15-26, 46-53,	1-108, 114-192,	11	32-98, 122-144,	15	229
	transglycosylase	59-68,	208-285,		157-323,
	precursor -	75-86, 93-104,	310-348,		336-351,
	Psychrobacters	116-122,	375-483		369-389,
	(strain	126-140,			433-440
	PRwf-1)	154-165,
		175-181,
		206-216,
		230-243,
		251-257,
		294-301,
		305-311,
		324-336,
		364-372,
		374-381,
		394-400,
		419-427,
		435-447,
		451-459
MCRH0372	Electron	4-10, 12-23,	1-41, 47-100	1	7-58	16	230
	transfer	27-40, 42-49,
	flavoprotein-	59-67,
	ubiquinone	94-99
	oxidoreductase
	(EC 1.5.5.1) -
	Psychrobacter
	arcticum
MCRH0391	Putative	4-22, 38-52,	1-29, 33-61,	4	24-38, 46-67,	17	231
	uncharacterized	110-117,	71-128,		128-177
	protein -	122-132,	138-189
	Psychrobacter	144-154,
	arcticum	167-175
MCRH0397	Ribose-	4-9, 13-29,	1-21, 42-140,	3	135-227	18	232
	phosphate	39-45, 50-64,	145-261,
	pyrophospho	67-77,	263-315
	kinase (EC	85-94, 103-120,
	2.7.6.1) -	127-146,
	Psychrobacters	148-156,
	(strain	163-178,
	PRwf-1)	180-192,
		204-211,
		213-238,
		241-256,
		266-295
MCRH0432	—	4-15, 33-58,	1-178	5	7-61	19	233
		67-120,
		129-139,
		144-171
MCRH0441	Excinuclease	18-24, 26-34,	1-43, 49-70,	18	288-408,	20	234
	ABC subunit	40-47,	100-164,		577-608,
	A -	53-70, 74-88,	173-192,		622-764,
	Psychrobacter	103-125,	198-258,		826-899,
	arcticum	128-137,	265-306,		923-948
		150-159,	315-371,
		164-174,	373-433,
		181-188,	438-461,
		201-209,	465-523,
		220-230,	525-741,
		232-238,	761-785,
		249-272,	787-808,
		278-287,	810-925,
		290-303,	937-953
		318-332,
		346-353,
		379-387,
		394-419,
		421-430,
		433-441,
		443-450,
		470-488,
		498-507,
		509-524,
		532-539,
		543-550,
		557-579,
		602-608,
		611-617,
		624-632,
		641-657,
		671-676,
		694-701,
		720-730,
		753-762,
		764-785,
		792-797,
		801-807,
		817-840,
		853-859,
		868-874,
		878-894,
		899-913,
		915-921,
		929-935
MCRH0448	Nicotinate-	10-23, 36-44,	1-185, 190-234,	1	182-251	21	235
	nucleotide	46-63,	240-254
	adenylyltransferase	70-79, 89-106,
	(EC	118-128,
	2.7.7.18) -	140-149,
	Psychrobacter	161-170,
	arcticum	197-207,
		233-245
MCRH0464	8-amino-7-	6-20, 26-32,	1-399	5	303-363	22	236
	oxononanoate	41-47, 50-72,
	synthase (EC	75-83,
	2.3.1.47) -	102-112,
	Acinetobacters	114-140,
	(strain	156-172,
	ADP1)	178-187,
		197-218,
		228-269,
		275-297,
		307-336,
		352-368,
		370-377,
		387-397
MCRH0477	Multi-sensor	8-38, 40-46,	1-76, 82-103,	8	888-947	23	237
	hybrid	53-70, 88-94,	105-156,
	histidine	127-136,	158-289,
	kinase	140-145,	295-474,
	precursor -	154-174,	506-557,
	Psychrobacters	186-191,	565-803,
	(strain	198-226,	806-857,
	PRwf-1)	236-243,	867-898,
		249-256,	914-988
		260-266,
		269-280,
		320-333,
		342-349,
		357-363,
		365-381,
		385-430,
		433-449,
		456-465,
		467-475,
		488-493,
		498-503,
		510-520,
		557-609,
		618-624,
		631-644,
		646-673,
		679-685,
		690-696,
		698-734,
		780-797,
		802-808,
		810-829,
		831-870,
		879-890,
		910-916,
		926-942,
		944-980
MCRH0498	Possible	16-23, 25-33,	10-182,	5	140-160,	24	238
	ATP-	41-53,	212-232,		232-300,
	dependent	56-63, 70-79,	236-358,		321-394,
	DEAD/DEAH	87-98,	364-409,		514-522
	box RNA-	100-111,	461-475,
	helicase -	120-128,	478-498,
	Psychrobacter	131-149,	542-577
	arcticum	160-171,
		214-230,
		232-242,
		247-255,
		259-266,
		273-282,
		284-292,
		308-347,
		355-364,
		370-376,
		379-386,
		554-563
MCRH0512	DNA-	9-35, 39-51,	8-241, 244-265,	7	152-279	25	239
	(Apurinic or	54-67, 71-78,	269-302
	apyrimidinic	93-102,
	site)lyase/	105-116,
	Formamidopyrimidine-	122-129,
	DN	138-147,
	glycosylase	155-162,
	(EC	168-192,
	4.2.99.18)	194-231,
	(EC 3.2.2.23) -	253-261,
	Psychrobacter	264-276,
	cryohalolentis	281-293
	(strain K5)
MCRH0525	Phenylalanyl-	35-56, 61-70,	1-22, 36-70,	6	115-232,	26	240
	tRNA	74-101,	72-128,		312-332,
	synthetase	120-129,	141-223,		539-555,
	beta subunit	152-165,	231-267,		558-620
	(EC 6.1.1.20) -	171-183,	270-334,
	Psychrobacter	192-224,	339-605,
	cryohalolentis	241-271,	613-652,
	(strain K5)	278-285,	655-778
		291-301,
		303-319,
		334-340,
		367-391,
		401-426,
		437-442,
		446-469,
		475-488,
		495-520,
		523-529,
		532-541,
		543-550,
		554-564,
		570-598,
		624-630,
		633-638,
		643-655,
		663-685,
		687-694,
		698-725,
		735-751,
		758-765,
		778-795
MCRH0546	Putative	15-39, 54-61	1-170, 175-230	2	162-217	27	241
	uncharacterized	65-71,	244-278
	protein -	84-89, 98-111
	Pseudoalteromonas	126-144
	haloplanktis	154-160,
	(strain TAC	189-196,
	125	199-206,
		218-224,
		259-275
MCRH0558	Nitric oxide	22-37, 45-57,	11-57, 72-88,	4	60-145,	28	242
	reductase	59-65,	99-122,		187-223
	large subunit-	82-93,	139-221,
	like protein -	102-112,	236-274,
	Psychrobacters	160-172,	279-328,
	(strain	200-207,	332-766
	PRwf-1)	229-236,
		244-268,
		275-322,
		327-344,
		346-369,
		376-414,
		428-451,
		459-490,
		495-527,
		531-555,
		561-582,
		600-612,
		616-622,
		625-635,
		638-669,
		683-709,
		726-732,
		735-763
MCRH0580	Ferredoxin-	4-12, 32-78,	1-208, 230-310,	6	186-238,	29	243
	dependent	81-89, 91-100,	315-438,		398-572
	glutamate	105-111,	440-456,
	synthase -	125-132,	467-580
	Psychrobacters	170-176,
	(strain	179-189,
	PRwf-1)	194-204,
		223-229,
		235-245,
		259-276,
		282-289,
		303-313,
		318-326,
		334-345,
		350-356,
		364-370,
		387-405,
		409-422,
		424-430,
		442-457,
		459-465,
		490-516,
		521-532,
		534-541,
		566-571
MCRH0587	Putative DNA	14-20, 28-35,	22-125,	6	59-74, 94-141,	30	244
	helicase -	41-47,	130-150,		143-203,
	Psychrobacter	52-61, 73-86,	157-177,		297-362
	cryohalolentis	92-103,	179-368,
	(strain K5)	124-141,	403-495
		143-158,
		172-180,
		185-191,
		193-221,
		239-250,
		262-273,
		280-290,
		304-324,
		328-357,
		367-376,
		378-384,
		394-401,
		420-428,
		439-458,
		460-469,
		474-492
MCRH0594	Dihydrouridine	8-17, 25-39,	1-170, 175-247,	4	17-28, 61-99,	31	245
	synthase,	41-67, 69-85,	261-323		221-240
	DuS -	93-115,
	Psychrobacters	123-143,
	(strain	145-157,
	PRwf-1)	166-174,
		178-185,
		188-199,
		204-210,
		221-238,
		245-252,
		257-268,
		284-290,
		294-306,
		312-320
MCRH0621	Phosphoribosylformylglycinamidine	5-14, 16-26,	8-53, 68-252,	15	291-408,	32	246
	synthase (EC	28-45, 54-60,	265-283,		411-425,
	6.3.5.3) -	72-82,	287-320,		500-552,
	Psychrobacters	98-104,	329-353,		719-741,
	(strain	106-114,	357-383,		975-1034,
	PRwf-1)	121-132,	389-470,		1089-1156,
		137-164,	475-495,		1203-1234,
		169-175,	507-527,		1273-1301
		188-194,	534-749,
		214-220,	773-870,
		233-239,	876-898,
		283-296,	906-928,
		302-310,	938-1150,
		336-347,	1153-1255,
		357-364,	1267-1287,
		400-407,	1291-1314
		419-425,
		433-439,
		443-452,
		464-470,
		492-499,
		511-521,
		528-534,
		536-548,
		564-571,
		577-595,
		597-611,
		615-623,
		637-649,
		652-670,
		681-700,
		716-724,
		726-738,
		766-792,
		806-833,
		839-851,
		853-872,
		880-899,
		905-912,
		919-927,
		935-956,
		958-970,
		980-988,
		990-995,
		1004-1013,
		1017-1031,
		1033-1039,
		1043-1055,
		1059-1068,
		1087-1093,
		1105-1114,
		1116-1122,
		1128-1134,
		1149-1158,
		1164-1172,
		1197-1204,
		1208-1215,
		1230-1244,
		1284-1290
MCRH0655	Acriflavin	9-20, 25-40,	6-382, 385-465	9	204-286,	33	247
	resistance	48-61, 70-84,			310-463
	protein -	89-113,
	Psychrobacters	116-145,
	(strain	161-181,
	PRwf-1)	183-193,
		196-212,
		217-229,
		235-246,
		251-276,
		284-290,
		300-325,
		338-349,
		365-376,
		387-393,
		401-421,
		429-438,
		448-458
MCRH0673	ATP-	9-47, 52-61,	1-463, 466-510,	10	7-54, 198-314,	34	248
	dependent	63-76, 82-88,	519-641,		333-381,
	DNA helicase	95-106,	647-702		404-452,
	RecG (EC	112-126,			523-549
	3.6.1.—) -	132-138,
	Psychrobacters	145-154,
	(strain	162-176,
	PRwf-1)	178-223,
		228-234,
		236-247,
		253-290,
		300-307,
		311-325,
		327-347,
		353-361,
		379-408,
		426-436,
		454-460,
		490-504,
		506-517,
		526-532,
		539-545,
		550-574,
		580-591,
		598-612,
		616-622,
		625-634,
		652-668,
		670-679,
		682-688
MCRH0682	Sulfate	4-20, 24-38,	8-88	11	15-72	35	249
	adenylate	40-64, 66-85
	transferase
	subunit 1 -
	Actinobacillus
	pleuropneumoniae
	serotype 3
	(strain JL03)
MCRH0704	S. 3-ketoacyl-	6-15, 28-33,	3-44, 64-172,	4	56-110,	36	250
	CoA thiolase	35-46, 54-65,	175-191,		143-224,
	(EC 2.3.1.16)	74-108,	202-348		252-308
	(Fatty acid	141-146,
	oxidation	172-178,
	complex	204-217,
	subunit beta)	221-231,
	(Beta-	242-248,
	ketothiolase)	251-257,
	(Acetyl-CoA	276-294,
	acyltransferase) -	298-304,
	Psychrobacters	316-332,
	(strain	341-353,
	PRwf-1)	356-363,
		369-382
MCRH0744	Prolylendopeptidase -	4-28, 49-60,	1-39, 46-64,	6	609-738	37	251
	Vibrio	66-71, 158-164,	80-100,
	parahaemolyticus	215-221,	105-130,
		254-275,	142-162,
		288-294,	166-233,
		318-326,	249-342,
		338-344,	352-467,
		366-380,	470-511,
		389-395,	539-591,
		400-421,	596-687,
		423-430,	689-859
		450-458,
		465-470,
		474-491,
		495-504,
		512-517,
		543-563,
		577-582,
		587-594,
		617-623,
		631-637,
		644-652,
		677-691,
		694-700,
		705-733,
		748-783,
		791-819,
		833-840,
		847-854,
		876-886
MCRH0756	—	7-31, 42-54,	1-147, 155-276,	1	87-164	38	252
		56-64, 94-110,	278-319,
		112-122,	329-387,
		128-136,	391-405
		172-195,
		198-224,
		256-263,
		281-287,
		298-310,
		340-346,
		356-372
MCRH0759	TonB-	4-20, 33-41,	1-20, 27-172,	12	16-32, 133-197,	39	253
	dependent	58-65, 78-86,	175-195,		277-385,
	receptor	93-100,	218-270,		556-573,
	precursor -	102-108,	276-308,		798-874
	Haemophilus	114-161,	334-388,
	somnus	194-200,	409-449,
	(strain 2336)	202-212,	451-712,
	(Histophilus	218-225,	741-824,
	somni (strain	230-235,	828-913
	2336))	243-250,
		269-278,
		291-301,
		312-317,
		329-340,
		352-375,
		416-422,
		427-434,
		443-453,
		468-476,
		481-491,
		510-532,
		537-554,
		560-566,
		574-588,
		612-623,
		653-663,
		678-702,
		721-728,
		742-748,
		770-778,
		796-805,
		807-817,
		824-832,
		856-864,
		871-878,
		885-900
MCRH0780	2-	7-14, 17-30,	25-79, 83-126,	9	53-163,	40	254
	oxoglutarate	35-40, 62-69,	151-284,		191-246,
	dehydrogenase	80-86,	292-408,		457-515,
	E1	88-99, 109-118,	415-511,		520-528,
	component	120-133,	532-577,		620-642,
	(EC 1.2.4.2) -	154-179,	594-627,		759-800,
	Psychrobacter	203-209,	634-735,		894-943
	cryohalolentis	216-226,	740-809,
	(strain K5)	237-245,	815-866,
		253-260,	885-916
		265-273,
		286-291,
		315-339,
		353-371,
		390-398,
		412-430,
		435-448,
		456-464,
		476-487,
		492-503,
		522-533,
		537-549,
		579-590,
		606-622,
		648-654,
		664-679,
		681-692,
		700-711,
		718-730,
		743-752,
		763-798,
		808-815,
		823-838,
		846-864,
		891-925,
		927-933,
		935-948
MCRH0790	Succinate	21-31, 48-58,	1-93, 98-116,	5	251-272,	41	255
	dehydrogenase	60-66,	118-149,		290-363,
	subunit A	83-89, 102-115,	174-199,		424-502
	(EC 1.3.5.1) -	144-154,	202-302,
	Psychrobacters	159-168,	319-393,
	(strain	175-186,	410-468,
	PRwf-1)	195-200,	493-578,
		206-218,	582-623
		244-250,
		259-272,
		293-299,
		301-307,
		324-330,
		339-348,
		358-369,
		380-391,
		404-421,
		429-437,
		456-467,
		481-486,
		495-502,
		509-533,
		536-550,
		553-571,
		575-580,
		599-620
MCRH0808	Putative	12-19, 30-48,	8-52, 80-171,	7	15-64, 77-168,	42	256
	uncharacterized	55-61,	173-201,		190-252
	protein -	66-74, 103-115,	208-237,
	Mannheimia	117-131,	240-258
	haemolytica	176-183,
	PHL213	192-197,
		200-222
MCRH0815	RNA binding	4-19, 31-58,	1-55, 59-164,	5	124-151,	43	257
	S1 domain	61-70, 84-96,	168-185,		326-341,
	protein -	104-112,	193-238,		567-720
	Psychrobacters	131-138,	244-333,
	(strain	143-165,	343-375,
	PRwf-1)	168-178,	378-517,
		197-208,	521-583,
		213-220,	602-656,
		255-268,	670-761,
		273-281,	788-808
		313-320,
		334-354,
		359-367,
		369-376,
		382-399,
		406-420,
		429-439,
		441-461,
		463-507,
		513-544,
		550-557,
		569-580,
		583-589,
		598-618,
		640-650,
		675-680,
		683-711,
		713-739,
		797-805
MCRH0853	Putative	5-12, 22-37,	1-105, 121-344	3	141-168,	44	258
	uncharacterized	47-75, 78-94,			256-313
	protein	102-107,
	(EC 5.3.4.1) -	110-118,
	Psychrobacter	124-130,
	arcticum	140-154,
		161-166,
		185-193,
		205-213,
		218-237,
		244-257,
		264-269,
		276-283,
		289-315,
		317-323,
		334-341
MCRH0867	Dihydrodipic	33-48, 56-63,	1-108, 110-265	5	4-75, 108-199	45	259
	olinate	65-79,
	reductase (EC	84-101,
	1.3.1.26) -	109-122,
	Psychrobacters	127-160,
	(strain	211-216,
	PRwf-1)	224-229,
		239-256
MCRH0911	Glutamate--	6-14, 24-40,	1-101, 115-152,	5	35-136,	46	260
	cysteine	47-60, 79-98,	154-203,		348-355
	ligase (EC	102-108,	210-314,
	6.3.2.2) -	114-120,	321-368,
	Psychrobacter	122-132,	375-412,
	arcticum	154-160,	414-434,
		168-179,	438-494,
		187-206,	497-529,
		221-226,	544-558
		231-253,
		255-268,
		296-314,
		316-337,
		341-347,
		357-378,
		383-407,
		418-425,
		443-452,
		456-462,
		485-495,
		503-510,
		512-523,
		533-543,
		550-555
MCRH0917	Putative	4-11, 14-22,	1-27, 29-48,	1	322-374	47	261
	UDP-N-	24-29, 33-40,	59-199,
	acetylmuramate:	42-52,	206-257,
	L-alanyl-	54-70, 87-111,	264-378,
	gamma-D-	121-128,	398-459
	glutamyl-	132-143,
	meso	149-168,
	diaminopimelate	180-190,
	ligase -	197-215,
	Psychrobacter	223-232,
	arcticum	244-251,
		296-319,
		328-349,
		363-368,
		378-399,
		414-431,
		434-440,
		451-457
MCRH1023	Putative	4-20, 28-34,	1-37, 52-81,	2	29-90, 125-197	48	262
	uncharacterized	36-42, 56-72,	93-152,
	protein -	80-86,	173-252,
	Acinetobacter	97-110,	266-406
	baumannii	118-127,
		132-138,
		145-150,
		173-179,
		181-195,
		232-244,
		250-263,
		270-276,
		308-314,
		324-331,
		336-343,
		355-364,
		398-403
MCRH1076	L-threonine	15-23, 31-38,	1-87, 104-194,	5	82-164,	49	263
	synthase (EC	51-66,	199-392,		236-325,
	4.2.3.1) -	89-105,	404-425,		392-453
	Psychrobacters	123-130,	443-466
	(strain	135-140,
	PRwf-1)	149-154,
		198-208,
		212-220,
		228-239,
		249-257,
		261-267,
		275-281,
		300-310,
		322-343,
		354-365,
		372-384,
		394-404,
		409-420,
		422-429,
		446-455,
		459-468
MCRH1163	Putative	4-10, 17-46,	1-160, 168-188,	15	126-294	50	264
	uncharacterized	51-80, 87-92,	200-263,
	protein -	99-121,	276-355
	Deinococcus	135-150,
	radiodurans	170-176,
		200-212,
		243-252,
		277-282,
		310-331,
		340-349
MCRH1198	DNA repair	4-18, 35-48,	1-62, 84-178,	8	53-62, 112-187,	51	265
	protein RecN -	52-67, 73-80,	182-264,		233-265,
	Psychrobacter	90-96,	295-396,		375-402
	cryohalolentis	108-147,	403-452,
	(strain K5)	156-171,	461-502,
		188-207,	520-568
		227-234,
		239-248,
		263-286,
		301-330,
		347-363,
		365-395,
		409-419,
		421-431,
		442-447,
		454-468,
		471-482,
		484-510,
		517-525,
		545-555
MCRH1227	MFS cyanate	13-31, 36-48,	14-409	2	23-124,	52	266
	efflux pump,	54-64,			134-172
	CynX -	67-83, 93-104,
	Psychrobacter	106-126,
	arcticum	128-137,
		144-154,
		161-169,
		179-196,
		205-221,
		232-255,
		257-263,
		265-305,
		307-324,
		326-340,
		354-367,
		372-378,
		383-402
MCRH1256	Putative	5-21, 26-35,	1-78, 81-112	1	41-103	53	267
	ComEA	37-44, 57-69,
	protein-	71-78,
	related	84-93, 95-101
	protein -
	Candidatus
	Cloacamonas
	acidaminovorans
MCRH1257	Poly(A)	13-19, 21-42,	1-47, 50-65,	23	14-77, 217-269,	54	268
	polymerase -	47-55,	80-108,		380-542
	Psychrobacters	57-66, 68-74,	121-135,
	(strain	77-82,	145-234,
	PRwf-1)	84-90, 92-101,	236-322,
		106-112,	326-379,
		147-169,	396-488,
		171-183,	502-522,
		185-200,	564-589
		221-230,
		240-252,
		269-275,
		284-296,
		299-308,
		311-322,
		332-342,
		344-358,
		374-382,
		386-391,
		403-412,
		445-451,
		462-477,
		484-494,
		557-563,
		565-574
MCRH1303	Putative	4-34, 36-44,	1-87, 89-172,	2	191-197,	55	269
	hemolysin-	51-57, 63-68,	175-193,		350-438
	related	70-80,	222-244,
	protein -	91-130,	247-366,
	Acinetobacter	136-173,	381-409,
	baumannii	186-193,	416-437
		195-201,
		207-213,
		226-234,
		242-266,
		268-284,
		290-308,
		315-332,
		368-378,
		386-402,
		413-437
MCRH1397	Thiamin-	11-24, 29-51,	10-66, 69-102,	6	2-98	56	270
	monophosphate	61-91,	111-145
	kinase (EC	98-116,
	2.7.4.16) -	119-129,
	Acinetobacters	132-142
	(strain
	ADP1)
MCRH1430	S-adenosyl-	16-49, 55-74,	1-333	6	32-89, 108-141,	57	271
	methyltransferase	78-88,			246-333
	MraW -	91-112,
	Psychrobacters	119-127,
	(strain	140-145,
	PRwf-1)	156-165,
		167-173,
		199-209,
		234-249,
		251-263,
		265-272,
		282-290,
		299-305,
		322-330
MCRH1438	Putative	19-43, 49-55,	1-65, 68-113	1	45-108	58	272
	uncharacterized	78-92
	protein -
	Psychrobacter
	cryohalolentis
	(strain K5)
MCRH1440	Peptidoglycan	15-23, 52-84,	23-105,	7	158-222,	59	273
	synthetase	97-103,	115-152,		327-423,
	FtsI (EC	110-133,	154-220,		427-526
	2.4.1.129) -	149-154,	226-263,
	Psychrobacters	160-183,	276-296,
	(strain	203-227,	302-368,
	PRwf-1)	233-241,	378-402,
		243-250,	414-477,
		313-330,	481-536,
		338-351,	539-581,
		373-382,	588-652
		384-396,
		401-407,
		425-452,
		469-474,
		476-482,
		490-513,
		519-528,
		533-545,
		591-613,
		619-649
MCRH1475	S. Translation	4-11, 19-29,	39-105,	10	78-130,	60	274
	initiation	43-52, 82-96,	132-176,		215-292,
	factor IF-2 -	101-110,	183-247,		655-713,
	Psychrobacters	138-144,	258-292,		818-880
	(strain	157-165,	300-324,
	PRwf-1)	258-268,	328-398,
		326-334,	405-506,
		336-344,	512-532,
		346-358,	544-653,
		377-383,	659-838,
		386-394,	840-885
		397-405,
		413-427,
		429-434,
		437-443,
		449-458,
		486-495,
		514-521,
		535-546,
		554-560,
		566-578,
		589-596,
		603-612,
		618-625,
		644-659,
		662-671,
		675-680,
		707-720,
		722-735,
		737-752,
		754-771,
		788-802,
		816-830,
		836-844,
		851-870,
		884-909
MCRH1486	S. Ribosome-	5-14, 21-27,	1-63, 81-141	4	58-141	61	275
	binding factor	29-37, 46-72,
	A -	83-96,
	Psychrobacter	113-121,
	arcticum	129-134
MCRH1493	OlpA -	4-9, 20-34,	1-53, 75-116,	7	92-234	62	276
	Moraxella	36-54, 88-94,	126-174,
	catarrhalis	124-148,	177-235
		155-161,
		182-202,
		204-211,
		220-232
MCRH1546	—	4-22, 31-38,	1-24, 28-49	24	7-65	63	277
		49-55, 82-91
MCRH1561	Sulphate	4-28, 30-83,	1-298, 301-566	4	14-31, 282-347	64	278
	transporter -	85-92, 94-123,
	Psychrobacters	125-157,
	(strain	163-196,
	PRwf-1)	216-234,
		240-258,
		260-289,
		307-313,
		315-323,
		328-378,
		392-424,
		428-440,
		458-472,
		474-482,
		491-506,
		508-530,
		539-564
MCRH1565	Probable zinc	7-17, 20-28,	1-158, 163-275	4	60-112,	65	279
	metallopeptidases,	30-37, 39-47,			149-246
	family	49-61,
	M48 -	77-96, 114-128,
	Psychrobacter	149-154,
	arcticum	166-180,
		182-200,
		240-248,
		256-271
MCRH1568	Phosphate	6-25, 31-37,	1-46, 48-196,	4	184-277	66	280
	ABC	43-57, 63-72,	208-231,
	transporter,	82-90,	234-278,
	periplasmic	96-107,	290-374
	phosphate-	109-118,
	binding	123-140,
	protein -	144-149,
	Psychrobacters	155-162,
	(strain	168-182,
	PRwf-1)	184-190,
		213-219,
		237-244,
		247-256,
		262-268,
		271-284,
		295-301,
		307-320,
		325-337,
		346-364
MCRH1577	Phosphate	27-46, 53-77,	2-210, 215-271	6	12-138	67	281
	ABC	103-123,
	transporter,	130-142,
	ATPase	153-159,
	subunit	171-179,
	precursor -	182-209,
	Psychrobacters	215-222,
	(strain	224-233,
	PRwf-1)	237-243,
		271-276
MCRH1586	Ppx/GppA	17-24, 38-45,	8-134, 141-167,	9	284-393,	68	282
	phosphatase	49-67,	175-224,		462-469
	(EC 3.6.1.40) -	80-86, 94-112,	231-473,
	Psychrobacters	119-141,	479-496
	(strain	154-159,
	PRwf-1)	165-171,
		183-200,
		218-226,
		234-252,
		263-291,
		328-336,
		338-349,
		357-385,
		397-405,
		423-440,
		445-453,
		457-466,
		470-477,
		487-493
MCRH1671	GTP-binding	4-9, 16-23,	1-20, 39-103	5	11-36	69	283
	protein Era -	56-64, 70-82,
	Psychrobacters	86-93
	(strain
	PRwf-1)
MCRH1677	Outer	5-38, 69-77,	1-49, 54-69,	3	65-116,	70	284
	membrane	83-99, 143-151,	96-110,		179-231,
	protein CD	154-160,	130-248,		328-395
	precursor	176-186,	251-270,
	(OmpCD) -	198-210,	312-368,
	Moraxella	226-268,	382-450
	catarrhalis	270-288,
		293-303,
		305-324,
		327-342,
		344-353,
		388-394,
		432-438
MCRH1696	Extracellular	8-23, 58-70,	1-27, 30-46,	3	3-91	71	285
	solute-	74-80, 85-91,	53-71, 77-171,
	binding	95-103,	181-264
	protein,	110-127,
	family 3	143-150,
	precursor -	153-160,
	Psychrobacters	164-171,
	(strain	189-201,
	PRwf-1)	218-224,
		228-233,
		236-242,
		248-255
MCRH1701	Extracellular	4-19, 30-49,	1-22, 33-51,	6	108-262	72	286
	solute-	57-83, 88-100,	55-99, 114-139,
	binding	111-119,	163-204,
	protein,	122-129,	220-250
	family 3	154-159,
	precursor -	168-173,
	Psychrobacters	189-200,
	(strain	222-229,
	PRwf-1)	237-243,
		251-259
MCRH1705	ABC	4-19, 26-51,	1-174, 176-277	1	47-117	73	287
	transporter	53-62, 68-79,
	related -	83-96,
	Psychrobacters	99-105,
	(strain	114-122,
	PRwf-1)	128-135,
		138-146,
		155-164,
		180-199,
		201-218,
		226-232,
		237-249,
		257-264
MCRH1720	DNA	12-17, 23-31,	9-61, 75-124,	11	59-133,	74	288
	topoisomerase	42-62,	132-288,		260-272,
	I (EC	81-88, 96-103,	299-547,		382-410,
	5.99.1.2) -	109-118,	592-608,		440-515,
	Psychrobacters	122-128,	612-660,		711-872
	(strain	136-146,	666-695,
	PRwf-1)	151-158,	733-753,
		188-203,	776-813,
		210-224,	818-854,
		229-238,	858-875
		270-287,
		296-308,
		310-333,
		341-354,
		361-368,
		383-395,
		404-420,
		423-435,
		443-456,
		460-503,
		510-517,
		521-526,
		541-548,
		565-570,
		575-586,
		606-617,
		625-637,
		648-655,
		683-696,
		702-713,
		722-728,
		740-748,
		779-786,
		789-801,
		807-812,
		820-829,
		849-854,
		862-868
MCRH1732	DNA	15-24, 28-56,	1-155, 162-181,	9	700-781,	75	289
	polymerase I	63-87,	186-321,		800-903
	(EC 2.7.7.7) -	98-104,	346-600,
	Psychrobacters	112-121,	608-760,
	(strain	125-133,	778-897,
	PRwf-1)	142-159,	910-953
		166-176,
		189-198,
		200-207,
		216-223,
		225-231,
		236-243,
		245-252,
		262-287,
		295-304,
		315-323,
		326-332,
		340-356,
		361-368,
		370-376,
		387-398,
		401-410,
		412-420,
		422-434,
		442-455,
		469-476,
		486-496,
		507-514,
		519-527,
		547-556,
		559-564,
		569-575,
		588-594,
		604-609,
		611-622,
		634-662,
		676-686,
		712-718,
		723-738,
		752-758,
		779-790,
		793-822,
		833-850,
		866-891,
		894-908,
		935-950
MCRH1742	S. Peptide	25-31, 41-54,	2-33, 36-217,	20	2-122, 192-306	76	290
	chain release	79-86,	231-251
	factor 3 (RF-	88-96, 111-125,	263-363,
	3) -	135-141,	367-427,
	Psychrobacters	145-152,	430-527
	(strain	159-182,
	PRwf-1)	188-195,
		208-214,
		257-267,
		275-297,
		306-312,
		326-333,
		342-358,
		370-377,
		399-414,
		420-428,
		434-439,
		446-492,
		499-507,
		509-521,
		527-533
MCRH1777	Uncharacterized	6-13, 18-26,	1-83, 100-280,	5	63-151,	77	291
	iron-	28-46, 49-56,	295-370,		220-291,
	regulated	62-69,	374-509		321-388
	membrane	74-84, 88-110,
	protein-like	116-132,
	protein -	135-149,
	Psychrobacters	156-178,
	(strain	181-191,
	PRwf-1)	202-223,
		247-260,
		274-281,
		295-303,
		336-344,
		368-375,
		377-390,
		395-404,
		409-433,
		450-458,
		470-506
MCRH1841	McmA -	4-11, 16-27,	7-194, 202-235,	30	96-113,	78	292
	Moraxella	31-41, 62-81,	261-383,		634-656,
	catarrhalis	101-108,	401-505,		658-734
		116-144,	524-617,
		169-185,	624-727,
		203-218,	735-749,
		228-234,	761-879,
		236-245,	898-973,
		255-273,	975-989
		283-294,
		298-304,
		306-313,
		315-340,
		342-350,
		357-371,
		378-390,
		402-416,
		421-436,
		444-456,
		458-464,
		481-487,
		496-508,
		510-531,
		536-541,
		544-551,
		556-569,
		576-590,
		600-614,
		627-636,
		644-651,
		664-670,
		675-688,
		700-720,
		727-770,
		775-797,
		802-818,
		828-835,
		864-884,
		887-892,
		899-907,
		918-928,
		940-958,
		965-980
MCRH1853	HI0933	4-12, 14-23,	1-100, 105-343,	3	76-101,	79	293
	family protein -	26-35, 51-78,	365-406		295-353
	Psychrobacters	83-94,
	(strain	100-106,
	PRwf-1)	109-119,
		121-135,
		140-149,
		153-177,
		179-202,
		212-224,
		229-239,
		241-252,
		260-312,
		330-336,
		342-352,
		368-383,
		393-403
MCRH1878	S. Glycyl-	4-28, 31-38,	1-84	1	9-69	80	294
	tRNA	65-81
	synthetase
	alpha subunit
	(EC 6.1.1.14)
	(Glycine--
	tRN ligase
	alpha subunit)
	(GlyRS) -
	Moraxella
	catarrhalis
MCRH1934	RNAse E (EC	4-11, 26-35,	1-104, 108-152,	11	378-625,	81	295
	3.1.4.—) -	55-74, 78-90,	161-190,		795-908
	Psychrobacters	109-115,	195-323,
	(strain	125-141,	337-366,
	PRwf-1)	164-169,	369-410,
		173-181,	415-449,
		193-203,	453-495,
		212-231,	532-552,
		253-266,	559-624,
		289-295,	677-697,
		297-313,	724-751,
		350-356,	784-802,
		380-388,	804-824,
		410-416,	834-931,
		425-433,	949-977,
		449-462,	979-999,
		466-473,	1060-1149
		478-497,
		562-584,
		586-606,
		614-619,
		718-724,
		727-738,
		745-752,
		782-789,
		812-821,
		827-834,
		844-854,
		864-874,
		902-908,
		920-927,
		952-957,
		966-973,
		982-988,
		1010-1019,
		1027-1034,
		1050-1057,
		1082-1088,
		1111-1125,
		1129-1135,
		1150-1163
MCRH2003	Phospholipid/	7-31, 36-49,	1-42, 58-233,	2	181-234	82	296
	glycerol	59-66, 78-85,	236-279,
	acyltransferase	89-95,	290-315
	precursor -	101-120,
	Psychrobacter	127-151,
	cryohalolentis	174-183,
	(strain K5)	185-194,
		207-217,
		221-230,
		238-247,
		258-268,
		290-297,
		309-315
MCRH2082	S. 30S	17-25, 60-72,	1-72, 74-120	4	22-110	83	297
	ribosomal	80-90,
	protein S11 -	97-109,
	Psychrobacters	111-117
	(strain
	PRwf-1)
MCRH2139	UDP-N-	4-31, 44-53,	1-35, 45-218,	3	105-193,	84	298
	acetylmuramoylalanine-	55-70, 76-99,	225-323,		327-355
	D-	109-115,	347-386,
	glutamate	123-130,	388-403,
	ligase (EC	136-142,	405-446,
	6.3.2.9) -	147-155,	448-469
	Acinetobacter	158-191,
	baumannii	199-220,
		223-235,
		243-257,
		264-281,
		286-299,
		313-323,
		350-365,
		373-391,
		397-441,
		455-466
MCRH2151	Probable	4-19, 47-53,	1-32, 75-89,	5	43-142,	85	299
	DNA/RNA	69-75, 81-87,	107-127,		300-314
	non-specific	109-122,	133-171,
	endonuclease	125-140,	188-310,
	(Mg-	142-156,	325-349
	dependent) -	191-199,
	Psychrobacter	219-238,
	arcticum	246-271,
		279-290,
		296-302,
		305-313,
		321-327,
		331-345
MCRH2184	Mg chelatase,	4-43, 49-56,	1-38, 43-62,	3	19-76, 195-221,	86	300
	subunit ChlI -	66-74, 80-94,	77-137,		271-292
	Psychrobacters	97-110,	140-173,
	(strain	116-136,	179-291,
	PRwf-1)	139-145,	328-376,
		151-163,	386-400,
		165-178,	406-504
		182-210,
		213-220,
		222-231,
		241-252,
		256-271,
		277-293,
		300-314,
		330-336,
		340-363,
		369-375,
		381-404,
		419-426,
		439-445,
		447-453,
		465-501
MCRH2216	Selenocysteine	4-19, 25-31,	10-99, 101-185,	1	74-161	87	301
	synthase	40-63, 71-80,	200-294,
	(EC 2.9.1.1) -	89-98,	297-347
	Haemophilus	101-119,
	influenzae	125-151,
	(strain	159-164,
	PittEE)	173-201,
		219-227,
		247-258,
		263-284,
		308-319,
		321-326
MCRH2239	Putative	12-18, 25-31,	1-28, 33-134,	15	25-59, 156-270,	88	302
	uncharacterized	37-43,	137-179,		272-335,
	protein -	76-94, 121-135,	181-221,		557-565
	Pseudomonas	146-176,	255-323,
	fluorescens	191-199,	333-439,
	(strain PfO-1)	205-212,	443-532,
		259-290,	537-692
		300-313,
		340-347,
		364-373,
		375-385,
		413-420,
		424-430,
		446-462,
		465-473,
		475-486,
		505-522,
		537-544,
		557-563,
		573-582,
		588-602,
		604-613,
		620-630,
		638-675,
		677-683
MCRH2257	S. 10 kDa	10-22, 24-54,	1-60, 88-136	33	17-135	89	303
	chaperonin	63-70,
	(Protein	77-86, 94-110,
	Cpn10)	112-133
	(groES
	protein) -
	Psychrobacters
	(strain
	PRwf-1)
MCRH2276	Monooxygenase -	7-24, 28-47,	4-47, 50-248,	5	61-67, 168-195,	90	304
	Neisseria	76-86, 92-103,	261-393		257-320
	meningitidis	122-129,
	serogroup C	135-141,
	(strain	152-157,
	053442)	162-173,
		192-205,
		210-225,
		230-236,
		270-286,
		289-297,
		299-314,
		316-328,
		334-341,
		349-356,
		376-382,
		384-395
MCRH2384	S. Threonyl-	4-9, 20-39,	15-126,	7	172-215,	91	305
	tRNA	42-48, 67-101,	129-166,		218-291,
	synthetase	129-135,	171-217,		417-440,
	(EC 6.1.1.3)	156-162,	227-307,		498-574
	(Threonine--	170-176,	309-361,
	tRNA ligase	178-189,	363-431,
	(ThrRS) -	192-197,	461-496,
	Psychrobacters	228-235,	501-637
	(strain	260-266,
	PRwf-1)	272-278,
		291-301,
		330-342,
		347-360,
		367-373,
		383-390,
		397-409,
		432-443,
		466-487,
		506-517,
		520-545,
		551-561,
		588-598,
		602-608,
		622-630
MCRH2399		8-28, 34-67	1-70	2	2-32	92	306
MCRH2415	Lytic	9-26, 33-40,	1-30, 33-99,	3	59-118,	93	307
	transglycosylase,	79-93, 98-105,	106-126,		160-215
	catalytic	112-121,	129-331,
	precursor -	128-137,	340-511,
	Psychrobacters	144-156,	522-565,
	(strain	160-167,	578-599,
	PRwf-1)	183-190,	608-667
		198-204,
		210-224,
		250-275,
		283-289,
		296-302,
		314-321,
		336-344,
		346-353,
		355-362,
		367-380,
		382-391,
		393-403,
		407-413,
		416-423,
		434-443,
		469-492,
		499-509,
		550-559,
		596-603,
		613-633,
		635-642,
		648-659
MCRH2484	Acetyl-	7-20, 25-33,	5-90, 113-250,	6	9-47, 145-198,	94	308
	coenzyme A	36-64, 74-83,	260-293		227-239
	carboxylase	117-124,
	carboxyl	126-137,
	transferase	146-159,
	subunit alpha -	164-173,
	Psychrobacter	181-186,
	arcticum	195-231,
		240-255,
		258-266,
		268-276,
		283-290
MCRH2496	Possible	4-24, 31-41,	1-101, 103-140,	2	189-242	95	309
	Peptidoglycan-	43-57, 61-68,	202-263,
	binding	72-78,	286-362
	LysM -	83-88, 115-121,
	Psychrobacter	123-135,
	arcticum	190-200,
		214-224,
		239-248,
		291-299,
		305-312,
		328-333,
		351-362
MCRH2506	Msp22	4-21, 110-126,	1-29, 31-62,	2	7-80	96	310
	(Fragment) -	128-134,	65-94, 99-149
	Moraxella	139-149
	catarrhalis
MCRH2519	S. 1-deoxy-	4-9, 23-29,	1-104, 121-135,	6	162-186,	97	311
	D-xylulose-5-	37-52, 57-73,	143-176,		309-397,
	phosphate	80-98,	182-274,		435-463,
	synthase (EC	101-119,	281-301,		503-565
	2.2.1.7) (1-	146-163,	306-385,
	deoxyxylulose	175-181,	393-502,
	5-phosphate	198-208,	505-648,
	synthase)	222-228,	654-709
	(DXP	243-249,
	synthase)	258-263,
	(DXPS) -	269-278,
	Psychrobacter	283-298,
	cryohalolentis	309-324,
	(strain K5)	329-339,
		342-353,
		356-363,
		365-374,
		389-395,
		397-404,
		408-415,
		424-429,
		432-450,
		457-490,
		503-524,
		526-556,
		568-575,
		584-592,
		596-619,
		621-646,
		653-673,
		675-694,
		699-706
MCRH2553	S. Cysteinyl-	5-21, 24-49,	1-89, 106-125,	5	166-220,	98	312
	tRNA	51-63, 65-76,	129-207,		369-473
	synthetase	111-117,	210-230,
	(EC 6.1.1.16)	133-145,	255-357,
	(Cysteine--	149-156,	360-453
	tRNA ligase	189-197,
	(CysRS) -	217-230,
	Psychrobacter	240-245,
	cryohalolentis	288-295,
	(strain K5)	297-316,
		321-335,
		339-346,
		369-382,
		401-421
MCRH2560	Putative	4-11, 22-37,	1-51, 59-102	2	41-100	99	313
	cytochrome c,	39-54, 62-70,
	class I	78-84,
	precursor -	94-100,
	Psychrobacter	106-111
	cryohalolentis
	(strain K5)
MCRH2592	S. Ribonuclease	16-26, 29-46,	1-35, 61-81,	12	43-75	100	314
	PH (EC	48-54,	88-116,
	2.7.7.56)	94-101,	139-205,
	(RNase PH)	103-110,	207-227
	(tRN	117-125,
	nucleotidyltransferase) -	134-148,
	Psychrobacters	156-180,
	(strain	190-196,
	PRwf-1)	221-239
MCRH2606	Outer	5-19, 21-27,	1-76, 83-155,	4	15-137	101	315
	membrane	30-35, 40-57,	158-191,
	protein G1b -	67-75,	218-253
	Moraxella	104-114,
	catarrhalis	121-130,
		152-161,
		167-175,
		187-205,
		219-227,
		244-250
MCRH2612	Probable	15-38, 40-49,	1-83, 85-115,	3	598-632,	102	316
	aminopeptidase	57-66,	136-179,		690-700,
	N (EC	68-76, 80-88,	192-223,		772-833
	3.4.11.2) -	91-109,	257-360,
	Psychrobacter	117-122,	375-486,
	arcticum	148-154,	501-527,
		161-169,	530-618,
		195-214,	626-644,
		220-230,	653-867
		233-241,
		263-269,
		285-293,
		301-312,
		324-340,
		349-367,
		372-386,
		398-407,
		426-434,
		436-443,
		446-452,
		456-476,
		478-498,
		510-517,
		523-550,
		558-565,
		589-604,
		610-619,
		634-639,
		659-668,
		680-688,
		698-704,
		732-739,
		741-754,
		758-767,
		783-796,
		800-812,
		816-826,
		842-860
MCRH2625	Periplasmic	4-12, 41-49,	1-68, 71-150,	6	37-125,	103	317
	protein -	61-66, 111-118,	193-226,		272-341
	Neisseria	123-128,	230-296,
	meningitidis	139-144,	305-406
	serogroup C	159-166,
	(strain	178-185,
	053442)	206-214,
		236-248,
		262-268,
		275-293,
		297-314,
		348-360,
		368-399
MCRH2627	GDSL-like	8-15, 18-54,	17-110,	5	62-107,	104	318
	lipase/ccylhydrolase	61-80, 109-114,	118-139,		123-182
	family protein -	143-149,	149-238,
	Pseudomonas	153-160,	247-365
	fluorescens	172-185,
	(strain Pf-5/	188-194,
	ATCC BAA-	196-207,
	477)	221-230,
		238-244,
		273-279,
		291-297,
		305-319,
		325-333,
		342-362
MCRH2641	Putative	15-28, 34-59,	1-32, 39-55,	6	50-100,	105	319
	uncharacterized	105-135,	76-122,		116-176,
	protein -	137-144,	124-279,		261-288
	Psychrobacter	155-184,	282-337
	cryohalolentis	187-212,
	(strain K5)	221-233,
		240-260,
		267-278,
		286-291,
		299-315,
		319-334
MCRH2669	Cytochrome	4-22, 43-56,	1-36, 57-112,	22	4-108	106	320
	c1 precursor -	71-108,	121-181,
	Psychrobacters	119-125,	190-241
	(strain	127-138,
	PRwf-1)	146-155,
		164-171,
		173-181,
		190-199,
		214-234
MCRH2682	S. tRNA	11-19, 24-31,	8-273, 283-299,	22	17-74, 155-189,	107	321
	uridine 5-	37-42,	309-346,		220-302,
	carboxymethylaminomethyl	61-71, 85-91,	367-490,		418-540,
	modification	94-100,	494-537,		550-612
	enzyme gid	105-115,	558-632
	(Glucose-	123-138,
	inhibited	140-148,
	division	153-161,
	protein A) -	164-173,
	Psychrobacter	185-192,
	arcticum	194-202,
		212-222,
		227-233,
		244-250,
		276-287,
		298-304,
		309-315,
		319-330,
		342-353,
		367-374,
		387-394,
		403-425,
		439-445,
		514-520,
		528-543,
		545-553,
		569-584,
		586-595,
		603-621
MCRH2754	Probable	4-20, 41-47,	1-62, 70-209,	3	140-149,	108	322
	Transcription	49-56, 68-86,	217-319		174-233,
	regulator,	95-101,			243-300
	LysR family -	108-121,
	Psychrobacter	123-137,
	arcticum	145-150,
		158-178,
		187-198,
		218-226,
		231-237,
		240-250,
		252-262,
		269-282,
		287-317
MCRH2759	4Fe—4S	16-21, 29-36,	1-15, 19-260,	16	242-272,	109	323
	ferredoxin,	43-61,	292-312,		435-457
	iron-sulfur	68-84, 89-136,	314-451,
	binding -	159-197,	465-483
	Psychrobacter	206-216,
	cryohalolentis	224-240,
	(strain K5)	244-251,
		270-286,
		292-307,
		313-323,
		327-332,
		339-364,
		388-395,
		402-409,
		423-440,
		448-461,
		464-476
MCRH2783	Cytochrome c	8-28, 30-35,	1-40, 44-96,	2	158-168,	110	324
	oxidase cbb3-	55-64, 69-79,	103-154,		286-334
	type, subunit	87-95,	158-177,
	III (EC	132-143,	181-202,
	1.9.3.1) -	161-177,	208-268,
	Psychrobacter	186-198,	272-355
	arcticum	222-227,
		247-258,
		266-288,
		340-352
MCRH2844	Oligopeptide-	5-24, 39-46,	1-33, 35-108,	6	162-181,	111	325
	binding	50-64, 79-89,	118-236,		331-422,
	protein oppA -	103-109,	245-260,		481-534,
	Streptococcus	116-133,	264-324,		591-669
	pyogenes	137-150,	329-358,
	serotype M2	159-167,	360-397,
	(strain	169-177,	448-485,
	MGAS10270)	184-202,	492-536,
		209-229,	541-599,
		245-254,	607-669
		272-282,
		294-301,
		340-356,
		368-378,
		388-395,
		429-444,
		450-463,
		484-490,
		536-545,
		547-557,
		565-601,
		615-627,
		648-655
MCRH2851	Putative	26-55, 58-73,	6-151	3	15-96	112	326
	uncharacterized	75-81,
	protein -	87-96, 131-140,
	Psychrobacters	150-158
	(strain
	PRwf-1)
MCRH2861	S. Leucyl-	4-9, 43-52,	27-88, 90-173,	18	24-123,	113	327
	tRNA	63-71, 97-104,	176-283,		243-316,
	synthetase	117-125,	303-404,		430-472,
	(EC 6.1.1.4)	130-136,	421-443,		512-571
	(Leucine--	143-161,	473-487,
	tRNA ligase)	167-179,	507-540,
	(LeuRS) -	185-191,	544-642,
	Psychrobacter	194-201,	645-673,
	cryohalolentis	205-211,	677-761,
	(strain K5)	219-225,	767-840,
		240-245,	843-878
		247-256,
		261-279,
		286-301,
		323-338,
		340-349,
		359-370,
		390-397,
		399-414,
		439-473,
		485-496,
		505-521,
		534-561,
		566-571,
		575-588,
		598-606,
		623-629,
		653-665,
		675-680,
		683-690,
		692-700,
		730-755,
		769-798,
		802-815,
		818-826,
		852-859,
		862-875
MCRH2874	Sulfatase	40-55, 59-119,	1-336, 344-379,	4	230-331,	114	328
	domain	145-180,	395-438,		454-460
	protein -	182-210,	440-457,
	Dichelobacter	215-230,	476-571
	nodosus	264-274,
	(strain	293-305,
	VCS1703A)	308-314,
		316-328,
		339-346,
		348-354,
		388-393,
		395-414,
		416-425,
		431-436,
		442-451,
		467-473,
		477-487,
		491-498,
		505-513,
		519-542,
		548-565
MCRH2891	Na+	5-24, 32-46,	1-321, 323-344,	3	341-374,	115	329
	symporter -	72-77, 83-98,	353-371,		406-416
	Marinomonass	100-106,	374-588
	(strain	113-135,
	MWYL1)	140-150,
		154-170,
		176-199,
		218-240,
		259-278,
		282-301,
		307-323,
		379-386,
		389-430,
		449-457,
		465-484,
		504-520,
		537-561,
		568-581
MCRH2904	Glycine	11-20, 27-37,	3-31, 36-63,	9	293-326,	116	330
	dehydrogenase	39-54,	66-80, 103-208,		691-787,
	(Decarboxylating)	67-73, 79-89,	231-299,		896-950
	beta	92-102,	307-399,
	subunit/	112-120,	445-517,
	glycine	125-138,	528-586,
	dehydrogenase	145-152,	597-618,
	(Decarboxylating)	172-178,	632-652,
	alpha	181-191,	661-742,
	subunit (EC	193-204,	745-781,
	1.4.4.2) -	213-220,	785-852,
	Psychrobacters	222-235,	855-893,
	(strain	241-257,	909-947
	PRwf-1)	281-288,
		298-306,
		331-359,
		361-403,
		420-426,
		434-456,
		462-470,
		477-484,
		497-504,
		520-527,
		535-548,
		566-575,
		584-592,
		606-613,
		624-629,
		635-651,
		654-660,
		683-694,
		697-703,
		714-725,
		728-757,
		767-779,
		787-794,
		801-814,
		834-843,
		858-867,
		869-875,
		888-899,
		912-933,
		936-946
MCRH2916	S. Aminomethyl	4-21, 29-46,	1-88	1	24-78	117	331
	transferase	61-77, 81-88
	(EC 2.1.2.10)
	(Glycine
	cleavage
	system
	protein) -
	Neisseria
	meningitidis
	serogroup B
MCRH2935	Putative	8-41, 48-53,	1-49, 69-149,	4	39-67, 77-105	118	332
	lipoprotein -	73-78, 81-98,	161-227,
	Bordetella	115-140,	230-306
	petrii (strain	143-149,
	ATCC BAA-	154-162,
	461/DSM	182-196,
	12804/	207-215,
	CCUG	221-226,
	43448)	230-239,
		257-264,
		268-276,
		287-303
MCRH3006	Aspartate	4-11, 16-23,	1-51, 93-245,	6	3-9, 107-183,	119	333
	semialdehyde	31-39, 64-79,	259-377		210-318
	dehydrogenase	82-89,
	(EC	98-105,
	1.2.1.11) -	111-117,
	Psychrobacter	136-146,
	arcticum	162-168,
		186-193,
		196-205,
		219-236,
		268-287,
		289-300,
		308-314,
		323-339,
		349-365,
		368-374
MCRH3022	Probable	4-28, 35-48,	1-26, 54-136,	2	14-88	120	334
	cytochrome	56-64, 91-125,	144-167
	C-type	135-143,
	biogenesis	150-159
	protein -
	Psychrobacter
	arcticum
MCRH3030	Putative	4-16, 21-39,	1-153, 178-240,	4	79-169,	121	335
	uncharacterized	53-83, 88-93,	254-282,		194-251,
	protein -	99-133,	284-349,		451-509
	Psychrobacter	152-164,	366-446,
	arcticum	167-187,	450-784,
		192-209,	794-813
		211-231,
		245-273,
		278-284,
		289-304,
		317-333,
		335-341,
		346-353,
		359-366,
		378-389,
		394-402,
		424-438,
		447-460,
		473-480,
		482-492,
		494-500,
		506-520,
		523-541,
		558-574,
		577-588,
		598-605,
		611-619,
		622-629,
		633-643,
		649-669,
		685-701,
		707-727,
		735-749,
		768-780,
		783-789,
		798-810
MCRH3040	Putative	4-30, 38-46,	1-52, 64-78,	4	25-108,	122	336
	uncharacterized	56-70, 80-90,	81-202,		505-560
	protein -	101-110,	221-297,
	Psychrobacter	117-123,	335-381,
	arcticum	149-157,	384-454,
		162-172,	464-552,
		178-191,	559-590,
		211-218,	598-620,
		230-256,	622-644,
		261-283,	646-668,
		292-310,	672-821,
		335-344,	833-898
		348-354,
		361-369,
		382-396,
		403-414,
		425-431,
		452-458,
		460-472,
		484-504,
		508-514,
		524-541,
		562-580,
		597-603,
		627-643,
		645-657,
		688-696,
		707-713,
		719-736,
		746-759,
		769-775,
		791-797,
		802-812,
		824-830,
		838-847,
		856-864,
		880-901,
		909-916
MCRH3120	2-	16-24, 37-46,	7-44, 51-79,	11	126-230,	123	337
	isopropylmalate	60-73,	81-114,		248-286,
	synthase -	79-90, 97-116,	116-263,		364-508,
	Psychrobacter	130-141,	285-307,		510-517
	cryohalolentis	143-150,	311-351,
	(strain K5)	155-161,	379-416,
		179-186,	427-482,
		193-206,	491-509,
		212-225,	535-570
		230-253,
		258-267,
		290-296,
		306-344,
		366-381,
		383-390,
		398-405,
		418-425,
		438-449,
		465-482,
		489-499,
		503-509,
		530-543,
		545-567
MCRH3128	UspA1 -	4-10, 13-21,	1-19, 24-55,	43	37-262,	124	338
	Moraxella	27-40, 90-95,	88-108,		272-351,
	catarrhalis	121-128,	118-153,		453-474,
		197-203,	172-190,		499-518,
		239-246,	198-281,		527-547,
		253-259,	299-361,		569-643,
		305-311,	369-392,		897-954
		322-336,	430-578,
		349-356,	661-689,
		358-364,	698-738,
		372-379,	795-850,
		393-408,	861-901,
		411-417,	904-924,
		427-434,	927-946
		457-467,
		481-490,
		492-497,
		504-513,
		533-542,
		561-571,
		592-600,
		641-646,
		661-667,
		671-682,
		711-720,
		722-734,
		763-771,
		779-796,
		833-838,
		855-865,
		873-879,
		886-896,
		901-908,
		913-924,
		931-939
MCRH3146	Putative	22-28, 39-49	4-20, 26-44	4	5-65	125	339
	uncharacterized
	protein -
	Alteromonadales
	bacterium
	TW-7
MCRH3327	DsrE-like	4-39, 52-63,	1-88, 94-125	2	56-113	126	340
	protein -	70-88, 97-112
	Psychrobacter
	cryohalolentis
	(strain K5)
MCRH3328	Glutamate-	10-29, 35-40,	1-30, 35-106,	5	231-286,	127	341
	ammonia-	46-62,	117-348,		394-429,
	ligase	77-84, 94-129,	357-381,		686-758
	adenylyltransferase	146-153,	390-551,
	(EC	158-164,	553-800,
	2.7.7.42) -	166-174,	804-835,
	Psychrobacter	196-202,	844-882,
	cryohalolentis	209-215,	893-937
	(strain K5)	225-240,
		250-258,
		272-287,
		305-313,
		318-335,
		339-353,
		362-381,
		384-390,
		395-424,
		439-452,
		454-463,
		467-476,
		482-496,
		504-529,
		535-562,
		564-573,
		576-588,
		596-602,
		604-619,
		625-658,
		664-675,
		685-696,
		714-727,
		746-758,
		770-777,
		780-789,
		793-799,
		845-861,
		863-873,
		890-901,
		905-910,
		921-934
MCRH3363	Aminotransferase	8-31, 35-70,	5-104, 122-260,	3	106-129,	128	342
	(EC	83-89, 91-102,	271-340,		215-224,
	2.6.1. -) -	108-116,	353-564,		454-522
	Psychrobacters	130-156,	566-603
	(strain	169-191,
	PRwf-1)	203-217,
		238-256,
		270-292,
		298-311,
		315-331,
		335-345,
		364-370,
		376-382,
		384-401,
		405-419,
		421-430,
		434-441,
		445-452,
		467-481,
		503-511,
		516-522,
		524-531,
		534-539,
		547-560,
		570-582
MCRH3380	Carbamoyl-	8-27, 29-37,	1-68, 84-100,	8	155-209,	129	343
	phosphate	42-49, 63-69,	152-182,		427-501,
	synthase,	74-80,	198-344,		658-688,
	large subunit	95-118,	348-413,		809-841,
	(EC 6.3.5.5) -	140-148,	416-552,		862-871,
	Acinetobacter	156-170,	558-621,		927-1010
	baumannii	189-195,	624-788,
		197-209,	800-892,
		218-223,	958-1060,
		227-235,	1064-1078
		247-255,
		268-275,
		294-299,
		305-313,
		316-333,
		347-359,
		369-376,
		381-386,
		399-410,
		423-432,
		436-455,
		462-475,
		524-537,
		578-588,
		597-605,
		616-622,
		624-634,
		636-644,
		648-655,
		657-665,
		693-698,
		706-723,
		726-732,
		736-778,
		782-805,
		820-832,
		835-846,
		848-870,
		882-909,
		927-933,
		951-957,
		962-971,
		976-982,
		986-1001,
		1016-1021,
		1042-1050,
		1054-1061,
		1066-1075
MCRH3399	Peptidase	15-36, 55-67,	2-36, 52-72,	9	21-78, 90-154,	130	344
	M23B	70-76,	82-152,		168-215
	precursor -	81-88, 110-125,	156-188,
	Psychrobacter	128-134,	191-225,
	cryohalolentis	136-188,	232-322
	(strain K5)	197-214,
		216-223,
		244-250,
		257-263,
		265-280,
		300-306,
		311-319
MCRH3401	PpiC-type	4-25, 32-41,	1-120, 126-160,	2	8-56, 181-269	131	345
	peptidyl-	51-58, 71-78,	173-257,
	prolyl cis-	81-89,	266-313,
	trans	101-123,	322-342,
	isomerase	130-138,	354-434
	precursor -	140-151,
	Psychrobacter	165-171,
	cryohalolentis	176-193,
	(strain K5)	202-214,
		244-259,
		266-282,
		285-293,
		295-301,
		316-325,
		329-334,
		344-350,
		381-390,
		405-415,
		423-431
MCRH3404	S. LPS-	4-46, 53-60,	1-91, 114-128,	14	103-238,	132	346
	assembly	74-86, 97-104,	130-149,		247-273,
	protein	121-128,	156-190,		451-470,
	precursor	149-160,	207-306,		687-734,
	(Organic	206-217,	308-330,		753-796
	solvent	252-258,	342-429,
	tolerance	274-280,	436-450,
	protein) -	287-294,	454-662,
	Psychrobacter	311-327,	666-937
	cryohalolentis	356-370,
	(strain K5)	378-383,
		394-403,
		425-432,
		434-447,
		473-484,
		491-502,
		543-556
		599-608
		610-623,
		641-657,
		671-684,
		698-707,
		719-733,
		748-758,
		772-779,
		798-808,
		814-820,
		830-843,
		854-866,
		872-884,
		891-899,
		901-907,
		920-932
MCRH3409	Aminoglycoside	35-41, 52-58,	4-104, 112-131,	5	36-58, 282-336	133	347
	phosphotransferase -	66-74,	150-214,
	Psychrobacter	78-87, 92-99,	216-345
	cryohalolentis	101-109,
	(strain K5)	120-132,
		153-164,
		176-182,
		186-194,
		208-238,
		246-262,
		272-284,
		291-311,
		317-322,
		328-343
MCRH3501	Phospholipase	5-27, 29-42,	1-35, 53-118,	4	22-120	134	348
	D/Transphosphatidylase -	57-71, 73-80,	123-280,
	Psychrobacters	92-99,	283-440,
	(strain	101-107,	442-542
	PRwf-1)	126-136,
		142-151,
		155-163,
		173-179,
		214-232,
		235-254,
		262-274,
		297-309,
		311-323,
		330-358,
		362-379,
		383-394,
		396-416,
		432-457,
		464-472,
		474-485,
		487-498,
		511-519,
		527-536
MCRH3534	ABC	4-15, 27-61,	1-149, 151-408,	4	324-391	135	349
	transporter	64-77, 84-97,	442-594
	related -	102-122,
	Psychrobacters	144-215,
	(strain	223-239,
	PRwf-1)	242-252,
		257-263,
		271-286,
		295-302,
		312-324,
		328-348,
		380-400,
		403-411,
		417-428,
		434-440,
		444-451,
		453-467,
		485-496,
		522-538,
		540-545,
		552-560,
		566-577,
		582-588
MCRH3547	Putative	21-41, 43-123,	1-437	6	84-149,	136	350
	uncharacterized	125-137,			163-176,
	protein	139-157,			260-321
	PM0287 -	171-190,
	Pasteurella	193-203,
	multocida	205-226,
		242-256,
		264-274,
		289-299,
		301-311,
		323-368,
		375-396,
		400-408,
		418-434
MCRH3567	Nucleoside-	4-11, 19-34,	1-65, 81-124,	1	157-215	137	351
	diphosphate-	44-65, 70-115,	127-206,
	sugar	130-140,	215-232
	epimerase-	142-168,
	like protein -	170-176,
	Psychrobacters	185-206,
	(strain	221-227
	PRwf-1)
MCRH3578	DNA gyrase	4-19, 44-51,	1-15, 21-119,	11	77-182	138	352
	subunit A	66-104,	123-213
	(EC 5.99.1.3) -	122-129,
	Psychrobacters	136-141,
	(strain	143-150,
	PRwf-1)	154-159,
		166-173,
		181-205,
		207-218
MCRH3622	L-serine	4-15, 17-34,	1-76, 82-168,	4	234-319	139	353
	dehydratase 1	40-51, 61-71,	179-209,
	(EC 4.3.1.17) -	84-89,	227-249,
	Psychrobacters	137-146,	253-473
	(strain	153-159,
	PRwf-1)	163-170,
		175-191,
		203-210,
		216-228,
		242-250,
		259-271,
		279-284,
		291-329,
		333-372,
		376-394,
		399-404,
		406-412,
		416-424,
		446-451
MCRH3635	Alanine	8-15, 17-26,	1-47, 68-99,	9	135-280	140	354
	racemase (EC	30-45, 47-74,	104-174,
	5.1.1.1) -	80-92,	178-378
	Psychrobacter	94-111,
	cryohalolentis	114-120,
	(strain K5)	124-133,
		148-173,
		175-181,
		183-196,
		198-208,
		212-231,
		238-244,
		248-262,
		266-285,
		290-302,
		308-317,
		327-332,
		334-341,
		346-355,
		357-365,
		373-380
MCRH3705	Na+/H+	12-28, 32-56,	1-224, 229-283,	1	350-406	141	355
	antiporter	61-72,	296-464
	NhaC -	74-89, 95-128,
	Psychrobacter	130-144,
	cryohalolentis	150-163,
	(strain K5)	177-189,
		196-214,
		218-282,
		294-305,
		313-382,
		384-398,
		404-420,
		426-454,
		456-461
MCRH3709	Peptidase	6-17, 22-31,	1-28, 30-82,	47	5-136, 172-260	142	356
	S11, D-	35-63, 67-85,	86-267,
	alanyl-D-	89-101,	271-307,
	alanine	128-147,	310-376
	carboxypeptidase 1	149-155,
	precursor -	161-170,
	Psychrobacters	172-179,
	(strain	187-198,
	PRwf-1)	228-234,
		240-247,
		284-291,
		298-308,
		326-337,
		344-350,
		357-366
MCRH3729	Probable	9-32, 36-52,	1-40, 49-69,	9	297-327,	143	357
	membrane-	72-90, 149-157,	72-92, 97-265,		539-627,
	bound lytic	165-177,	271-369,		712-776
	murein	188-196,	373-452,
	transglycosylase	223-233,	456-476,
	D -	237-245,	496-527,
	Psychrobacter	264-273,	535-555,
	arcticum	280-293,	567-637,
		298-307,	652-819
		309-348,
		352-362,
		366-374,
		381-396,
		405-413,
		416-421,
		428-435,
		438-444,
		449-457,
		464-476,
		491-555,
		579-597,
		602-628,
		646-652,
		663-670,
		674-692,
		739-747,
		762-771,
		779-789,
		809-816
MCRH3742	Putative DNA	5-16, 21-27,	1-15, 53-86,	6	107-184,	144	358
	repair protein	39-46, 60-77,	89-225,		256-311
	radA -	92-104,	237-320,
	Psychrobacter	112-118,	331-398,
	arcticum	129-136,	406-467
		141-164,
		167-176,
		178-214,
		220-228,
		234-251,
		284-301,
		303-310,
		315-328,
		336-353,
		362-375,
		380-390,
		393-409,
		411-417,
		430-438,
		448-459,
		461-468
MCRH3759	—	13-34, 36-42,	1-38, 41-140	3	4-89	145	359
		49-62,
		86-92, 124-130,
		132-145
MCRH3844	Putative	16-22, 45-51,	1-34, 79-113,	13	108-194,	146	360
	uncharacterized	80-85,	129-325,		370-410,
	protein -	88-108,	327-360,		411-442,
	Mannheimia	115-127,	363-383,		956-1046,
	succiniciproducens	139-157,	385-514,		1055-1236
	(strain	160-176,	530-565,
	MBEL55E)	184-189,	577-701,
		198-204,	704-941,
		227-234,	944-981,
		249-270,	985-1005,
		277-295,	1012-1032,
		314-325,	1049-1088,
		331-337,	1097-1183,
		344-367,	1188-1320
		397-424,
		432-500,
		512-521,
		524-530,
		555-563,
		569-583,
		586-617,
		622-653,
		661-673,
		679-696,
		710-738,
		754-759,
		764-786,
		793-808,
		813-820,
		831-837,
		844-850,
		864-889,
		891-899,
		912-926,
		961-970,
		975-982,
		1016-1024,
		1027-1034,
		1051-1059,
		1096-1102,
		1134-1147,
		1158-1177,
		1179-1185,
		1188-1215,
		1235-1250,
		1271-1284,
		1308-1315
MCRH3884	Putative	20-32, 67-73,	7-94, 98-116,	5	113-132,	147	361
	uncharacterized	84-91,	121-196,		167-245
	protein -	102-108,	205-224,
	Mannheimia	148-163,	240-254
	haemolytica	198-209,
	PHL213	232-237
MCRH3895	Transcriptional	7-16, 36-43,	1-77, 97-156,	23	92-164	148	362
	regulator -	69-77, 101-114,	172-215,
	Pseudomonas	136-150,	221-240
	phage F116	155-161,
		183-195,
		197-206,
		210-218,
		225-237
MCRH3907	Putative	17-23, 52-62,	2-35, 81-179,	14	2-126, 245-300	149	363
	uncharacterized	75-87,	181-264,
	protein -	107-112,	267-288,
	Psychrobacters	120-135,	292-376
	(strain	140-146,
	PRwf-1)	170-176,
		188-206,
		210-220,
		228-255,
		273-282,
		304-314,
		324-331,
		344-355,
		363-370
MCRH3939	Possible	4-9, 11-17,	1-113, 117-183,	15	63-136,	150	364
	membrane-	24-85, 89-103,	186-275,		248-305,
	bound lytic	114-119,	294-362,		458-466
	murein	126-135,	370-448,
	transglycosylase -	151-158,	468-485
	Psychrobacter	169-175,
	arcticum	191-203,
		226-237,
		247-256,
		261-266,
		278-283,
		306-316,
		321-331,
		333-340,
		350-358,
		361-392,
		397-408,
		412-419
MCRH3966	Probable	4-23, 41-47,	1-39, 43-79,	5	132-199,	151	365
	Insulinase-	52-59, 62-75,	88-160,		214-286
	like	86-92,	165-236,
	peptidase,	98-104,	241-426,
	family M16 -	106-112,	434-470
	Psychrobacter	126-143,
	arcticum	152-157,
		160-166,
		183-192,
		196-201,
		207-214,
		227-235,
		238-245,
		248-265,
		276-285,
		304-314,
		324-342,
		350-356,
		360-375,
		390-403,
		411-435,
		437-446,
		453-461
MCRH3969	Probable	8-31, 35-48,	1-331, 352-386,	2	80-156,	152	366
	Insulinase-	75-98, 100-106,	404-488		237-303
	like	128-134,
	peptidase,	151-166,
	family M16 -	168-177,
	Psychrobacter	202-223,
	arcticum	252-257,
		265-277,
		288-302,
		306-316,
		371-377,
		385-396,
		408-413,
		429-445,
		472-480
MCRH3996	KpsF/GutQ	4-10, 22-32,	1-203, 205-339	8	31-111,	153	367
	family protein	39-46, 52-69,			205-228
	(EC 5.3.1.13) -	90-96,
	Psychrobacters	104-117,
	(strain	126-142,
	PRwf-1)	148-161,
		163-174,
		178-195,
		200-210,
		216-222,
		225-236,
		238-246,
		248-265,
		279-285,
		313-322,
		324-336
MCRH4056	Pyruvate	6-26, 33-63,	16-83, 126-172,	14	81-97, 197-371,	154	368
	dehydrogenase	66-74, 125-148,	175-210,		470-500
	complex	153-185,	245-325,
	dihydrolipoamide	225-230,	329-448,
	acetyltransferas	234-250,	469-557
	(EC	253-278,
	2.3.1.61) -	286-299,
	Psychrobacters	312-319,
	(strain	335-346,
	PRwf-1)	348-361,
		383-401,
		414-419,
		423-430,
		432-440,
		447-454,
		470-478,
		488-508,
		519-533,
		537-554
MCRH4147	SSU	4-9, 11-32,	1-73, 112-209,	11	495-559	155	369
	ribosomal	44-56, 68-74,	231-294,
	protein S1P -	82-98,	296-314,
	Psychrobacters	104-109,	326-381,
	(strain	131-140,	388-414,
	PRwf-1)	144-164,	423-457,
		176-182,	462-484,
		188-194,	495-514,
		206-212,	522-544,
		219-225,	547-568
		228-235,
		237-244,
		254-271,
		277-287,
		294-302,
		312-318,
		324-331,
		341-350,
		352-358,
		399-405,
		411-419,
		438-447,
		452-459,
		465-470,
		475-484,
		486-506,
		514-523,
		525-532,
		537-543
MCRH4174	ABC	4-26, 29-36,	1-36, 40-114,	7	54-144,	156	370
	transporter	45-52, 57-63,	125-255,		543-627
	related -	68-75,	266-395,
	Psychrobacters	77-88, 93-108,	413-511,
	(strain	114-120,	530-557,
	PRwf-1)	138-143,	564-583,
		148-155,	605-640
		161-179,
		198-211,
		214-224,
		229-242,
		244-253,
		268-274,
		302-311,
		319-331,
		335-349,
		364-374,
		377-384,
		388-394,
		423-436,
		441-446,
		452-471,
		478-501,
		506-514,
		522-546,
		558-579,
		589-604,
		623-631
MCRH4206	Ribonucleoside-	5-10, 24-30,	1-63, 73-369,	5	600-685,	157	371
	diphosphate	37-53, 62-94,	371-428,		741-753
	reductase	101-111,	432-454,
	class Ia alpha	118-125,	457-567,
	subuni (EC	145-151,	573-635,
	1.17.4.1) -	156-162,	655-698,
	Psychrobacters	164-186,	707-744
	(strain	191-198,
	PRwf-1)	204-212,
		224-233,
		244-253,
		263-269,
		277-297,
		304-322,
		334-341,
		355-372,
		379-384,
		393-406,
		412-425,
		427-435,
		437-449,
		461-471,
		483-506,
		515-527,
		535-543,
		546-559,
		562-570,
		576-584,
		588-601,
		640-647,
		651-673,
		681-689,
		712-734
MCRH4291	Chromosome	13-19, 27-38,	1-47, 49-66,	5	14-57, 76-167,	158	372
	segregation	78-87,	70-96, 102-285,		191-245
	DNA-binding	105-129,	287-322,
	protein -	134-142,	330-346
	Psychrobacter	151-179,
	arcticum	184-197,
		206-214,
		225-235,
		246-265,
		274-314,
		322-330,
		334-343
MCRH4304	Putative	15-21, 26-45,	1-64, 73-192,	5	86-169,	159	373
	uncharacterized	49-60,	209-327,		318-384,
	protein -	62-69, 77-87,	329-418,		502-520,
	Psychrobacter	96-104,	421-529,		529-602
	arcticum	108-114,	534-586,
		117-130,	599-646,
		135-144,	650-682,
		149-160,	703-793
		174-181,
		186-193,
		203-218,
		228-234,
		236-242,
		257-264,
		285-326,
		332-342,
		344-349,
		353-370,
		373-425,
		428-437,
		447-457,
		472-483,
		490-505,
		516-526,
		546-554,
		556-570,
		572-585,
		601-609,
		613-624,
		626-640,
		647-663,
		665-671,
		678-689,
		691-697,
		707-719,
		721-751,
		769-778
MCRH4322	S. Adenylosuccinate	4-10, 19-26,	1-37, 41-119,	6	93-182,	160	374
	synthetase	28-34, 41-60,	133-251,		360-391
	(EC 6.3.4.4)	62-81,	255-277,
	(IMP--	89-97, 99-115,	291-355,
	aspartate	143-156,	359-411
	ligase (AdSS)	162-181,
	(AMPSase) -	186-193,
	Psychrobacter	197-207,
	cryohalolentis	225-231,
	(strain K5)	233-240,
		254-266,
		283-296,
		309-323,
		325-338,
		341-352,
		363-373,
		388-413,
		420-426
MCRH4367	L-aspartate	5-37, 48-77,	2-44, 85-108,	9	34-88, 104-164,	161	375
	oxidase (EC	82-89, 91-102,	127-242,		168-227,
	1.4.3.16) -	119-127,	245-259,		235-259,
	Limnobacters	129-138,	271-288,		279-301,
	MED105	145-179,	291-329,		345-377
		186-194,	339-433,
		197-203,	443-457,
		205-212,	468-517
		219-235,
		243-250,
		273-289,
		294-304,
		309-349,
		352-363,
		377-388,
		391-399,
		407-415,
		418-443,
		449-463,
		471-477,
		479-494,
		496-503
MCRH4378	Putative	4-24, 32-39,	15-131,	7	9-96, 181-194	162	376
	nicotinate-	44-51, 60-70,	139-285
	nucleotide	86-96,
	pyrophosphorylase	98-105,
	(EC	108-117,
	2.4.2.19) -	122-138,
	Neisseria	142-156,
	gonorrhoeae	167-181,
	(strain ATCC	192-212,
	700825/FA	214-223,
	1090)	231-240,
		254-278
MCRH4392	Msp75	12-31, 41-47,	9-27, 31-92,	2	72-139	163	377
	(Fragment) -	50-73,	94-134,
	Moraxella	86-99, 103-109,	147-199,
	catarrhalis	141-147,	202-268,
		152-167,	271-291,
		180-195,	303-333,
		201-214,	336-465,
		216-237,	480-498
		240-246,
		251-265,
		275-281,
		289-294,
		301-314,
		320-333,
		342-349,
		351-361,
		367-389,
		402-408,
		426-435,
		459-469
MCRH4469	Molybdate	23-34, 41-52,	17-154,	2	137-270	164	378
	transport	74-86,	183-272
	protein (ABC	92-99, 107-121,
	superfamily,	123-130,
	peri_bind) -	154-159,
	Acinetobacter	162-169,
	baumannii	193-227,
		229-243,
		249-255,
		260-269
MCRH4491	Respiratory	4-29, 81-88,	1-52, 60-99,	4	419-508	165	379
	nitrate	106-128,	102-161,
	reductase beta	177-200,	166-189,
	subunit -	207-214,	203-294,
	Psychrobacters	221-234,	312-429,
	(strain	240-250,	436-468,
	PRwf-1)	255-277,	487-512
		279-285,
		291-324,
		326-347,
		349-362,
		376-392,
		400-412,
		415-423,
		425-440,
		444-449,
		453-460
MCRH4496	Respiratory	4-11, 42-49,	2-25, 30-82,	7	200-260,	166	380
	nitrate	51-68, 91-97,	95-287,		562-610,
	reductase	111-122,	304-370,		815-858,
	alpha subunit	130-142,	444-491,		892-903,
	apoprotein -	156-178,	507-662,		961-1060,
	Psychrobacter	186-230,	677-698,		1065-1073
	arcticum	247-252,	704-746,
		278-297,	763-811,
		303-331,	818-920,
		337-344,	926-970,
		355-360,	989-1127,
		383-391,	1130-1198,
		415-421,	1203-1239
		439-444,
		452-463,
		468-481,
		516-524,
		537-542,
		558-570,
		576-582,
		590-597,
		621-633,
		655-661,
		667-673,
		680-688,
		728-734,
		764-771,
		776-789,
		803-810,
		825-841,
		844-853,
		877-891,
		893-907,
		924-933,
		947-960,
		966-974,
		984-989,
		998-1011,
		1018-1024,
		1028-1034,
		1044-1053,
		1084-1093,
		1104-1110,
		1114-1121,
		1146-1154,
		1166-1173,
		1189-1196,
		1202-1210,
		1212-1218,
		1220-1228,
		1243-1248
MCRH4526	Hemoglobin	25-41, 50-56	1-52, 55-157	15	75-155,	167	381
	utilization	58-70,	189-250		197-242,
	protein	101-111,	269-311		257-412,
	(Fragment) -	118-126,	358-576		457-547,
	Moraxella	138-147,	582-774,		715-788,
	catarrhalis	161-168,	783-837,		874-936
		242-252,	855-924,
		271-291,	930-961
		320-329,
		336-342,
		353-364,
		369-375,
		383-389,
		418-432,
		473-484,
		499-514,
		520-532,
		537-545,
		548-560,
		567-575,
		588-606,
		612-622,
		628-634,
		647-658,
		666-673,
		686-693,
		700-706,
		714-720,
		734-744,
		754-759,
		786-798,
		820-827,
		852-859,
		864-885,
		892-899,
		908-915,
		921-933,
		941-946,
		950-957
MCRH4552	S. UPF0082	29-36, 49-55	1-43, 55-73,	9	20-87	168	382
	protein	92-100,	108-155,
	Psyc_0938 -	109-116,	164-249
	Psychrobacter	127-135,
	arcticum	140-148,
		153-158,
		169-176,
		179-191,
		214-221,
		227-237,
		240-246
MCRH4568	Protein	5-19, 30-41,	1-39, 57-119,	4	15-83	169	383
	tyrosine	44-49, 64-70,	131-166
	phosphatase -	72-80,
	Reinekea	82-88, 95-100,
	sMED297	115-120,
		141-163
MCRH4629	NADH-	18-53, 64-75,	1-110, 114-160,	9	155-173,	170	384
	quinone	91-98,	169-189,		239-280,
	oxidoreductase	103-109,	216-238,		501-575,
	(EC	112-119,	244-364,		689-727,
	1.6.99.5) -	154-167,	378-409,		766-789,
	Psychrobacters	173-181,	422-446,		826-880
	(strain	200-208,	453-475,
	PRwf-1)	226-241,	487-570,
		261-268,	590-640,
		283-289,	643-713,
		294-309,	722-809,
		344-371,	816-952,
		374-382,	969-1017
		388-398,
		416-423,
		425-439,
		442-453,
		456-472,
		491-497,
		502-513,
		515-524,
		527-542,
		547-562,
		599-608,
		610-620,
		623-632,
		636-656,
		666-675,
		683-692,
		697-703,
		709-716,
		747-769,
		778-785,
		792-805,
		829-836,
		863-870,
		877-905,
		913-922,
		937-950,
		952-985,
		991-999
MCRH4641	NADH-	29-35, 40-64,	43-130,	13	19-144,	171	385
	quinone	66-75,	136-211,		219-359,
	oxidoreductase,	115-122,	242-297,		438-452
	F subunit	141-173,	314-333,
	(EC 1.6.99.5) -	179-187,	338-387,
	Psychrobacters	199-206,	396-416,
	(strain	209-215,	422-459
	PRwf-1)	234-240,
		244-251,
		259-272,
		297-303,
		319-325,
		332-348,
		358-368,
		372-382,
		388-397,
		399-405,
		431-439,
		446-454
MCRH4697	Transferrin	4-23, 30-35,	1-28, 74-140,	7	16-72, 526-619,	172	386
	binding	77-84, 92-97,	151-185,		625-694
	protein B -	105-113,	194-218,
	Moraxella	115-120,	225-298,
	catarrhalis	131-137,	300-355,
		140-151,	358-443,
		200-211,	445-463,
		226-239,	468-553,
		241-247,	567-595,
		255-261,	597-621,
		271-278,	631-676,
		304-309,	681-699
		395-400,
		429-435,
		443-453,
		478-493,
		521-530,
		535-553,
		563-568,
		571-581,
		623-628,
		660-667,
		693-700
MCRH4706	Putative	4-18, 26-42,	1-49, 59-247,	7	244-462	173	387
	uncharacterized	58-79, 83-89,	251-321,
	protein -	99-108,	338-404,
	Moraxella	110-118,	410-430,
	catarrhalis	125-140,	438-493
		143-156,
		168-175,
		177-189,
		211-227,
		262-273,
		281-291,
		356-363,
		383-402,
		464-472
MCRH4712	Hypothetical	6-28, 57-65,	1-43, 46-68,	4	13-105	174	388
	periplasmic	72-81, 87-99	83-104
	protein -
	Neisseria
	meningitidis
	serogroup C
	(strain
	053442)
MCRH4716	Transferrin	4-12, 32-55,	1-91, 102-123,	14	219-278,	175	389
	binding	67-81, 92-100,	130-170,		280-417,
	protein A -	119-126,	173-290,		599-696,
	Moraxella	143-151,	307-327,		750-759
	catarrhalis	153-163,	334-354,
		182-188,	359-396,
		201-210,	416-484,
		238-244,	507-563,
		250-256,	567-590,
		278-283,	600-618,
		290-296,	623-648,
		303-308,	661-686,
		312-317,	692-813,
		345-354,	822-887,
		356-367,	905-984,
		378-388,	994-1076,
		423-434,	1079-1093
		442-449,
		458-465,
		476-488,
		494-501,
		510-520,
		530-538,
		551-563,
		570-577,
		582-601,
		628-635,
		700-714,
		719-725,
		744-752,
		761-769,
		778-786,
		790-796,
		821-839,
		851-867,
		872-878,
		920-945,
		957-975,
		1022-1031,
		1038-1047,
		1084-1090
MCRH4730	Methionine-	6-39, 46-61,	1-33, 35-63,	3	151-220	176	390
	R-sulfoxide	67-72, 75-93,	71-209,
	reductase (EC	107-116,	217-237,
	1.8.4.11) -	129-164,	246-260,
	Psychrobacters	169-176,	283-313,
	(strain	181-189,	317-397,
	PRwf-1)	226-243,	399-431,
		245-255,	445-476,
		267-274,	479-558
		277-305,
		319-326,
		342-363,
		375-385,
		390-404,
		419-428,
		439-446,
		448-465,
		478-488,
		507-516,
		524-530,
		546-555
MCRH4740	Lytic	5-14, 25-50,	9-175, 183-302	7	104-209,	177	391
	transglycosylase,	84-91, 99-111,			229-292
	catalytic	119-125,
	precursor -	129-144,
	Psychrobacters	188-197,
	(strain	200-208,
	PRwf-1)	228-234,
		243-250,
		262-269,
		275-282,
		294-299
MCRH4750	RNA	8-27, 29-42,	1-71	1	8-61	178	392
	polymerase	45-62
	sigma factor -
	Psychrobacters
	(strain
	PRwf-1)
MCRH4908	Probable	13-39, 41-47,	3-118, 120-171,	8	261-330,	179	393
	penicillin-	56-75,	183-290,		337-365,
	binding	78-87, 100-107,	293-313,		587-709
	protein	121-127,	316-343,
	transpeptidase	146-157,	345-367,
	domain -	164-171,	373-555,
	Psychrobacter	173-183,	561-651,
	arcticum	201-207,	655-730,
		210-216,	737-773
		221-231,
		244-252,
		255-263,
		281-289,
		291-309,
		348-374,
		390-401,
		424-433,
		435-445,
		458-474,
		509-530,
		534-545,
		548-565,
		582-591,
		597-602,
		605-620,
		622-628,
		663-672,
		686-691,
		699-711,
		728-742,
		745-758,
		773-786
MCRH4917	Acid	4-25, 41-54,	1-30, 37-57,	2	506-517,	180	394
	phosphatase	92-98, 104-111,	64-86, 93-191,		532-590
	autotransporter -	122-131,	193-217,
	Moraxella	144-151,	231-277,
	catarrhalis	154-160,	296-361,
		200-207,	365-394,
		215-233,	398-529,
		243-253,	534-735,
		259-270,	742-785,
		278-297,	788-912
		299-310,
		328-337,
		345-352,
		373-389,
		398-405,
		419-425,
		438-447,
		472-479,
		483-489,
		492-501,
		505-519,
		531-546,
		550-555,
		568-574,
		581-589,
		595-600,
		622-631,
		683-689,
		714-725,
		733-738,
		747-759,
		761-766,
		772-779,
		795-801,
		814-821,
		856-868,
		899-907,
		923-929,
		931-937
MCRH4930	Adhesin -	4-10, 16-25,	1-30, 32-88,	488	60-298,	181	395
	Moraxella	31-59, 66-75,	143-330,		308-571,
	catarrhalis	90-99,	337-359,		616-842,
		102-107,	379-434,		971-1120,
		136-149,	480-583,		1126-1252,
		151-159,	604-715,		1359-1652,
		185-202,	719-736,		1688-1980,
		204-215,	744-761,		2051-2106
		224-237,	768-800,
		240-248,	805-850,
		254-262,	874-889,
		272-278,	898-948,
		310-316,	961-1053,
		337-349,	1058-1110,
		390-396,	1135-1150,
		400-409,	1157-1183,
		420-429,	1189-1230,
		478-484,	1235-1255,
		502-517,	1259-1277,
		533-539,	1308-1377,
		570-584,	1383-1422,
		614-621,	1428-1450,
		625-634,	1453-1472,
		645-654,	1474-1501,
		662-672,	1507-1591,
		677-686,	1596-1660,
		719-733,	1669-1703,
		747-753,	1709-1767,
		762-772,	1788-1848,
		782-792,	1861-1876,
		802-811,	1922-1941,
		820-826,	1944-1979,
		832-841,	1998-2061,
		856-862,	2069-2102,
		912-918,	2115-2136
		931-937,
		964-970,
		989-994,
		1006-1011,
		1017-1023,
		1036-1043,
		1053-1064,
		1071-1077,
		1082-1090,
		1103-1109,
		1128-1135,
		1137-1143,
		1156-1165,
		1169-1174,
		1197-1204,
		1213-1222,
		1255-1273,
		1305-1311,
		1315-1322,
		1340-1346,
		1352-1359,
		1386-1393,
		1403-1412,
		1441-1447,
		1459-1468,
		1470-1479,
		1493-1500,
		1511-1520,
		1525-1537,
		1544-1557,
		1564-1569,
		1580-1585,
		1591-1596,
		1627-1633,
		1642-1656,
		1690-1697,
		1707-1713,
		1715-1721,
		1751-1761,
		1773-1781,
		1791-1796,
		1804-1810,
		1846-1851,
		1860-1870,
		1876-1882,
		1910-1917,
		1947-1953,
		1962-1970,
		2008-2019,
		2026-2036,
		2043-2051,
		2085-2090,
		2105-2115,
		2132-2142
MCRH4983	Heat shock	17-25, 29-38,	1-98, 108-194,	10	16-287	182	396
	protein 70 -	41-60,	205-473,
	Psychrobacter	97-105,	490-510,
	cryohalolentis	111-117,	517-557,
	(strain K5)	124-159,	559-615
		172-181,
		186-192,
		200-207,
		212-229,
		241-257,
		267-298,
		305-314,
		316-322,
		335-341,
		345-355,
		357-364,
		367-375,
		377-382,
		387-400,
		407-413,
		417-428,
		437-443,
		448-454,
		456-463,
		469-477,
		483-489,
		495-508,
		513-524,
		540-551,
		585-602
MCRH5027	Alanyl-tRNA	10-33, 35-41,	1-31, 33-74,	16	34-89, 124-209,	183	397
	synthetase	50-55,	85-118,		227-245,
	(EC 6.1.1.7) -	60-73, 81-90,	138-158,		271-338,
	Psychrobacters	106-114,	165-184,		402-453,
	(strain	121-136,	204-359,		496-596
	PRwf-1)	147-158,	375-402,
		179-191,	408-478,
		212-219,	491-511,
		229-236,	531-640,
		266-275,	653-745,
		285-307,	756-861,
		312-318,	871-889
		334-345,
		351-359,
		361-336,
		373-390,
		398-419,
		450-464,
		475-483,
		495-506,
		524-530,
		534-546,
		555-565,
		572-583,
		596-603,
		623-640,
		658-664,
		675-685,
		690-696,
		702-711,
		714-723,
		742-748,
		765-772,
		783-790,
		811-823,
		835-842,
		848-854,
		874-881
MCRH5073	Flavin	19-31, 39-48,	1-32, 34-74,	7	119-185,	184	398
	reductase (EC	50-61,	93-115,		241-349
	1.5.1.30) -	70-76, 85-96,	134-190,
	Mannheimia	98-106,	192-255,
	haemolytica	114-127,	266-339
	PHL213	129-159,
		166-184,
		195-210,
		215-224,
		234-240,
		247-254,
		259-265,
		267-284,
		286-295,
		297-307,
		310-318,
		320-330,
		340-347
MCRH5107	Putative	16-31, 45-52,	1-62, 85-154,	5	188-255,	185	399
	uncharacterized	62-68,	172-222,		319-334
	protein	89-149,	224-248,
	precursor -	154-164,	252-328
	Psychrobacters	175-185,
	(strain	187-193,
	PRwf-1)	195-207,
		222-230,
		253-279,
		287-295,
		299-323
MCRH5149	Surface	7-15, 38-65,	1-105, 115-293,	1	23-98	186	400
	antigen (D15)	67-76, 80-103,	300-342,
	precursor -	123-139,	344-418,
	Psychrobacters	146-152,	432-562,
	(strain	181-188,	567-598,
	PRwf-1)	196-206,	613-656,
		223-229,	670-713,
		234-245,	757-803
		259-267,
		285-296,
		323-333,
		340-358,
		387-393,
		401-409,
		417-427,
		431-439,
		441-450,
		472-477,
		483-496,
		508-524,
		591-598,
		602-610,
		616-624,
		627-641,
		655-664,
		666-679,
		687-694,
		696-709,
		714-726,
		759-766,
		768-778,
		782-793,
		804-810
MCRH5176	S. Uridylate	9-18, 35-57,	2-174, 176-241	2	26-112	187	401
	kinase (EC	65-74, 82-87,
	2.7.4.22)	92-98,
	(UK)	105-117,
	(Uridine	128-136,
	monophosphate	147-154,
	kinase	157-165,
	(UMP kinase)	181-187,
	(UMPK) -	189-218,
	Psychrobacter	224-232
	cryohalolentis
	(strain K5)
MCRH5178	Transketolase	4-13, 24-35,	1-40, 46-117,	4	13-84, 395-470	188	402
	(EC 2.2.1.1) -	49-56, 71-77,	127-171,
	Acinetobacter	81-98,	186-201,
	baumannii	103-109,	203-318,
		134-150,	332-352,
		158-169,	356-390,
		176-186,	411-543,
		188-195,	560-669
		222-229,
		249-262,
		273-282,
		335-341,
		358-366,
		375-395,
		415-423,
		431-448,
		468-477,
		486-492,
		500-517,
		526-539,
		553-558,
		563-571,
		573-579,
		581-603,
		606-641,
		649-657,
		659-669
MCRH5224	Valyl-tRNA	43-56, 95-108,	1-91, 93-195,	12	30-60, 107-144,	189	403
	synthetase -	139-145,	199-284,		235-255,
	Psychrobacter	158-178,	324-357,		439-492,
	cryohalolentis	186-199,	385-405,		620-673,
	(strain K5)	204-216,	409-499,		767-814,
		224-231,	506-604,		864-969
		234-247,	621-678,
		251-275,	688-749,
		282-290,	753-806,
		303-309,	815-882,
		312-329,	887-927,
		331-341,	933-953
		349-355,
		379-405,
		410-416,
		429-436,
		452-457,
		463-474,
		476-483,
		485-491,
		507-531,
		546-563,
		573-585,
		628-635,
		663-669,
		687-700,
		718-725,
		731-742,
		758-769,
		788-795,
		800-811,
		824-831,
		841-852,
		859-865,
		869-881,
		885-891,
		896-904,
		941-951,
		955-972
MCRH5239	Putative	6-31, 59-73,	6-51, 54-151,	2	319-382	190	404
	uncharacterized	76-87, 98-104,	161-194,
	protein	107-118,	203-357,
	precursor -	126-145,	361-474,
	Psychrobacters	156-165,	477-497,
	(strain	168-177,	501-538,
	PRwf-1)	185-193,	541-615
		206-214,
		217-237,
		245-252,
		256-276,
		288-304,
		314-331,
		333-340,
		366-378,
		387-406,
		409-423,
		428-437,
		457-463,
		480-488,
		509-519,
		536-542,
		550-563,
		571-593,
		606-612
MCRH5260	Putative	6-12, 17-31,	1-53	8	2-60	191	405
	amino-acid	33-48
	transport
	protein -
	Neisseria
	gonorrhoeae
	(strain ATCC
	700825/FA
	1090)
MCRH5275	Isoleucyl-	54-61, 64-86,	2-97, 111-196,	5	29-54, 261-267,	192	406
	tRNA	91-109,	209-321,		635-691
	synthetase	111-120,	327-349,
	(EC 6.1.1.5) -	127-133,	353-367,
	Psychrobacters	135-143,	377-408,
	(strain	147-152,	418-491,
	PRwf-1)	166-176,	500-664,
		179-186,	691-772,
		189-207,	788-1019,
		212-220,	1024-1049,
		226-231,	1054-1077,
		235-242,	1079-1095
		250-267,
		271-279,
		294-308,
		316-336,
		347-353,
		362-368,
		380-389,
		391-398,
		406-418,
		441-463,
		471-500,
		503-522,
		524-560,
		562-570,
		572-582,
		584-590,
		634-639,
		643-652,
		657-663,
		666-673,
		697-707,
		714-721,
		727-734,
		743-751,
		758-776,
		796-806,
		852-867,
		870-896,
		898-905,
		923-945,
		948-955,
		957-962,
		964-969,
		982-988,
		1000-1006,
		1008-1014,
		1030-1042,
		1056-1068,
		1075-1086
MCRH5294	Major outer	4-16, 18-41,	1-139, 145-180,	34	118-193,	193	407
	membrane	47-54, 106-113,	186-261,		209-355,
	protein -	120-131,	271-291,		505-657
	Moraxella	144-150,	308-328,
	catarrhalis	165-171,	332-365,
		192-202,	372-394,
		220-227,	400-440,
		229-239,	453-530,
		245-259,	533-585,
		314-327,	588-631,
		358-365,	639-687,
		377-384,	695-746
		396-402,
		414-430,
		436-442,
		457-466,
		474-480,
		498-519,
		524-531,
		535-541,
		564-569,
		581-597,
		622-628,
		653-676,
		681-686,
		719-727
MCRH5308	Putative	6-27, 50-56,	1-44, 46-115,	2	161-212,	194	408
	thiol:disulfide	62-72, 80-86,	132-192,		218-247
	interchange	98-111,	222-242,
	protein	118-127,	246-264
	precursor -	138-168,
	Psychrobacter	176-184,
	cryohalolentis	189-201,
	(strain K5)	205-245,
		256-263
MCRH5342	RND efflux	6-28, 32-41,	1-49, 51-166,	6	5-82, 101-118,	195	409
	system, outer	46-66, 74-84,	170-203,		260-376
	membrane	92-108,	208-368,
	lipoprotein,	110-117,	382-486,
	NodT -	137-146,	489-507
	Psychrobacter	152-170,
	cryohalolentis	173-200,
	(strain K5)	226-264,
		266-275,
		284-301,
		308-313,
		316-335,
		355-364,
		374-388,
		394-404,
		406-416,
		424-431,
		437-452,
		456-477
MCRH5355	Secretion	4-13, 16-26,	1-67, 73-274,	8	119-189,	196	410
	protein HlyD	28-34, 39-45,	288-429,		200-246,
	precursor -	47-63,	434-465		399-436
	Psychrobacter	68-121,
	cryohalolentis	130-145,
	(strain K5)	150-162,
		165-171,
		176-203,
		215-240,
		242-249,
		251-261,
		282-290,
		293-309,
		316-322,
		334-352,
		360-367,
		397-410,
		412-422,
		430-439
MCRH5441	Putative	4-12, 15-22,	1-42, 57-82,	3	30-110	197	411
	uncharacterized	25-34, 36-47,	87-123,
	protein -	54-79,	127-498
	Psychrobacter	92-99, 106-114,
	arcticum	129-136,
		150-160,
		168-184,
		186-215,
		222-229,
		239-249,
		286-293,
		299-314,
		322-327,
		333-358,
		371-378,
		387-406,
		413-422,
		435-443,
		448-455,
		461-471,
		474-480,
		489-495
MCRH5451	Alcohol	4-29, 42-68,	1-52, 57-80	4	15-86	198	412
	dehydrogenase	70-79
	class III/S-
	(Hydroxymethyl)glutathion
	dehydrogenase -
	Actinobacillus
	succinogenes
	(strain ATCC
	55618/
	130Z)
MCRH5471	Probable	8-51, 78-86,	1-129, 131-247,	5	187-241,	199	413
	penicillin-	94-99, 107-113,	263-335,		319-394,
	binding	140-148,	343-364,		411-485,
	protein 1A -	156-162,	370-639,		657-679
	Psychrobacter	173-181,	652-697,
	arcticum	189-197,	712-779
		213-229,
		264-270,
		274-281,
		283-293,
		308-321,
		347-360,
		399-411,
		425-436,
		442-449,
		466-482,
		486-498,
		516-521,
		527-534,
		557-565,
		586-594,
		604-617,
		622-630,
		652-674,
		720-731,
		742-754,
		773-783
MCRH5547	Phosphoribosylaminoimidazole	7-14, 36-46,	16-67, 69-91,	2	48-105	200	414
	carboxylase,	48-57, 74-81,	96-134,
	catalytic	85-108,	137-177
	subuni (EC	113-131,
	4.1.1.21) -	141-154,
	Psychrobacters	168-179
	(strain
	PRwf-1)
MCRH5559	Putative	4-20, 28-33,	1-85, 92-148	9	15-142	201	415
	uncharacterized	39-55, 57-65,
	protein -	68-82,
	Moraxella	84-92, 117-123
	nonliquefaciens
MCRH5574	Dihydroorotase	4-9, 17-23,	1-18, 37-57,	5	7-100, 152-243	202	416
	(EC	37-53, 71-89,	68-278,
	3.5.2.3) -	104-116,	306-400
	Psychrobacters	123-128,
	(strain	133-138,
	PRwf-1)	145-170,
		173-205,
		213-232,
		234-252,
		260-268,
		275-280,
		287-311,
		316-345,
		357-364
MCRH5581	Sel1 domain	5-19, 38-45,	1-27, 44-147,	2	22-73, 224-290	203	417
	protein	77-83, 89-99,	159-255,
	repeat-	108-119,	262-280,
	containing	125-132,	288-302
	protein -	144-153,
	Candidatus	161-171,
	Amoebophilus	180-191,
	asiaticus	221-227,
	5a2	233-243,
		274-286,
		293-299
MCRH5639	Aconitate	8-15, 17-39,	2-17, 28-146,	43	157-185,	204	418
	hydratase 2	48-55, 57-77,	152-170,		341-483
	(EC 4.2.1.3) -	87-95,	198-214,
	Acinetobacter	99-109,	235-270,
	baumannii	119-132,	279-326,
		146-152,	333-368,
		171-176,	381-407,
		196-203,	435-608,
		220-229,	624-650,
		231-245,	656-673,
		253-259,	711-779,
		273-285,	782-799,
		293-300,	809-872
		318-324,
		331-344,
		367-374,
		395-401,
		409-414,
		434-471,
		483-490,
		509-517,
		519-534,
		537-544,
		554-574,
		584-590,
		599-609,
		615-625,
		632-639,
		665-672,
		676-686,
		688-697,
		702-708,
		714-720,
		730-742,
		744-752,
		780-786,
		790-800,
		812-836
MCRH5640	Aconitate	4-57, 60-67,	5-114	5	38-112	205	419
	hydratase 2	80-96, 98-118
	(EC 4.2.1.3) -
	Acinetobacter
	baumannii
MCRH5644	Aconitate	11-34, 43-51,	9-30, 40-168	12	13-78	206	420
	hydratase 2	67-108,
	(EC 4.2.1.3) -	116-138,
	Acinetobacter	151-165
	baumannii
MCRH5729	S. UvrABC	18-24, 40-47,	7-67, 71-131,	3	504-552	207	421
	system	51-62,	134-231,
	protein C	67-74, 87-94,	233-260,
	(Protein	102-109,	276-340,
	uvrC)	112-121,	342-385,
	(Excinuclease	141-147,	388-443,
	ABC subunit	152-164,	447-475,
	C) -	175-198,	509-616
	Psychrobacter	200-224,
	cryohalolentis	229-235,
	(strain K5)	245-260,
		262-293,
		310-323,
		326-349,
		390-414,
		423-431,
		440-447,
		456-462,
		466-476,
		481-493,
		497-504,
		512-538,
		545-550,
		560-573,
		584-591,
		596-602,
		607-613
MCRH5753	Aminobutyrate	11-17, 38-49,	7-106	4	16-107	208	422
	aminotransferase -	52-58,
	Moraxella	67-79, 98-104
	catarrhalis
MCRH5785	Putative	5-17, 32-46,	3-38, 55-69,	4	22-96, 124-131,	209	423
	uncharacterized	54-60, 89-102,	72-135,		143-172,
	protein -	112-127,	165-301		209-275
	Neisseria	152-161,
	gonorrhoeae	169-178,
	(strain ATCC	186-193,
	700825/FA	228-240,
	1090)	247-256,
		259-266,
		274-296
MCRH5787	Replication	17-26, 32-44	1-43	7	28-49	210	424
	restart DNA
	helicase PriA -
	Psychrobacter
	arcticum
MCRH5838	Ubiquitous	11-17, 26-32,	73-125,	83	6-428, 493-608	211	425
	surface	72-77,	133-152,
	protein A2H -	162-168,	163-246,
	Moraxella	204-211,	264-330,
	catarrhalis	218-224,	337-376,
		234-243,	594-635,
		270-276,	660-680,
		287-301,	693-711,
		314-333,	715-816
		339-345,
		360-375,
		392-398,
		403-409,
		419-434,
		438-444,
		454-468,
		473-479,
		489-503,
		508-514,
		524-539,
		543-549,
		559-574,
		578-584,
		607-616,
		618-628,
		664-669,
		710-715,
		718-730,
		734-740,
		747-763,
		766-772,
		774-785,
		787-796
MCRH5853	Putative	14-29, 32-58,	1-58, 67-95,	6	127-190,	212	426
	isocitrate	63-76,	123-168,		537-604,
	dehydrogenase	78-86, 89-100,	176-240,		697-727
	(EC	102-110,	242-284,
	1.1.1.42) -	131-140,	287-317,
	Neisseria	148-154,	325-390,
	meningitidis	188-201,	398-418,
	serogroup C/	210-219,	423-499,
	serotype 2a	230-243,	510-582,
	(strain ATCC	245-254,	598-631,
	700532-	262-281,	635-683,
	FAM18)	298-307,	692-734
		316-324,
		340-352,
		373-396,
		407-414,
		440-453,
		456-477,
		480-487,
		495-508,
		515-529,
		537-544,
		552-560,
		564-572,
		587-592,
		605-619,
		625-632,
		638-644,
		660-674,
		684-690,
		697-719,
		732-738
MCRH5872	Alpha/beta	13-27, 38-44,	1-106, 125-251,	6	42-51, 97-267	213	427
	hydrolase	48-55,	257-306,
	fold -	57-69, 72-81,	310-356
	Psychrobacter	97-108,
	cryohalolentis	126-141,
	(strain K5)	152-160,
		165-177,
		180-186,
		197-241,
		289-296,
		309-328,
		334-339,
		341-353
MCRH5882	Probable	4-10, 12-21,	1-124	1	9-86	214	428
	HesA/MoeB/	29-35, 44-56,
	ThiF family	59-67,
	protein -	74-86, 91-98,
	Psychrobacter	103-114
	arcticum
The 214 antigenic ORFs were identified by screening bacterial libraries displaying the fragmented genome of Mcat RH4 with human immunoglobulin.
aFrom the whole genome sequence of Mcat RH4, determined by Intercell AG (Vienna, Austria).
bPrediction of sequences longer than 5 amino acids capable of inducing an antibody response was performed with the program ANTIGENIC (Kolaskar, A. S. and Tongaonkar, P. C., 1990).
cPrediction of sequences capable of inducing a human class II-restricted T cell response was performed with the program “MHC-II binding predictions” from IEDB Analysis (http://www.immuneepitope.org/). The prediction was performed for the eleven human MHC II types HLADRB1*0101, *0301, *0401, *0404, *0405, *0701, *0802, *1101, *1302, *1501 and HLADRB5*0101 with a consensus approach of three methods (ARB, SMM_align, Sturniolo). Epitopes with a consensus percentile up to 5 in any of the MHC II types were mapped (Zhang et al., 2008; Wang et al., 2008).
dNumber of clones selected by human immunoglobulin from bacterial display libraries matching the ORF.
eLocation of immunogenic region, based on sequence alignment with the clones selected by human immunoglobulin from bacterial display libraries matching the ORF.
fSequence identification numbers for nucleotide (SEQ ID NOs 1 to 214) and protein sequences (SEQ ID NOs 215 to 428).

TABLE 5
List of 47 Mcat isolates used for gene
distribution (PCR genotyping) analysis.
Internal	Internal
Intercell	Intercell	Supplier		Geograph-
reference	reference	isolate		ical
number A	number B	number	Species	origin
RH4	RH4	Not	M. catarrhalis
		applicable
45	I, E9	2554135	M. catarrhalis	Spain
39	I, E3	2560117	M. catarrhalis	Portugal
2	I, A2	1090124	M. catarrhalis	Australia
4	I, A4	1090127	M. catarrhalis	Australia
5	I, A5	1091216	M. catarrhalis	Australia
40	I, E4	3681122	M. catarrhalis	South Africa
33	I, D6	2084130	M. catarrhalis	Japan
34	I, D7	2085119	M. catarrhalis	Japan
35	I, D8	3079119	M. catarrhalis	Japan
41	I, E5	1070122	M. catarrhalis	South Korea
42	I, E6	1071124	M. catarrhalis	South Korea
43	I, E7	2070120	M. catarrhalis	South Korea
56	I, G2	3696117	M. catarrhalis	Taiwan
57	I, G3	3696119	M. catarrhalis	Taiwan
58	I, G4	3696126	M. catarrhalis	Taiwan
62	I, G8	2660116	M. catarrhalis	Turkey
63	I, G9	2660119	M. catarrhalis	Turkey
65	I, H2	2660124	M. catarrhalis	Turkey
6	I, A6	1510233	M. catarrhalis	Belgium
16	I, B7	1502130	M. catarrhalis	France
17	I, B8	3502122	M. catarrhalis	France
20	I, C2	3502129	M. catarrhalis	France
21	I, C3	3517132	M. catarrhalis	Germany
22	I, C4	3518116	M. catarrhalis	Germany
23	I, C5	3519121	M. catarrhalis	Germany
25	I, C7	3522120	M. catarrhalis	Germany
27	I, C9	3650122	M. catarrhalis	Hungary
28	I, D1	3650134	M. catarrhalis	Hungary
29	I, D2	1530120	M. catarrhalis	Italy
30	I, D3	2530126	M. catarrhalis	Italy
31	I, D4	3530121	M. catarrhalis	Italy
46	I, F1	3552130	M. catarrhalis	Spain
47	I, F2	3553117	M. catarrhalis	Spain
51	I, F6	3590123	M. catarrhalis	Sweden
52	I, F7	3590127	M. catarrhalis	Sweden
54	I, F9	3590135	M. catarrhalis	Sweden
7	I, A7	1041218	M. catarrhalis	Brazil
8	I, A8	3041116	M. catarrhalis	Brazil
9	I, A9	3041117	M. catarrhalis	Brazil
12	I, B3	1023257	M. catarrhalis	Canada
13	I, B4	2022135	M. catarrhalis	Canada
14	I, B5	3020116	M. catarrhalis	Canada
66	I, H3	1001118	M. catarrhalis	United States
67	I, H4	1001207	M. catarrhalis	United States
68	I, H5	1009124	M. catarrhalis	United States
69	I, H6	1009125	M. catarrhalis	United States
The Mcat strain RH4 also used to construct the libraries was isolated from the blood of an infected patient (Christensen et al., 1994) and obtained from Arne Forsgren (Malmö University Hospital, Sweden). Other strains were obtained from GR Micro.

TABLE 6
Conservation of 214 open reading frames coding for antigenic proteins in 47 Mcat isolates
			Positives
SEQ ID NOs			of 47
(nt, aa)	MC ORF	Common Name	tested
1, 215	MCRH0005	Putative uncharacterized protein - Psychrobacter s(strain PRwf-1)	47
2, 216	MCRH0052	S. UDP-N-acetylmuramate--L-alanine ligase (EC 6.3.2.8) (UDP-N	47
		acetylmuramoyl-L-alanine synthetase) - Psychrobacter arcticum
3, 217	MCRH0079	SSU ribosomal protein S2P - Psychrobacter cryohalolentis (strain K5)	47
4, 218	MCRH0082	SMC protein (Fragment) - Terfezia boudieri	47
5, 219	MCRH0103	C-terminal processing peptidase-1. Serine peptidase. MEROPS famil	45
		S41A - Psychrobacter arcticum
6, 220	MCRH0125	S. DNA-directed RNA polymerase subunit beta′ (EC 2.7.7.6) (RNAP	47
		subuni beta′) (Transcriptase subunit beta′) (RNA polymerase subunit
		beta′) - Psychrobacter s(strain PRwf-1)
7, 221	MCRH0139	S. DNA-directed RNA polymerase subunit beta (EC 2.7.7.6) (RNAP	47
		subuni beta) (Transcriptase subunit beta) (RNA polymerase subunit beta) -
		Psychrobacter arcticum
8, 222	MCRH0176	—	47
9, 223	MCRH0211	S. Shikimate dehydrogenase (EC 1.1.1.25) - Psychrobacter cryohalolentis	47
		(strain K5)
10, 224	MCRH0249	Anthranilate synthase, component I (EC 4.1.3.27) - Psychrobacter	47
		cryohalolentis (strain K5)
11, 225	MCRH0267	Putative uncharacterized protein - Moraxella catarrhalis	47
12, 226	MCRH0268	Lactoferrin binding protein A - Moraxella catarrhalis	46
13, 227	MCRH0278	Lactoferrin binding protein B - Moraxella catarrhalis	29
14, 228	MCRH0342	Putative uncharacterized protein - Psychrobacter arcticum	41
15, 229	MCRH0357	Lytic murein transglycosylase precursor - Psychrobacter s(strain PRwf-1)	47
16, 230	MCRH0372	Electron transfer flavoprotein-ubiquinone oxidoreductase (EC 1.5.5.1) -	47
		Psychrobacter arcticum
17, 231	MCRH0391	Putative uncharacterized protein - Psychrobacter arcticum	46
18, 232	MCRH0397	Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) - Psychrobacter	46
		s(strain PRwf-1)
19, 233	MCRH0432	—	47
20, 234	MCRH0441	Excinuclease ABC subunit A - Psychrobacter arcticum	47
21, 235	MCRH0448	Nicotinate-nucleotide adenylyltransferase (EC 2.7.7.18) - Psychrobacter	47
		arcticum
22, 236	MCRH0464	8-amino-7-oxononanoate synthase (EC 2.3.1.47) - Acinetobacter s(strain	46
		ADP1)
23, 237	MCRH0477	Multi-sensor hybrid histidine kinase precursor - Psychrobacter s(strain	47
		PRwf-1)
24, 238	MCRH0498	Possible ATP-dependent DEAD/DEAH box RNA-helicase -	47
		Psychrobacter arcticum
25, 239	MCRH0512	DNA-(Apurinic or apyrimidinic site) lyase/Formamidopyrimidine-DN	47
		glycosylase (EC 4.2.99.18) (EC 3.2.2.23) - Psychrobacter cryohalolentis
		(strain K5)
26, 240	MCRH0525	Phenylalanyl-tRNA synthetase beta subunit (EC 6.1.1.20) -	47
		Psychrobacter cryohalolentis (strain K5)
27, 241	MCRH0546	Putative uncharacterized protein - Pseudoalteromonas haloplanktis (strain	47
		TAC 125)
28, 242	MCRH0558	Nitric oxide reductase large subunit-like protein - Psychrobacter s(strain	47
		PRwf-1)
29, 243	MCRH0580	Ferredoxin-dependent glutamate synthase - Psychrobacter s(strain PRwf-1)	46
30, 244	MCRH0587	Putative DNA helicase - Psychrobacter cryohalolentis (strain K5)	47
31, 245	MCRH0594	Dihydrouridine synthase, DuS - Psychrobacter s(strain PRwf-1)	47
32, 246	MCRH0621	Phosphoribosylformylglycinamidine synthase (EC 6.3.5.3) -	47
		Psychrobacter s(strain PRwf-1)
33, 247	MCRH0655	Acriflavin resistance protein - Psychrobacter s(strain PRwf-1)	47
34, 248	MCRH0673	ATP-dependent DNA helicase RecG (EC 3.6.1.—) - Psychrobacter s(strain	47
		PRwf-1)
35, 249	MCRH0682	Sulfate adenylate transferase subunit 1 - Actinobacillus	47
		pleuropneumoniae serotype 3 (strain JL03)
36, 250	MCRH0704	S. 3-ketoacyl-CoA thiolase (EC 2.3.1.16) (Fatty acid oxidation comple	47
		subunit beta) (Beta-ketothiolase) (Acetyl-CoA acyltransferase) -
		Psychrobacter s(strain PRwf-1)
37, 251	MCRH0744	Prolyl endopeptidase - Vibrio parahaemolyticus	43
38, 252	MCRH0756	—	47
39, 253	MCRH0759	TonB-dependent receptor precursor - Haemophilus somnus (strain 2336)	44
		(Histophilus somni (strain 2336))
40, 254	MCRH0780	2-oxoglutarate dehydrogenase E1 component (EC 1.2.4.2) -	47
		Psychrobacter cryohalolentis (strain K5)
41, 255	MCRH0790	Succinate dehydrogenase subunit A (EC 1.3.5.1) - Psychrobacter s(strain	47
		PRwf-1)
42, 256	MCRH0808	Putative uncharacterized protein - Mannheimia haemolytica PHL213	47
43, 257	MCRH0815	RNA binding S1 domain protein - Psychrobacter s(strain PRwf-1)	34
44, 258	MCRH0853	Putative uncharacterized protein (EC 5.3.4.1) - Psychrobacter arcticum	47
45, 259	MCRH0867	Dihydrodipicolinate reductase (EC 1.3.1.26) - Psychrobacter s(strain	47
		PRwf-1)
46, 260	MCRH0911	Glutamate--cysteine ligase (EC 6.3.2.2) - Psychrobacter arcticum	42
47, 261	MCRH0917	Putative UDP-N-acetylmuramate: L-alanyl-gamma-D-glutamyl-meso	46
		diaminopimelate ligase - Psychrobacter arcticum
48, 262	MCRH1023	Putative uncharacterized protein - Acinetobacter baumannii	47
49, 263	MCRH1076	L-threonine synthase (EC 4.2.3.1) - Psychrobacter s(strain PRwf-1)	43
50, 264	MCRH1163	Putative uncharacterized protein - Deinococcus radiodurans	46
51, 265	MCRH1198	DNA repair protein RecN - Psychrobacter cryohalolentis (strain K5)	47
52, 266	MCRH1227	MFS cyanate efflux pump, CynX - Psychrobacter arcticum	47
53, 267	MCRH1256	Putative ComEA protein-related protein - Candidatus Cloacamonas	47
		acidaminovorans
54, 268	MCRH1257	Poly(A) polymerase - Psychrobacter s(strain PRwf-1)	47
55, 269	MCRH1303	Putative hemolysin-related protein - Acinetobacter baumannii	47
56, 270	MCRH1397	Thiamin-monophosphate kinase (EC 2.7.4.16) - Acinetobacter s(strain	46
		ADP1)
57, 271	MCRH1430	S-adenosyl-methyltransferase MraW - Psychrobacter s(strain PRwf-1)	33
58, 272	MCRH1438	Putative uncharacterized protein - Psychrobacter cryohalolentis (strain K5)	47
59, 273	MCRH1440	Peptidoglycan synthetase FtsI (EC 2.4.1.129) - Psychrobacter s(strain	47
		PRwf-1)
60, 274	MCRH1475	S. Translation initiation factor IF-2 - Psychrobacter s(strain PRwf-1)	45
61, 275	MCRH1486	S. Ribosome-binding factor A - Psychrobacter arcticum	46
62, 276	MCRH1493	OlpA - Moraxella catarrhalis	47
63, 277	MCRH1546	—	46
64, 278	MCRH1561	Sulphate transporter - Psychrobacter s(strain PRwf-1)	44
65, 279	MCRH1565	Probable zinc metallopeptidases, family M48 - Psychrobacter arcticum	47
66, 280	MCRH1568	Phosphate ABC transporter, periplasmic phosphate-binding protein -	38
		Psychrobacter s(strain PRwf-1)
67, 281	MCRH1577	Phosphate ABC transporter, ATPase subunit precursor - Psychrobacter	44
		s(strain PRwf-1)
68, 282	MCRH1586	Ppx/GppA phosphatase (EC 3.6.1.40) - Psychrobacter s(strain PRwf-1)	47
69, 283	MCRH1671	GTP-binding protein Era - Psychrobacter s(strain PRwf-1)	47
70, 284	MCRH1677	Outer membrane protein CD precursor (OmpCD) - Moraxella catarrhalis	47
71, 285	MCRH1696	Extracellular solute-binding protein, family 3 precursor - Psychrobacter	47
		s(strain PRwf-1)
72, 286	MCRH1701	Extracellular solute-binding protein, family 3 precursor - Psychrobacter	44
		s(strain PRwf-1)
73, 287	MCRH1705	ABC transporter related - Psychrobacter s(strain PRwf-1)	46
74, 288	MCRH1720	DNA topoisomerase I (EC 5.99.1.2) - Psychrobacter s(strain PRwf-1)	47
75, 289	MCRH1732	DNA polymerase I (EC 2.7.7.7) - Psychrobacter s(strain PRwf-1)	47
76, 290	MCRH1742	S. Peptide chain release factor 3 (RF-3) - Psychrobacter s(strain PRwf-1)	47
77, 291	MCRH1777	Uncharacterized iron-regulated membrane protein-like protein -	47
		Psychrobacter s(strain PRwf-1)
78, 292	MCRH1841	McmA - Moraxella catarrhalis	47
79, 293	MCRH1853	HI0933 family protein - Psychrobacter s(strain PRwf-1)	47
80, 294	MCRH1878	S. Glycyl-tRNA synthetase alpha subunit (EC 6.1.1.14) (Glycine--tRN	47
		ligase alpha subunit) (GlyRS) - Moraxella catarrhalis
81, 295	MCRH1934	RNAse E (EC 3.1.4.—) - Psychrobacter s(strain PRwf-1)	47
82, 296	MCRH2003	Phospholipid/glycerol acyltransferase precursor - Psychrobacter	47
		cryohalolentis (strain K5)
83, 297	MCRH2082	S. 30S ribosomal protein S11 - Psychrobacter s(strain PRwf-1)	47
84, 298	MCRH2139	UDP-N-acetylmuramoylalanine-D-glutamate ligase (EC 6.3.2.9) -	47
		Acinetobacter baumannii
85, 299	MCRH2151	Probable DNA/RNA non-specific endonuclease (Mg-dependent) -	44
		Psychrobacter arcticum
86, 300	MCRH2184	Mg chelatase, subunit ChlI - Psychrobacter s(strain PRwf-1)	47
87, 301	MCRH2216	Selenocysteine synthase (EC 2.9.1.1) - Haemophilus influenzae (strain	21
		PittEE)
88, 302	MCRH2239	Putative uncharacterized protein - Pseudomonas fluorescens (strain PfO-1)	27
89, 303	MCRH2257	S. 10 kDa chaperonin (Protein Cpn10) (groES protein) - Psychrobacter	47
		s(strain PRwf-1)
90, 304	MCRH2276	Monooxygenase - Neisseria meningitidis serogroup C (strain 053442)	46
91, 305	MCRH2384	S. Threonyl-tRNA synthetase (EC 6.1.1.3) (Threonine--tRNA ligase	47
		(ThrRS) - Psychrobacter s(strain PRwf-1)
92, 306	MCRH2399	—	47
93, 307	MCRH2415	Lytic transglycosylase, catalytic precursor - Psychrobacter s(strain PRwf-1)	47
94, 308	MCRH2484	Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha -	46
		Psychrobacter arcticum
95, 309	MCRH2496	Possible Peptidoglycan-binding LysM - Psychrobacter arcticum	47
96, 310	MCRH2506	Msp22 (Fragment) - Moraxella catarrhalis	47
97, 311	MCRH2519	S. 1-deoxy-D-xylulose-5-phosphate synthase (EC 2.2.1.7) (1-	47
		deoxyxylulose 5-phosphate synthase) (DXP synthase) (DXPS) -
		Psychrobacter cryohalolentis (strain K5)
98, 312	MCRH2553	S. Cysteinyl-tRNA synthetase (EC 6.1.1.16) (Cysteine--tRNA ligase	47
		(CysRS) - Psychrobacter cryohalolentis (strain K5)
99, 313	MCRH2560	Putative cytochrome c, class I precursor - Psychrobacter cryohalolentis	47
		(strain K5)
100, 314	MCRH2592	S. Ribonuclease PH (EC 2.7.7.56) (RNase PH) (tRN	45
		nucleotidyltransferase) - Psychrobacter s(strain PRwf-1)
101, 315	MCRH2606	Outer membrane protein G1b - Moraxella catarrhalis	47
102, 316	MCRH2612	Probable aminopeptidase N (EC 3.4.11.2) - Psychrobacter arcticum	46
103, 317	MCRH2625	Periplasmic protein - Neisseria meningitidis serogroup C (strain 053442)	47
104, 318	MCRH2627	GDSL-like lipase/ccylhydrolase family protein - Pseudomonas	47
		fluorescens (strain Pf-5/ATCC BAA-477)
105, 319	MCRH2641	Putative uncharacterized protein - Psychrobacter cryohalolentis (strain K5)	47
106, 320	MCRH2669	Cytochrome c1 precursor - Psychrobacter s(strain PRwf-1)	47
107, 321	MCRH2682	S. tRNA uridine 5-carboxymethylaminomethyl modification enzyme gid	47
		(Glucose-inhibited division protein A) - Psychrobacter arcticum
108, 322	MCRH2754	Probable Transcription regulator, LysR family - Psychrobacter arcticum	46
109, 323	MCRH2759	4Fe—4S ferredoxin, iron-sulfur binding - Psychrobacter cryohalolentis	47
		(strain K5)
110, 324	MCRH2783	Cytochrome c oxidase cbb3-type, subunit III (EC 1.9.3.1) -	43
		Psychrobacter arcticum
111, 325	MCRH2844	Oligopeptide-binding protein oppA - Streptococcus pyogenes serotype	47
		M2 (strain MGAS10270)
112, 326	MCRH2851	Putative uncharacterized protein - Psychrobacter s(strain PRwf-1)	47
113, 327	MCRH2861	S. Leucyl-tRNA synthetase (EC 6.1.1.4) (Leucine--tRNA ligase)	47
		(LeuRS) - Psychrobacter cryohalolentis (strain K5)
114, 328	MCRH2874	Sulfatase domain protein - Dichelobacter nodosus (strain VCS1703A)	47
115, 329	MCRH2891	Na+ symporter - Marinomonas s(strain MWYL1)	45
116, 330	MCRH2904	Glycine dehydrogenase (Decarboxylating) beta subunit/glycin	47
		dehydrogenase (Decarboxylating) alpha subunit (EC 1.4.4.2) -
		Psychrobacter s(strain PRwf-1)
117, 331	MCRH2916	S. Aminomethyltransferase (EC 2.1.2.10) (Glycine cleavage system	47
		protein) - Neisseria meningitidis serogroup B
118, 332	MCRH2935	Putative lipoprotein - Bordetella petrii (strain ATCC BAA-461/DSM	47
		12804/CCUG 43448)
119, 333	MCRH3006	Aspartate semialdehyde dehydrogenase (EC 1.2.1.11) - Psychrobacter	47
		arcticum
120, 334	MCRH3022	Probable cytochrome C-type biogenesis protein - Psychrobacter arcticum	42
121, 335	MCRH3030	Putative uncharacterized protein - Psychrobacter arcticum	47
122, 336	MCRH3040	Putative uncharacterized protein - Psychrobacter arcticum	47
123, 337	MCRH3120	2-isopropylmalate synthase - Psychrobacter cryohalolentis (strain K5)	44
124, 338	MCRH3128	UspA1 - Moraxella catarrhalis	44
125, 339	MCRH3146	Putative uncharacterized protein - Alteromonadales bacterium TW-7	47
126, 340	MCRH3327	DsrE-like protein - Psychrobacter cryohalolentis (strain K5)	47
127, 341	MCRH3328	Glutamate-ammonia-ligase adenylyltransferase (EC 2.7.7.42) -	46
		Psychrobacter cryohalolentis (strain K5)
128, 342	MCRH3363	Aminotransferase (EC 2.6.1.—) - Psychrobacter s(strain PRwf-1)	47
129, 343	MCRH3380	Carbamoyl-phosphate synthase, large subunit (EC 6.3.5.5) -	47
		Acinetobacter baumannii
130, 344	MCRH3399	Peptidase M23B precursor - Psychrobacter cryohalolentis (strain K5)	46
131, 345	MCRH3401	PpiC-type peptidyl-prolyl cis-trans isomerase precursor - Psychrobacter	47
		cryohalolentis (strain K5)
132, 346	MCRH3404	S. LPS-assembly protein precursor (Organic solvent tolerance protein) -	47
		Psychrobacter cryohalolentis (strain K5)
133, 347	MCRH3409	Aminoglycoside phosphotransferase - Psychrobacter cryohalolentis	46
		(strain K5)
134, 348	MCRH3501	Phospholipase D/Transphosphatidylase - Psychrobacter s(strain PRwf-1)	47
135, 349	MCRH3534	ABC transporter related - Psychrobacter s(strain PRwf-1)	47
136, 350	MCRH3547	Putative uncharacterized protein PM0287 - Pasteurella multocida	46
137, 351	MCRH3567	Nucleoside-diphosphate-sugar epimerase-like protein - Psychrobacter	47
		s(strain PRwf-1)
138, 352	MCRH3578	DNA gyrase subunit A (EC 5.99.1.3) - Psychrobacter s(strain PRwf-1)	47
139, 353	MCRH3622	L-serine dehydratase 1 (EC 4.3.1.17) - Psychrobacter s(strain PRwf-1)	47
140, 354	MCRH3635	Alanine racemase (EC 5.1.1.1) - Psychrobacter cryohalolentis (strain K5)	47
141, 355	MCRH3705	Na+/H+ antiporter NhaC - Psychrobacter cryohalolentis (strain K5)	46
142, 356	MCRH3709	Peptidase S11, D-alanyl-D-alanine carboxypeptidase 1 precursor -	47
		Psychrobacter s(strain PRwf-1)
143, 357	MCRH3729	Probable membrane-bound lytic murein transglycosylase D -	47
		Psychrobacter arcticum
144, 358	MCRH3742	Putative DNA repair protein radA - Psychrobacter arcticum	46
145, 359	MCRH3759	—	47
146, 360	MCRH3844	Putative uncharacterized protein - Mannheimia succiniciproducens	26
		(strain MBEL55E)
147, 361	MCRH3884	Putative uncharacterized protein - Mannheimia haemolytica PHL213	35
148, 362	MCRH3895	Transcriptional regulator - Pseudomonas phage F116	1
149, 363	MCRH3907	Putative uncharacterized protein - Psychrobacter s(strain PRwf-1)	47
150, 364	MCRH3939	Possible membrane-bound lytic murein transglycosylase - Psychrobacter	45
		arcticum
151, 365	MCRH3966	Probable Insulinase-like peptidase, family M16 - Psychrobacter arcticum	46
152, 366	MCRH3969	Probable Insulinase-like peptidase, family M16 - Psychrobacter arcticum	47
153, 367	MCRH3996	KpsF/GutQ family protein (EC 5.3.1.13) - Psychrobacter s(strain PRwf-1)	44
154, 368	MCRH4056	Pyruvate dehydrogenase complex dihydrolipoamide acetyltransferas (EC	46
		2.3.1.61) - Psychrobacter s(strain PRwf-1)
155, 369	MCRH4147	SSU ribosomal protein S1P - Psychrobacter s(strain PRwf-1)	47
156, 370	MCRH4174	ABC transporter related - Psychrobacter s(strain PRwf-1)	46
157, 371	MCRH4206	Ribonucleoside-diphosphate reductase class Ia alpha subuni (EC	46
		1.17.4.1) - Psychrobacter s(strain PRwf-1)
158, 372	MCRH4291	Chromosome segregation DNA-binding protein - Psychrobacter arcticum	41
159, 373	MCRH4304	Putative uncharacterized protein - Psychrobacter arcticum	47
160, 374	MCRH4322	S. Adenylosuccinate synthetase (EC 6.3.4.4) (IMP--aspartate ligase	8
		(AdSS) (AMPSase) - Psychrobacter cryohalolentis (strain K5)
161, 375	MCRH4367	L-aspartate oxidase (EC 1.4.3.16) - Limnobacter sMED105	46
162, 376	MCRH4378	Putative nicotinate-nucleotide pyrophosphorylase (EC 2.4.2.19) -	46
		Neisseria gonorrhoeae (strain ATCC 700825/FA 1090)
163, 377	MCRH4392	Msp75 (Fragment) - Moraxella catarrhalis	47
164, 378	MCRH4469	Molybdate transport protein (ABC superfamily, peri_bind) -	47
		Acinetobacter baumannii
165, 379	MCRH4491	Respiratory nitrate reductase beta subunit - Psychrobacter s(strain PRwf-1)	47
166, 380	MCRH4496	Respiratory nitrate reductase alpha subunit apoprotein - Psychrobacter	45
		arcticum
167, 381	MCRH4526	Hemoglobin utilization protein (Fragment) - Moraxella catarrhalis	46
168, 382	MCRH4552	S. UPF0082 protein Psyc_0938 - Psychrobacter arcticum	38
169, 383	MCRH4568	Protein tyrosine phosphatase - Reinekea sMED297	47
170, 384	MCRH4629	NADH-quinone oxidoreductase (EC 1.6.99.5) - Psychrobacter s(strain	47
		PRwf-1)
171, 385	MCRH4641	NADH-quinone oxidoreductase, F subunit (EC 1.6.99.5) - Psychrobacter	46
		s(strain PRwf-1)
172, 386	MCRH4697	Transferrin binding protein B - Moraxella catarrhalis	45
173, 387	MCRH4706	Putative uncharacterized protein - Moraxella catarrhalis	45
174, 388	MCRH4712	Hypothetical periplasmic protein - Neisseria meningitidis serogroup C	46
		(strain 053442)
175, 389	MCRH4716	Transferrin binding protein A - Moraxella catarrhalis	47
176, 390	MCRH4730	Methionine-R-sulfoxide reductase (EC 1.8.4.11) - Psychrobacter s(strain	47
		PRwf-1)
177, 391	MCRH4740	Lytic transglycosylase, catalytic precursor - Psychrobacter s(strain PRwf-1)	45
178, 392	MCRH4750	RNA polymerase sigma factor - Psychrobacter s(strain PRwf-1)	46
179, 393	MCRH4908	Probable penicillin-binding protein transpeptidase domain -	47
		Psychrobacter arcticum
180, 394	MCRH4917	Acid phosphatase autotransporter - Moraxella catarrhalis	47
181, 395	MCRH4930	Adhesin - Moraxella catarrhalis	47
182, 396	MCRH4983	Heat shock protein 70 - Psychrobacter cryohalolentis (strain K5)	46
183, 397	MCRH5027	Alanyl-tRNA synthetase (EC 6.1.1.7) - Psychrobacter s(strain PRwf-1)	47
184, 398	MCRH5073	Flavin reductase (EC 1.5.1.30) - Mannheimia haemolytica PHL213	41
185, 399	MCRH5107	Putative uncharacterized protein precursor - Psychrobacter s(strain PRwf-1)	44
186, 400	MCRH5149	Surface antigen (D15) precursor - Psychrobacter s(strain PRwf-1)	47
187, 401	MCRH5176	S. Uridylate kinase (EC 2.7.4.22) (UK) (Uridine monophosphate kinase	47
		(UMP kinase) (UMPK) - Psychrobacter cryohalolentis (strain K5)
188, 402	MCRH5178	Transketolase (EC 2.2.1.1) - Acinetobacter baumannii	47
189, 403	MCRH5224	Valyl-tRNA synthetase - Psychrobacter cryohalolentis (strain K5)	46
190, 404	MCRH5239	Putative uncharacterized protein precursor - Psychrobacter s(strain PRwf-1)	46
191, 405	MCRH5260	Putative amino-acid transport protein - Neisseria gonorrhoeae (strain	47
		ATCC 700825/FA 1090)
192, 406	MCRH5275	Isoleucyl-tRNA synthetase (EC 6.1.1.5) - Psychrobacter s(strain PRwf-1)	47
193, 407	MCRH5294	Major outer membrane protein - Moraxella catarrhalis	47
194, 408	MCRH5308	Putative thiol: disulfide interchange protein precursor - Psychrobacter	47
		cryohalolentis (strain K5)
195, 409	MCRH5342	RND efflux system, outer membrane lipoprotein, NodT - Psychrobacter	47
		cryohalolentis (strain K5)
196, 410	MCRH5355	Secretion protein HlyD precursor - Psychrobacter cryohalolentis (strain K5)	47
197, 411	MCRH5441	Putative uncharacterized protein - Psychrobacter arcticum	47
198, 412	MCRH5451	Alcohol dehydrogenase class III/S-(Hydroxymethyl)glutathion	47
		dehydrogenase - Actinobacillus succinogenes (strain ATCC 55618/
		130Z)
199, 413	MCRH5471	Probable penicillin-binding protein 1A - Psychrobacter arcticum	47
200, 414	MCRH5547	Phosphoribosylaminoimidazole carboxylase, catalytic subuni (EC	47
		4.1.1.21) - Psychrobacter s(strain PRwf-1)
201, 415	MCRH5559	Putative uncharacterized protein - Moraxella nonliquefaciens	47
202, 416	MCRH5574	Dihydroorotase (EC 3.5.2.3) - Psychrobacter s(strain PRwf-1)	38
203, 417	MCRH5581	Sel1 domain protein repeat-containing protein - Candidatus	28
		Amoebophilus asiaticus 5a2
204, 418	MCRH5639	Aconitate hydratase 2 (EC 4.2.1.3) - Acinetobacter baumannii	47
205, 419	MCRH5640	Aconitate hydratase 2 (EC 4.2.1.3) - Acinetobacter baumannii	47
206, 420	MCRH5644	Aconitate hydratase 2 (EC 4.2.1.3) - Acinetobacter baumannii	47
207, 421	MCRH5729	S. UvrABC system protein C (Protein uvrC) (Excinuclease ABC subunit	47
		C) - Psychrobacter cryohalolentis (strain K5)
208, 422	MCRH5753	Aminobutyrate aminotransferase - Moraxella catarrhalis	47
209, 423	MCRH5785	Putative uncharacterized protein - Neisseria gonorrhoeae (strain ATCC	46
		700825/FA 1090)
210, 424	MCRH5787	Replication restart DNA helicase PriA - Psychrobacter arcticum	47
211, 425	MCRH5838	Ubiquitous surface protein A2H - Moraxella catarrhalis	46
212, 426	MCRH5853	Putative isocitrate dehydrogenase (EC 1.1.1.42) - Neisseria meningitidis	46
		serogroup C/serotype 2a (strain ATCC 700532 - FAM18)
213, 427	MCRH5872	Alpha/beta hydrolase fold - Psychrobacter cryohalolentis (strain K5)	47
214, 428	MCRH5882	Probable HesA/MoeB/ThiF family protein - Psychrobacter arcticum	47
A total number of 47 Mcat isolates, including the RH4 isolate also used for library construction as a positive PCR control, were tested by PCR with oligonucleotides specific for the genes encoding relevant antigens.
The table lists the number of positive PCR results for the 47 Mcat isolates tested.

TABLE 7
PCR primer sequences used for the analysis
of the conservation in 47 Mcat isolates of
the 214 immunogenic Mcat ORFs identified by
screening bacterial display libraries of the
fragmented genome of Mcat RH4 with human
immunoglobulin
The table provides sequences for the
oligonucleotide primers used to examine
the conservation of 214 antigenic Mcat
ORFs, by PCR. The table lists a total
of 214 PCR primer pairs, i.e. 428
oligonucleotide sequences.
		Oligonucleo-
		tide name	PCR
SEQ		(ORF	pro-
ID	DNA oligonucleotide	name_oligo	duct
NO	sequence (5′-3′)	sense)	size
429	CTAATACCAAAATACCCGTTTG	MCRH0005_fwd	1277
430	CAGGCTCAATCACCTCAAAG	MCRH0005_rev
431	CAATCATGAAAACATCAATGG	MCRH0052_fwd	1055
432	GTGAAATGGGTTTTTGTGC	MCRH0052_rev
433	GGACTTTTTGATAAGAATTTGC	MCRH0079_fwd	1197
434	GATATCACCGCCTGCTTC	MCRH0079_rev
435	GATAATTATGACAAAAGCTGGTTG	MCRH0082_fwd	1123
436	GATGTCATTGATTTGCTCATCC	MCRH0082_rev
437	CAGCCATGATACCCAAAAAG	MCRH0103_fwd	1088
438	CTTATTTGTGATCGGCTTTG	MCRH0103_rev
439	CATGCGTACTTTCCATGTC	MCRH0125_fwd	957
440	ATCTGACGAACGATGACTTC	MCRH0125_rev
441	CTCAGGTATGACCAAGCTCG	MCRH0139_fwd	1094
442	GACGAACACTAACCATCTCTTC	MCRH0139_rev
443	CATTGTCAAACTGTCAGAAGG	MCRH0176_fwd	787
444	GTTTAGACCTTCATCACGCTC	MCRH0176_rev
445	GCTTGCTGGATTAGCATACC	MCRH0211_fwd	924
446	CAGATCAGCCAAGTTTTACG	MCRH0211_rev
447	CTTCAGCCCATCAAAGACG	MCRH0249_fwd	1088
448	GCCTTTTGACCGAAGATG	MCRH0249_rev
449	ACTTAGGGCAAGTTTACTTTGG	MCRH0267_fwd	657
450	ATGTATTAAACACCCGCTACAC	MCRH0267_rev
451	GAAGAAAAAGACGCCTGTGG	MCRH0268_fwd	1033
452	GGGTGTTTAATACATTGTTGATACC	MCRH0268_rev
453	CCACAAAAACGGTGTATTTTAG	MCRH0278_fwd	956
454	GACGATATCACCTCTCTCATCG	MCRH0278_rev
455	GACCCAATGTGGTAATCGTC	MCRH0342_fwd	1100
456	GCCAAATTTATTGAACCTAAGC	MCRH0342_rev
457	GGCGTCTTTGGTAGACAGTG	MCRH0357_fwd	1006
458	CGTGTATTATCACCGATGATG	MCRH0357_rev
459	GTATTTGGCAAGCAGTTCG	MCRH0372_fwd	782
460	CATAATCCAAGCCATTTTATCC	MCRH0372_rev
461	GCTTGTTCTTGGTAATCACG	MCRH0391_fwd	1046
462	GCTTTGACAATGAGATTTTGG	MCRH0391_rev
463	CCAAGATTTTTCTATTTAACCAAG	MCRH0397_fwd	1293
464	GTTGATAGATTGGCATACCG	MCRH0397_rev
465	CGCTCATATACAATTTGCTG	MCRH0432_fwd	927
466	CAAAACTGTCCTCAATCTGC	MCRH0432_rev
467	GATATTGGCGTTGGAGCAG	MCRH0441_fwd	811
468	CCACCTCATGCAGTGCTTC	MCRH0441_rev
469	CTTTCAACATAACGGCGTTG	MCRH0448_fwd	1172
470	CACAAATAACGATGCGTTCAG	MCRH0448_rev
471	CTTACCTGCCATTTGTGATG	MCRH0464_fwd	1088
472	GCATCGGTTAAAAGACCAC	MCRH0464_rev
473	GCTTGGTGTTGAGGTTGTTAC	MCRH0477_fwd	1055
474	CATCAAGTCGATCGTATCATTC	MCRH0477_rev
475	CCATTGATAAGATCCCAAGG	MCRH0498_fwd	1225
476	GACGATTGTCATCGATACTGC	MCRH0498_rev
477	CATATGAAGTTTTATGTTGAGGTG	MCRH0512_fwd	1184
478	GGCAGATATGCTTGATGAGC	MCRH0512_rev
479	GTGGTATGGTACATCCACAAG	MCRH0525_fwd	1260
480	GGTCAACCGATCAAAATGTG	MCRH0525_rev
481	CAAAAACTTGAAAGCACAAAAG	MCRH0546_fwd	1156
482	GCAGGCACTCAAAAGTAGG	MCRH0546_rev
483	CTTAAGACATGAGCCTAACTGG	MCRH0558_fwd	1077
484	CTTAATGGGTCTTGTTTTGGTAC	MCRH0558_rev
485	CTGAAATTGCATTGACTCGTG	MCRH0580_fwd	1076
486	GATAAACACCACCCAAAACC	MCRH0580_rev
487	GGACGCTATAGGCTGTCTAATC	MCRH0587_fwd	1102
488	GGCTAAGATTGAAGATTGATTG	MCRH0587_rev
489	CGACTATGTCGTCTCAACTGC	MCRH0594_fwd	1399
490	GTAAAACAAGGCGAGTATTTGG	MCRH0594_rev
491	CAACAGGGCAGGTATCGG	MCRH0621_fwd	997
492	CCAACATATTGATCTTGGGTAAC	MCRH0621_rev
493	GATTTTTTATTGCCTCGCT	MCRH0655_fwd	802
494	GCTAATACTTGCCTCACCTG	MCRH0655_rev
495	GGTTAGTCGCTACGGTACACAG	MCRH0673_fwd	782
496	GCACCTGATTTTTTCTGATGG	MCRH0673_rev
497	GCACGCTCATCAAACCAAC	MCRH0682_fwd	721
498	CACGCAATATGGTTACTGGTG	MCRH0682_rev
499	GCATTGTGCGATAAGTACG	MCRH0704_fwd	1442
500	GTCAAAATGCGCATCTTG	MCRH0704_rev
501	CGGTTTATAAATACAGCCCATC	MCRH0744_fwd	1158
502	CTTTCTGAGCGTTATTATCATGG	MCRH0744_rev
503	CTTAGGCGTCTGAATGAAACC	MCRH0756_fwd	1083
504	GCTTGACCAACATTACCGC	MCRH0756_rev
505	GTACAACAACCTCAGGCGAG	MCRH0759_fwd	564
506	GCCCTTCTTTGGTCAAATAC	MCRH0759_rev
507	CCCTTGTTATCTTTATCCAAATG	MCRH0780_fwd	1102
508	CAAGGTGTGATGGGTTAAATG	MCRH0780_rev
509	CATGGACATGACGCACTTAG	MCRH0790_fwd	1099
510	CGCTTTAGGCGAAGTAAAAC	MCRH0790_rev
511	CAAACGTAAATAATCCGCTTC	MCRH0808_fwd	1112
512	CAAATCGGCAAATCATAAGC	MCRH0808_rev
513	CCGTTATCAGAATTGGTCAAG	MCRH0815_fwd	1227
514	CGATGTCAGCATCTTCGTG	MCRH0815_rev
515	CCGAGGGTAAATATCATCAAG	MCRH0853_fwd	1371
516	GTTGATGCCCCCATTTATC	MCRH0853_rev
517	GTATTGGGCTTGAATCACCC	MCRH0867_fwd	1099
518	GGATACTCGCTATGGTGGAC	MCRH0867_rev
519	CCATTGACTACACCGAAGG	MCRH0911_fwd	1064
520	CAAAATCAGCATAAGGCGTTC	MCRH0911_rev
521	CAGCTCATATTTAGGCAAGC	MCRH0917_fwd	1112
522	GATTGGGCCATTTTGATAC	MCRH0917_rev
523	CACGCATCATGAATGAAATC	MCRH1023_fwd	1067
524	CTCTTTCATCCAGCACAAAC	MCRH1023_rev
525	CGGTAATGGATTGGTATTATC	MCRH1076_fwd	1332
526	GCAACTTTTACACCATCAGC	MCRH1076_rev
527	GCTTGATAGTTACCAAAGTCTTATTG	MCRH1163_fwd	1446
528	CGCATCACAAAACATCTTGC	MCRH1163_rev
529	GTTAAAAAGGCACTCAAAGCAC	MCRH1198_fwd	1084
530	GATGTGCAAGGCTCAATAAG	MCRH1198_rev
531	GTTGATGGGGTAGAATTTGC	MCRH1227_fwd	1081
532	GCCATAAAATGGCAAGTGG	MCRH1227_rev
533	GCTAAGCGGTAATCAAAAGC	MCRH1256_fwd	791
534	CTCTTTTTGGTTTGTTTTGG	MCRH1256_rev
535	GTATCGCAGCAGACAAAAGTG	MCRH1257_fwd	1037
536	GTTTGAAGGCTTGTACGGC	MCRH1257_rev
537	GTCGTTTGTTTTGGTAACACTG	MCRH1303_fwd	1207
538	CAAAATTTGTAACAGCAGATGG	MCRH1303_rev
539	CCACCCTTGTACTGTTAAATCC	MCRH1397_fwd	846
540	CTGGTCAGCTGTGTGGATACC	MCRH1397_rev
541	GATAAACTTCCCACAACTCTTTG	MCRH1430_fwd	1254
542	GTTTTGGTTGTGATGATTTTTG	MCRH1430_rev
543	GCTTGCGGTGATTAGTTTTC	MCRH1438_fwd	801
544	GATCAAATATCGCCAAGCATC	MCRH1438_rev
545	CGCTTAACAATTGACTCACG	MCRH1440_fwd	1216
546	CAAAATTGGTCAAATTGACAC	MCRH1440_rev
547	GGTTAATGCGACGGTTGTC	MCRH1475_fwd	1875
548	CGTTTACCTGCATTATCAGCAC	MCRH1475_rev
549	CAAAGTAACCCAATGTAACTTTAAG	MCRH1486_fwd	1011
550	CAGCAGTATCTGTTTTCACGC	MCRH1486_rev
551	GATAGCTTGCTTGAGTTTGATG	MCRH1493_fwd	1053
552	CGCATTGCTAAAGCTTACCTAC	MCRH1493_rev
553	CCCTAACTTATTTTTATTTTCATCAC	MCRH1546_fwd	900
554	GTGGCAGTGCCAGTTCTTG	MCRH1546_rev
555	CTAAGCCAGCATTTGATCG	MCRH1561_fwd	1292
556	GACAGAGCTGATGATGACTGC	MCRH1561_rev
557	GGTTCTATAAACGATGATGATTTG	MCRH1565_fwd	1074
558	GCATATGGCTTTCATTTTGC	MCRH1565_rev
559	CAAAAGCCTTGTCATCAAATTG	MCRH1568_fwd	1487
560	CAAGTCGCTTTTGGCGAC	MCRH1568_rev
561	GATCACTTTTGCGGTATTATTG	MCRH1577_fwd	1165
562	GAATAAGGCTTTTGGGTAAGG	MCRH1577_rev
563	GCCCAAAAAGAAATCATTAAG	MCRH1586_fwd	1097
564	GCTCTACCAACTGAGCTATTCC	MCRH1586_rev
565	CCATATCTTTACGGGCATC	MCRH1671_fwd	751
566	CACATGCTTGGACAAAAGC	MCRH1671_rev
567	CTGCTCAAGCTGGTGTGAC	MCRH1677_fwd	1261
568	GTCGTTGTTTTGCTACCAGTG	MCRH1677_rev
569	GCCAGCCTAAAAACTAATGG	MCRH1696_fwd	1113
570	CAGTTGATTGCTGTGGTTTG	MCRH1696_rev
571	CCATTGAAGAGATTGGCACAC	MCRH1701_fwd	1051
572	CTCATTGCTACAAGCAGCAAG	MCRH1701_rev
573	GGTGTCTTTTTGACATAACTGTC	MCRH1705_fwd	1324
574	CATTAAATGGTGGGTAGGC	MCRH1705_rev
575	GATACGCACAGGTTCAACG	MCRH1720_fwd	987
576	CACCAATGCAGCTTGCTAC	MCRH1720_rev
577	GCAGATTATTCACAAATTGAGC	MCRH1732_fwd	944
578	CCCATAGCAAAGGAGAACC	MCRH1732_rev
579	GAAATATGGCTTAGATAAAATTATAGC	MCRH1742_fwd	798
580	CTTGTTATCCACAAAACTGTATACC	MCRH1742_rev
581	GTATCACAGGCTTGGCTTGG	MCRH1777_fwd	1075
582	CCCATATGAGTATCGGTGATGAG	MCRH1777_rev
583	GGCAGATGAGGTGATTAATAAC	MCRH1841_fwd	650
584	CAAAAACCAGCTTGTGAGATAC	MCRH1841_rev
585	CGAATTTTTGGGTGATTTG	MCRH1853_fwd	1501
586	GAACAAGAATTGGCACTCATC	MCRH1853_rev
587	GCGCCTTGCCATGTATATC	MCRH1878_fwd	792
588	GGCATGAATATCATCAAAGC	MCRH1878_rev
589	GATATCAATTCATCAAAATCCACC	MCRH1934_fwd	1128
590	CTCACCACGCACAATCTCAC	MCRH1934_rev
591	GCATTTTTCAGCAGTTTTTG	MCRH2003_fwd	1238
592	GGGTCAATAAAGAGGTGATGC	MCRH2003_rev
593	GATGATTCACAACTGGATGC	MCRH2082_fwd	859
594	GAAAACTGACGCTCTAATACACC	MCRH2082_rev
595	GGCTTGATAATTGCAGAAATC	MCRH2139_fwd	1107
596	CAAGTGCAGATAAGGCATTG	MCRH2139_rev
597	GCTCACTCATCTGAGCTATCG	MCRH2151_fwd	1377
598	GCAAAATCTTGTAAAGCCATC	MCRH2151_rev
599	GATGAAATTTTGGCACTTGAG	MCRH2184_fwd	1244
600	GAAAATCAGCAGGAAATGACAC	MCRH2184_rev
601	CCCTATTTTCCCCTATTTTGG	MCRH2216_fwd	1420
602	GACTGATGGATAATTTTCGTTG	MCRH2216_rev
603	GCGTATGGATTTTTGAAGGTG	MCRH2239_fwd	778
604	CAGTCATTGAGTACTGCCAAATAG	MCRH2239_rev
605	CATCTGTTTTGTACCTGCTTGAC	MCRH2257_fwd	851
606	CCAAATGATTTATCAATCACCAC	MCRH2257_rev
607	CTCAGCTTTCTGAAGATTTGC	MCRH2276_fwd	1494
608	GATTTGATGAAATATTATTCAAGTGC	MCRH2276_rev
609	GATGTCTCAAAATTCATATTATACGC	MCRH2384_fwd	1265
610	GATTAAACACCTGCACATGACAAG	MCRH2384_rev
611	GAATTCTACTCCCCTCTCACC	MCRH2399_fwd	666
612	CTGAAGAGTTTGATCATGGCTC	MCRH2399_rev
613	CTGTTGCTGGATTTTTGATG	MCRH2415_fwd	1069
614	CTAAGTTCGTCTTGAGCAATG	MCRH2415_rev
615	CTTGACGAGCTTACATCAAGTAC	MCRH2484_fwd	1260
616	GCATAAGATTCTCCATAGCCAG	MCRH2484_rev
617	GGTCATATCACACAAATTTTATCC	MCRH2496_fwd	1372
618	CCAATCAGTCCATAAATAAGATG	MCRH2496_rev
619	CAGATATGAACCCAAGCAAGG	MCRH2506_fwd	1050
620	GYCAAGATTATTTGGCACTG	MCRH2506_rev
621	CATGCTGTGCTAAATACCCC	MCRH2519_fwd	1102
622	CAGGTTTAATTTTGCCTGAAG	MCRH2519_rev
623	GTATTATGGAAAACCGCCAAG	MCRH2553_fwd	1074
624	GACAAGTTGCACAGCTTTGAG	MCRH2553_rev
625	GTGCTTAATATCGCCCAATC	MCRH2560_fwd	969
626	CAGCATACGCTGAAAAACTG	MCRH2560_rev
627	GGTTTTTGGCATTTACAATAAAAC	MCRH2592_fwd	1130
628	GCCTTTGAATATTAAACACAAATACC	MCRH2592_rev
629	CCTATTTATTTACATAAACGCATAG	MCRH2606_fwd	1061
630	CATGTTATCTGACTGACATTCG	MCRH2606_rev
631	CATTCAGCTGTCTGATGGTG	MCRH2612_fwd	1301
632	CTTAAGCGTTGGCTTATTTTTG	MCRH2612_rev
633	GCAGTACGCTGACTATTAAAGC	MCRH2625_fwd	1065
634	CTTTGGTAAACTCACCCAAAAC	MCRH2625_rev
635	CTTGGTTCATCTACCCCATC	MCRH2627_fwd	1386
636	GCGGTTATTATTGAGAGCTTG	MCRH2627_rev
637	GCATTGGGTGTGAGTCTATG	MCRH2641_fwd	1427
638	CATCAGCCATATCCGCC	MCRH2641_rev
639	CATGTTATCAAAAATTGCATTAG	MCRH2669_fwd	1133
640	GCTATTAATTCCATGATCAGC	MCRH2669_rev
641	CCACATTACCCGTCCAGG	MCRH2682_fwd	843
642	GTCATCGCCAAAAGACTGAC	MCRH2682_rev
643	GATGACTTGATACCACAAAGATG	MCRH2754_fwd	1263
644	CAAAACTGGCGATTAATTCAG	MCRH2754_rev
645	CCGCTTTAATGCTATTTATGG	MCRH2759_fwd	1013
646	GCTGACAGGCAAATCGTAC	MCRH2759_rev
647	CGTTTATCACCTTTATCGGTATC	MCRH2783_fwd	1405
648	CCGGTTACAAAGATAAATTTCG	MCRH2783_rev
649	CCTATACCGATGGCTCAGATC	MCRH2844_fwd	1376
650	CTTTGTTCAAAAACTGCAACC	MCRH2844_rev
651	GAAAATCATCAAATATGCAACAC	MCRH2851_fwd	992
652	CTTGTAATTGGGTATGATTGAATG	MCRH2851_rev
653	GCATCATCATCAAGCTTTCG	MCRH2861_fwd	1057
654	GATTGAATAAGTAAAATCCAACTCC	MCRH2861_rev
655	CCATGCCTTTGATGAATTTG	MCRH2874_fwd	1383
656	CATGGTCATCTAAATCCACCAAC	MCRH2874_rev
657	CAGAGTGGATGAAAAACTGG	MCRH2891_fwd	1060
658	GATTGGCATAACAAGACCTG	MCRH2891_rev
659	CTAAAAGCCAAATGTGATGAAC	MCRH2904_fwd	1194
660	CGAAAAATTACCAGAATGTAAAG	MCRH2904_rev
661	GGTCTCTCCTGTGAGTTTGG	MCRH2916_fwd	800
662	GTCATGCCAGCAGATGAG	MCRH2916_rev
663	CAGTGATAACATACATAAAAGTTGC	MCRH2935_fwd	1191
664	GCATATTATGCCGATAAAATCTC	MCRH2935_rev
665	CTAAAAATAAACGCCCAAAGAC	MCRH3006_fwd	1389
666	CAACTCAGATGTACTGTGCTTTC	MCRH3006_rev
667	CAAGATCCGTTTGTTTATTCTG	MCRH3022_fwd	960
668	GATAAGATGCCTCATCAATAACC	MCRH3022_rev
669	GAAGCCAATCTGCTATTTGG	MCRH3030_fwd	1098
670	GATACCACATCCTGAAATTTGG	MCRH3030_rev
671	CTCGGTTTCAAGTTTTGCTG	MCRH3040_fwd	1126
672	CTTTATCATCAGCACCTTCACC	MCRH3040_rev
673	CTGTACACCCAAGGCATTG	MCRH3120_fwd	937
674	GACACTCATCAGTCATTTACGC	MCRH3120_rev
675	GTAAATTTGGTTTGGTGGATG	MCRH3128_fwd	1068
676	GCTGTGAGCCATTAACAGC	MCRH3128_rev
677	GGTGCTCCTAGGATAAATGC	MCRH3146_fwd	689
678	GAACTGCATCGTCCTAGCTC	MCRH3146_rev
679	CCATATTCTCCCAAAAATGC	MCRH3327_fwd	895
680	CCAAACTGCCTGATTGTAATG	MCRH3327_rev
681	GCCGATGTATTAGGTTTGC	MCRH3328_fwd	1199
682	CTACYAAGGCACCATCTAGC	MCRH3328_rev
683	GATGCCTGATTATCCTTTGTG	MCRH3363_fwd	1116
684	CTGATAAAAAGCGTTCAGTTTG	MCRH3363_rev
685	CAAAGAGGTGGTGATTGGTG	MCRH3380_fwd	1212
686	GTTGCATAATTTTCCTTTCAATG	MCRH3380_rev
687	CTGAATTTACAGTTAAAACGGC	MCRH3399_fwd	1398
688	CAAGAATTACGCTCAAGTGC	MCRH3399_rev
689	CAGCCGTAGCCTCAAATAG	MCRH3401_fwd	1075
690	GCGATATCACTGCTTTGATTG	MCRH3401_rev
691	CATATTTGACTACCGCTATTCG	MCRH3404_fwd	868
692	CTGCTTATTTTATCCATTTGACTG	MCRH3404_rev
693	GCAATCAATGTTTGACGACG	MCRH3409_fwd	1435
694	GTGGCTCACCTTGTTCAATAG	MCRH3409_rev
695	GGGTGAGTTTACCACCAATC	MCRH3501_fwd	1086
696	CAGCATCTGAGCTGTCTTTG	MCRH3501_rev
697	GCTCAGCAAACCAAAATAAC	MCRH3534_fwd	1199
698	CCAAGGATTGGTCAAAGAAC	MCRH3534_rev
699	CAGGTTTACCTGTGACAATGG	MCRH3547_fwd	1071
700	GATGGCTCAAAAACAAAAGC	MCRH3547_rev
701	CACACCGCTTGGACAATATG	MCRH3567_fwd	1196
702	CAATTGGACTGACCGAATCAC	MCRH3567_rev
703	CGACCAGCATTAGCAATCTG	MCRH3578_fwd	1001
704	GATTGACATTCGTGATGAATTTG	MCRH3578_rev
705	CAATGAAACTGCCCTAAGAG	MCRH3622_fwd	1073
706	TGGTGATATTGAGGGTTTCC	MCRH3622_rev
707	CATTATTATCACCAAGACAAAGATG	MCRH3635_fwd	1519
708	CCAACGATTATTATCATAAAAAGC	MCRH3635_rev
709	CTTGTTGGCGTTGATTTATTC	MCRH3705_fwd	1086
710	CAGTGGTCATCGCAAGTAAC	MCRH3705_rev
711	CAATAGGGCGTTTGTATGC	MCRH3709_fwd	1534
712	CTGACCCCAATCTTCAAAATAG	MCRH3709_rev
713	GCTCGTGGTGAGACTAAAATG	MCRH3729_fwd	1231
714	CACATAATGCCAAACTTAAAGC	MCRH3729_rev
715	GCTGTGAAAAGCAGTATTAAACC	MCRH3742_fwd	1329
716	CCATAACTTTAACACCGCCTAC	MCRH3742_rev
717	GCTTTACTTTACGGCATTGG	MCRH3759_fwd	915
718	CCAATGGCATCTTGATTTG	MCRH3759_rev
719	CGCTGAGAAATATGCTTATTATACC	MCRH3844_fwd	974
720	CCATTTGGGTATCGGCTTG	MCRH3844_rev
721	CAAGAAAGTAGCGTTGATGAG	MCRH3884_fwd	1058
722	CAATCGCCTTAAAAGATGAC	MCRH3884_rev
723	GTTGCCACGGTAAATTTGC	MCRH3895_fwd	1146
724	CTTGTGACCATATACCACAAACC	MCRH3895_rev
725	CTGATATTTTTATCGCTGTCAATAC	MCRH3907_fwd	1564
726	GCTGCCCAGTGCTTCAAC	MCRH3907_rev
727	GACGGGCGTATCTGCAG	MCRH3939_fwd	836
728	CTAGGCTTGATAACGCACTG	MCRH3939_rev
729	CTGTGACATTTGGCTTTAGTG	MCRH3966_fwd	1067
730	CTATTGGCCTGCGTATAGC	MCRH3966_rev
731	CAAAAGCATTGGACAAAGTC	MCRH3969_fwd	1163
732	GGCATTAAAATCACCACCTG	MCRH3969_rev
733	GACTTATCCAACCCAAAATGAAG	MCRH3996_fwd	1396
734	CTTCGACCTGTGATAATGGC	MCRH3996_rev
735	GTGATGATGAGCAAACAGACG	MCRH4056_fwd	1029
736	CAATCTGCTTGATACCTTTATCTTG	MCRH4056_rev
737	GGACTGAGTCTGGTGAAGAAGC	MCRH4147_fwd	689
738	GACCTACCTCAATCACAAATCAAG	MCRH4147_rev
739	GGTATCATTGGCAAAAATGG	MCRH4174_fwd	1069
740	GTAAAAACGCCCATTCAAAC	MCRH4174_rev
741	CAGCACCTTTGGATAACATC	MCRH4206_fwd	1163
742	CAACTTGTTTTTGCATGGC	MCRH4206_rev
743	CTGATGATAAGGCGGTGAG	MCRH4291_fwd	1455
744	CAATAATGAATGGCGTTTTG	MCRH4291_rev
745	CGAAGCGTTGAAATTTATGG	MCRH4304_fwd	1077
746	GATATGGGTAATGTTTTTGACC	MCRH4304_rev
747	CTCAAACTGTGCCAAGAATG	MCRH4322_fwd	989
748	CCGATAAATAATCCAAAAAATC	MCRH4322_rev
749	CAAAGGTGAAATTGCGGATAC	MCRH4367_fwd	1199
750	GCTTAATCTGAGCGACATCC	MCRH4367_rev
751	CAAAACCTGATAGAAGCCATTG	MCRH4378_fwd	1213
752	CTATGATGTAAGTTCGGATAAGTGC	MCRH4378_rev
753	GTACGCCTTTTGCACTCAG	MCRH4392_fwd	1079
754	CGATAAAAGGTGCATTACCG	MCRH4392_rev
755	GATTTTTAAAGCCCCATCG	MCRH4469_fwd	1274
756	CTAAGGGCAAAAACACAAGG	MCRH4469_rev
757	CATCTCAGGCTGCCCTTAC	MCRH4491_fwd	1089
758	CTGTCATATTTGGCTTGTGC	MCRH4491_rev
759	CTGGGTTCATCAGGTAAAGG	MCRH4496_fwd	1291
760	CAATTTCTTTGTTGCCATTG	MCRH4496_rev
761	GGCCAATTTTCGTACGC	MCRH4526_fwd	862
762	GTTCAGCCCAACGGTTG	MCRH4526_rev
763	GATCAGATTTATCCTCTTTTTACCTG	MCRH4552_fwd	1136
764	GTATTCCTTTATTCTACCGTTGTTTT	MCRH4552_rev
765	GAGCTACAAGTCGGCGATAC	MCRH4568_fwd	994
766	CTGCTTGGCAATAAAACATTAC	MCRH4568_rev
767	GAGGACATTACCCAAACGG	MCRH4629_fwd	1291
768	CTAAGTCCTTAGGTTGCATGG	MCRH4629_rev
769	GCTGATGGCAGGTTTACCC	MCRH4641_fwd	772
770	CCACAAATATATCGCCCCG	MCRH4641_rev
771	GGAGCTGGCAGGTAAATTC	MCRH4697_fwd	1021
772	GCAATAATAATGGCAGATAATG	MCRH4697_rev
773	CATTATTTATGGACAAACAAAATG	MCRH4706_fwd	1127
774	GTTTGTGATGGACAAAATACGG	MCRH4706_rev
775	CCGTCTATGATTGATATAATTTGTG	MCRH4712_fwd	802
776	GATAAGCATTATGGTCGCTATG	MCRH4712_rev
777	GGTAAAGATTGGGGCGTG	MCRH4716_fwd	863
778	GCATTGTTATCCACCTCTTTTAC	MCRH4716_rev
779	GGCATAGTCGCATCATTTAAG	MCRH4730_fwd	1055
780	GTTTATTGACGCTGGTTGG	MCRH4730_rev
781	CCTAACAATAAGCAAAACTCTTG	MCRH4740_fwd	1185
782	GCAAAATAAAAGGCAAAAAC	MCRH4740_rev
783	CCATCAGACTCACCATCTTC	MCRH4750_fwd	828
784	GGCATAAGCTTTTAGGCTTTC	MCRH4750_rev
785	GTTAATGTCGGTATGCAGCTTG	MCRH4908_fwd	1048
786	GAAGTATCTTGGTGGTCAACG	MCRH4908_rev
787	GGCGAATTAACCTTATCTGG	MCRH4917_fwd	1082
788	GTTAAATTATCATGATTGGCTTG	MCRH4917_rev
789	GGGTAAAGGCACAAAAGACAC	MCRH4930_fwd	1057
790	GGCTTTGTACAACTGGCTACC	MCRH4930_rev
791	CAAAACTTTTACCATACCAAGATG	MCRH4983_fwd	1211
792	CGCTCAAAATATGCTGTAACG	MCRH4983_rev
793	GTTCACCCACTTGCTGACAT	MCRH5027_fwd	1035
794	CACATGCAGACCGCCAC	MCRH5027_rev
795	GATATCAGCCATATTGATAAGCAC	MCRH5073_fwd	1372
796	GAGCGAGTRGACACACGATAATG	MCRH5073_rev
797	CGATGGTATTTGGTATGAAGC	MCRH5107_fwd	1361
798	GTAAATCTCCCCACGATGAC	MCRH5107_rev
799	GGGCAAATCTATGAATGAAGC	MCRH5149_fwd	1110
800	CAAAGCGATATTGCTCACC	MCRH5149_rev
801	GTTAAAAATGATGATGGTTAGATTG	MCRH5176_fwd	1076
802	CTGATAAAATCCCTGGATGG	MCRH5176_rev
803	GAAGCAGCTTGGAATAAACAG	MCRH5178_fwd	1233
804	GCCCTTGCAAAATTAGGAC	MCRH5178_rev
805	GGCAATGTGCTTGACCC	MCRH5224_fwd	1191
806	CACCTGCTTCTCCACTTCTG	MCRH5224_rev
807	CGAGATTTGTGGCAATTTAG	MCRH5239_fwd	1293
808	CGTGGTATCTTTGACATCATC	MCRH5239_rev
809	CCAAAAATAATCGGTACGGTC	MCRH5260_fwd	733
810	CATGGTTATAAAACAACGGATG	MCRH5260_rev
811	CAAGTACCCATCATCACAGG	MCRH5275_fwd	1059
812	CGTATTGGCAAGTAAAAAGC	MCRH5275_rev
813	GCGATTGTTACCAAAACCTTAG	MCRH5294_fwd	793
814	CAAAATTAATATTAGCCCCTTTG	MCRH5294_rev
815	CCAATGACTTTTGGGATATG	MCRH5308_fwd	1166
816	CATAAGTTCGTGTTTTGTGCTAG	MCRH5308_rev
817	CTTTTGGTGTGGGTGTTGTG	MCRH5342_fwd	1049
818	GGCATCTATCGCTGGTAGTAG	MCRH5342_rev
819	GTGCCAAGTCTATGTCATTGC	MCRH5355_fwd	1131
820	CCAGACTCAATCTGTTCACG	MCRH5355_rev
821	CCAAAACGGTTGAAGAATG	MCRH5441_fwd	1106
822	GTTAAAACCCTCCATGAATGC	MCRH5441_rev
823	GCAGCTCACTACGACCTTTC	MCRH5451_fwd	663
824	CTAACCTTTGTTCGGCAGTG	MCRH5451_rev
825	GATAATGACGCTCAAACTGG	MCRH5471_fwd	1087
826	CTGGCGTATATTGACCAGAC	MCRH5471_rev
827	GCACGCCAAGCATCTTATC	MCRH5547_fwd	981
828	CACACCGTCAGCATCTTC	MCRH5547_rev
829	CCATACAGGAGTGCATTTTC	MCRH5559_fwd	832
830	CGAACTCTCTATTCCATTGTTC	MCRH5559_rev
831	GTTTGCTCAGGCTCAAAATAATC	MCRH5574_fwd	1496
832	GGTCAATGATGATGGCATTTG	MCRH5574_rev
833	GAACAACGGCTAAAACTTGC	MCRH5581_fwd	1352
834	CGCTTGCCAGCGTACC	MCRH5581_rev
835	GATGGCGGTGTGTGTTTG	MCRH5639_fwd	824
836	CCTTTGAATTTGACCAGTACAG	MCRH5639_rev
837	GTTGGGGCTTTGATGACAC	MCRH5640_fwd	849
838	CCCAAGCCTACTAAAAGCAG	MCRH5640_rev
839	GGTTCATGATAAAATCAGGTAGG	MCRH5644_fwd	843
840	GCCCTGACATTCCGCTAC	MCRH5644_rev
841	GAGTGATGATTTGCCTGATG	MCRH5729_fwd	1108
842	GTGCAGTCGTTAAAGATGACG	MCRH5729_rev
843	GAACCCATATGGTCAGCTTC	MCRH5753_fwd	779
844	GCCATTAAACTTGCCAAAAC	MCRH5753_rev
845	GTTGCTGCACTGCCTAAAG	MCRH5785_fwd	1384
846	GAGGTGCGATAAGACTGTCG	MCRH5785_rev
847	GATAAGACCATCAAGTGCAGAG	MCRH5787_fwd	667
848	CTTAGTATGTTTGGCGAGACC	MCRH5787_rev
849	CGGATATTGCTAATAACATCAAC	MCRH5838_fwd	1130
850	CGACCAAACGCTGATCG	MCRH5838_rev
851	GTGATCGATTTTTGTCGTGAG	MCRH5853_fwd	1288
852	CTATTGCTTCGATGACTCGTTAC	MCRH5853_rev
853	CGAATATGGCAAAAAGCTG	MCRH5872_fwd	1459
854	GCCGTTTCAATGATAGTTGC	MCRH5872_rev
855	CATCAGCATACCCACAGTTG	MCRH5882_fwd	1093
856	GGCATTGCCCATAGTTTTAG	MCRH5882_rev

EXAMPLES

Example 1

General Screening Procedure for the Identification of Antigenic Bacterial Proteins According to the Present Invention

The approach, which has been employed for the present invention, is based on the interaction of proteins or peptides encoded by Mcat with the antibodies present in human sera. The antibodies produced against Mcat by the human immune system and present in human sera are indicative of the in vivo expression of the antigenic proteins and their immunogenicity. In addition, the antigenic proteins, as identified by the bacterial surface display expression libraries using pools of pre-selected sera, are processed in a second and third round of screening by individual selected or generated sera. Thus the present invention supplies an efficient, relevant, comprehensive set of antigens as a pharmaceutical composition, especially a vaccine preventing infections caused by Mcat.

In the antigen identification program for identifying a comprehensive set of antigens according to the present invention, at least two different bacterial surface expression libraries from Mcat are screened with several serum pools or plasma fractions (antibody pools). The antibody pools are derived from a serum collection, which has been tested against antigenic compounds of Mcat, such as whole cell, total extracts. Sera determined to have high ELISA titre have to react with multiple proteins in immunoblotting in order to be considered relevant in the screening method applied for the present invention.

The expression libraries as used in the present invention should allow expression of all potential antigens, e.g. derived from all secreted and surface proteins of Mcat. Bacterial surface display libraries will be represented by a recombinant library of a bacterial host displaying a (total) set of expressed peptide sequences of Mcat on two selected outer membrane proteins (LamB and FhuA) at the bacterial host membrane (Georgiou, G., 1997; Etz, H. et al., 2001). One of the advantages of using recombinant expression libraries is that the identified serum-reactive antigens may be instantly produced by expression of the coding sequences of the screened and selected clones expressing the antigens without further recombinant DNA technology or cloning steps necessary.

The comprehensive set of antigens identified by the described program according to the present invention may be analysed further by one or more additional rounds of screening. Therefore individual antibody preparations or antibodies generated against selected peptides, which were identified as immunogenic can be used. According to a preferred embodiment the individual antibody preparations for the second round of screening are derived from healthy adults and/or challenged adults who show an antibody titre above a certain minimum level, for example an antibody titre being higher than 80 percentile, preferably higher than 90 percentile, especially higher than 95 percentile of the human (patient or healthy individual) sera tested. Using such high titre individual antibody preparations in the second screening round allows a very selective identification of the antigens and fragments thereof from Mcat.

Following the comprehensive screening procedure, the selected antigenic proteins, expressed as recombinant proteins, or—in case they can not be expressed in prokaryotic expression systems—in vitro translated products, or synthetically produced antigenic peptides can be tested in a second screening by a series of ELISA and Western blotting assays for the assessment of their immunogenicity with a large human serum collection (minimum ˜20 healthy and patients sera).

It is important that the individual antibody preparations (which may also be the selected serum) allow a selective identification of the most promising candidates of all the serum-reactive antigens from all the promising candidates from the first round. Therefore, preferably at least 10 individual antibody preparations (i.e. antibody preparations (e.g. sera) from at least 10 different individuals exposed to the chosen pathogen) should be used in identifying these antigens in the second screening round. Of course, it is possible to use also less than 10 individual preparations, however, selectivity of the step may not be optimal with a low number of individual antibody preparations. On the other hand, if a given antigen (or an antigenic fragment thereof) is recognized by at least 10 individual antibody preparations, preferably at least 30, especially at least 50 individual antibody preparations (in this case 22), identification of the antigen is also selective enough for a proper identification. Serum-reactivity may of course be tested with as many individual preparations as possible (e.g. with more than 100 or even with more than 1,000).

Therefore, the relevant portion of the antibody preparations according to the method of the present invention should preferably be at least 10, more preferably at least 30, especially at least 50 individual antibody preparations. Alternatively (or in combination) antigens may preferably be also identified with at least 20%, preferably at least 30%, especially at least 40% of all individual antibody preparations used in the second screening round.

According to a preferred embodiment of the present invention, the sera from which the individual antibody preparations for the second round of screening are prepared (or which are used as antibody preparations), are selected by their titre against Mcat (e.g. against a preparation of this pathogen, such as a lysate, cell wall components and recombinant proteins). Preferably, some are selected with high IgG titres, calculated from the OD reading at a given dilution when the whole organism (total lysate or whole cells) is used as antigen in the ELISA.

The recognition of linear epitopes recognized by serum antibodies can be based on sequences as short as 4-5 amino acids. Of course it does not necessarily mean that these short peptides are capable of inducing the given antibody in vivo. For that reason the defined epitopes, polypeptides and proteins are further to be tested in animals (mainly in mice) for their capacity to induce antibodies against the selected proteins in vivo.

The preferred antigens for extracellular bacteria are located on the cell surface or secreted, and are therefore accessible extracellularly. Antigens from bacteria with an intracellular stage in host cells may also be derived from intracellular locations, but need to be presented on the host cell as antigens. Antibodies against cell wall proteins are expected to serve multiple purposes: to inhibit adhesion, to interfere with nutrient acquisition, to inhibit immune evasion and to promote phagocytosis (Foxwell et al., 1998; Murphy 2005; Rao et al., 1999; Cripps and Otczyk 2006). Antibodies against secreted proteins are beneficial in neutralisation of their function as toxin or virulence component. It is also known that bacteria communicate with each other through secreted proteins. Neutralizing antibodies against these proteins will interrupt growth-promoting cross-talk between or within infection causing pathogen species. Bioinformatic analyses (signal sequences, cell wall localisation signals, and transmembrane domains) proved to be very useful in assessing cell surface localisation or secretion. The experimental approach includes the isolation of antibodies with the corresponding epitopes and proteins from human serum, and the generation of immune sera in mice against recombinant proteins selected on the basis of results from the bacterial surface display screens. These sera are then used in a third round of screening as reagents in at least one of the following assays: cell surface staining of Mcat grown under different conditions (FACS or microscopy), determination of neutralizing capacity (toxin, adherence), and promotion of opsonisation and serum bactericidal activity.

For that purpose, recombinant proteins made on the basis of results from screening of the E. coli libraries are injected into mice and immune sera are taken and tested in the relevant in vitro assay. Alternatively, specific antibodies may be purified from human or mouse sera using peptides or proteins as substrate.

According to the antigen identification method used herein, the present invention can surprisingly provide a set of comprehensive novel nucleic acids and novel proteins, antigens and fragments thereof of Mcat, among other things, as described herein. The nucleotide sequences according to the present invention encoding such antigens have preferably a nucleotide sequence which is individually set forth in SEQ ID NOs 1 to 214 (Table 4), whereby the corresponding encoded amino acid sequences have an amino acid sequence as set forth in SEQ ID NOs 215 to 428 (Table 4).

All linear fragments of a particular antigen may be identified by analysing the entire sequence of the protein antigen by a set of peptides overlapping by 1 amino acid with a length of at least 10 amino acids. Subsequently, non-linear epitopes can be identified by analysis of the antigen with sera using the expressed full-length protein or domain polypeptides thereof. Assuming that a distinct domain of a protein is sufficient to form the 3D structure independent from the native protein, the analysis of the respective recombinant or synthetically produced domain polypeptide with serum would allow the identification of conformational epitopes within the individual domains of multi-domain proteins. For those antigens where a domain possesses linear as well as conformational epitopes, competition experiments with peptides corresponding to the linear epitopes may be used to confirm the presence of conformational epitopes.

Subsequent sequencing of a larger number of randomly picked clones (approx. 600 clones per screen) led to the identification of the gene and the corresponding peptide or protein sequence that was specifically recognized by the human serum antibodies used for screening. The frequency with which a specific clone is selected reflects at least in part the abundance and/or affinity of the specific antibodies in the serum used for selection and recognizing the epitope presented by this clone. Table 4 summarizes the data obtained for the two performed screens.

It is further worth noticing that a large number of the genes identified by the bacterial surface display screen encode proteins of Mcat, which have no assigned function or may even constitute proteins, which have not been predicted by previous bioinformatic analysis. Thus, many of these candidates constitute novel antigenic proteins of Mcat.

Example 2

Characterization and Selection of Human Serum Samples Based on Anti-Mcat Antibodies and Preparation of Antibody Screening Reagents

Experimental Procedures

Human Serum Collection.

From 167 otitis media patients, middle ear fluid, sinus fluid and serum were obtained. Routine clinical examination and microbiology were used to establish clinical status and infection status with Mcat. Serum samples were also collected from 49 children with history of asthma but without history of otitis media for the last months, and a total of 13 serum samples were collected from children with no recent episode of acute otitis but with a history of recurrent otitis media. Finally, 85 serum samples were obtained from patients with respiratory allergies, and 54 serum samples were collected from healthy exposed individuals without oropharyngeal Mcat colonization, to serve as a reference for functional (protective) antibodies.

Serum samples were stored at −80° C. for long term storage, or at 4° C. with the addition of 0.05% sodium azide for short term storage for use in ELISA.

Enzyme-Linked Immunosorbent Assay (ELISA).

Mcat strain RH4 was grown in BHI liquid medium at 37° C., 5% CO₂ until a late log phase was reached. Cells were harvested by centrifugation (1,000×g, 10 min, 4° C.) and washed twice with PBS. Bacteria were re-suspended in PBS containing protease inhibitors and then lysed on ice by sonication (2 min, pulse 5, 100% power). The supernatant was collected by centrifugation (1,000×g, 15 min, 4° C.). The protein concentration was determined by Bradford assay.

ELISA plates (Maxisorb, Millipore) were coated with 5-10 μg/ml total protein diluted in coating buffer (0.1 M sodium carbonate pH 9.2). Two dilutions of sera (10,000×, 50,000×) were made in 1% BSA, PBS. Highly specific Horseradish Peroxidase (HRP)-conjugated anti-human IgG secondary antibodies (Southern Biotech, U.S.A.) were used according to the manufacturer's recommendations (dilution: 1,000×). Antigen-antibody complexes were quantified by measuring the conversion of the substrate (ABTS) to colored product based on OD405 nm readings by automatic ELISA reader (TECAN SUNRISE, Austria).

Immunoblotting.

Bacteria were solubilised by adding SDS-PAGE sample buffer containing SDS and 2-mercaptoethanol, and heat-denatured. Approximately 5 μg total proteins were separated by SDS-PAGE using the BioRad Mini-Protean 3 Cell electrophoresis system and proteins were transferred to nitrocellulose or PVDF membranes. After overnight blocking in 5% milk, human sera were added at between 1:1,000 and 1:5,000 dilution, and HRP labelled anti-human IgG was used for detection.

Purification of Antibodies for Genomic Screening.

For purification of immunoglobulin (IgGs), human serum pools were heat-inactivated at 56° C. for 30 min and centrifuged to remove precipitated proteins. The supernatant was passed through a 0.2 lam filter. Antibodies against E. coli proteins were removed by incubating the heat-inactivated sera with whole cell E. coli cells (DH5alpha, transformed with pHIE11, grown under the same condition as used for bacterial surface display). E. coli cells in the logarithmic growth phase were collected by centrifugation from a 100 ml culture, washed twice with PBS and re-suspended in PBS to a final volume of approximately 100 μl. One ml serum and 100 μA of bacterial cell suspension are mixed and incubated for 6 to 15 hrs at 4° C. under continuous agitation (Rotator Drive STR4, Stuart Scientific). After centrifugation at 8,000 rpm at 4° C. for 10 min, the supernatant is transferred into a new tube and depletion repeated twice using the same volume of E. coli cell suspension. Finally, bacterial cells are removed by centrifugation and the serum filtered through a 0.22 μm Spin-x® filter (Costar, U.S.A.).

For IgG purification, samples were diluted 1:3 with Binding Buffer (Pierce, U.S.A.) and applied to equilibrated Ultralink Immobilized Protein G columns (Pierce, #53125). A volume of binding solution containing 1 ml serum was applied to 1 ml Protein G beads. After washing the column with 20 bed volumes of Binding Buffer, bound IgGs were recovered by elution with Elution Buffer (Pierce, U.S.A.). The eluted fractions were adjusted immediately to physiologic pH by adding 1/10 of the volume of Neutralization Buffer. Samples with the highest absorbance were pooled and dialyzed against PBS.

The efficiency of depletion and purification was checked by 12% SDS-PAGE and the protein concentration was determined by comparison to a BSA standard on the same SDS-PAGE analysis. Pooled IgG fractions were biotinylated according to the manufacturer's instructions (EZ-Link Sulfo-NHS-LC-Biotin, Pierce) and the biotinylation controlled by qualitative Western blot analysis.

Results

Human sera, collected from otitis media patients, people suffering from respiratory allergies and healthy individuals, were analysed by ELISA on Moraxella catarrhalis (Mcat) lysate. The high titer sera were further analysed by Western blot (FIG. 1).

As expected with a complex Mcat lysate antigen, all the sera reacted with several bands from 250 to 10 kDa (FIG. 1). The reactivity of P4092 against Mcat antigens was particularly weak (FIG. 1). Two bands at approximately 90 kDa and 70 kDa were recognized by almost all sera (FIG. 1). In paired serum samples from the same patient, there was little if any difference between sera taken early and late in the course of otitis media (FIG. 1). Nevertheless, the convalescent serum from P4060 exhibited stronger reactivity to a band at approximately 70 kDa, and the convalescent serum from P4101 had reactivity with a doublet band at approximately 65 kDa, which was not present in the acute serum from this patient (FIG. 1). The low degree of differences between acute and convalescent sera could be due to otitis media being a multifactorial disease, with several viral as well as bacterial pathogens potentially being involved, which could reduce immune responses against Mcat.

The sera giving strong signals in Western blot analysis with Mcat antigen were selected to make serum pools to select bacterial display libraries (FIG. 1). In total, 4 different serum pools were generated based on the available clinical diagnosis: Pool IC20, sera IC58B, IC84B, IC85B, IC86B and IC89B (healthy individuals). Due to the limited serum volume, IC54A serum was used instead of IC84B serum for peptide ELISA. Pool P37, sera P3918, P3923, P3941, P3943 (children with asthma, age 5-17 years) and P3965 (a child with recurrent OM). Pool P36, sera P3792, P3801, P3819, P3832, P3861 (patients with respiratory allergies). Pool P39, individual sera P4060.2, P4070.2, P4072.2, P4101.2 and P4115.2 (patients recovering from otitis media). P4120 convalescent serum (P4120.2) was not part of the pool used for genomic peptide display library screening, but P4120 acute and convalescent serum pair was included in peptide ELISA due to the fact that Moraxella was isolated from this patient's clinical sample.

The serum pools were then preadsorbed on E. coli to remove anti-E. coli antibodies, including antibodies against the FhuA and LamB proteins used as scaffolds in the surface display libraries, and thereby reduce nonspecific binding in the subsequent MACS screening. Following preadsorption, human IgGs were purified, and biotinylated.

The IgG concentrations in each serum pool obtained after biotinylation were: Pool IC20, 0.38 mg/ml. Pool P36, 0.38 mg/ml. Pool P37, 0.21 mg/ml. Pool P39, 0.30 mg/ml.

These serum pools were used for MACS screening of the Mcat RH4 surface display libraries.

Example 3

Generation of Highly Random, Frame-Selected, Small-Fragment, Genomic DNA Libraries of Moraxella catarrhalis (Mcat)

Experimental Procedures

Preparation of genomic DNA from Moraxella catarrhalis RH4. Genomic DNA from Mcat RH4 was prepared using standard protocols. Briefly, cells from a 50 ml culture in BHI medium (Brain heart infusion medium) were harvested by centrifugation and resuspended in 8 ml TE buffer. After addition of 1 ml 10% SDS and 0.5 ml proteinase K (20 mg/ml) the solution was incubated at 37° C. for 1 h, followed by repeated phenol/chloroform extractions (at least twice). Genomic DNA was precipitated by adding 1/10 volumes of 3 M sodium acetate (pH 5.3) and 2 volumes of absolute ethanol, transferred to 70% ethanol using a sterile loop and centrifuged. The pellet was dried, redissolved in 1 ml TE buffer containing 0.2 mg/ml RNaseA and incubated at 37° C. for 30 min to remove residual RNA. The DNA was ethanol precipitated again and finally dissolved in 0.5 to 1 ml Tris/HCl buffer (10 mM, pH 8.5).

Preparation of small genomic DNA fragments from Mcat RH4. Depending on the desired size different methods were used for the random fragmentation of genomic DNA. Fragments ranging in size between 150 and 300 bp were obtained by sonication of approx. 100 μg DNA in 5×50 μl Tris/HCl (10 mM, pH 8.5) using a cup-horn sonicator (Bandelin Sonoplus UV 2200 sonicator equipped with a BB5 cup horn, 10 sec. pulses at 100% power output). Fragments of 50 to 150 bp in size were obtained by mild DNase I treatment using Novagen's shotgun cleavage kit. According to the manufacturer's instructions, preliminary tests using different DNase I dilutions were performed and evaluated on a 2% TAE agarose gel. The determined optimum conditions were applied for cleavage of approx. 100 μg DNA. This treatment resulted in total fragmentation of genomic DNA into near 50-150 bp fragments.

Following fragmentation, the DNA was separated on 5% TBE polyacrylamide gels or 2% TAE agarose gels (after DNase cleavage only), the areas corresponding to DNA fragments of 50-150 bp and 150-300 bp, respectively, were cut out and the DNA isolated using the QiaExII kit (Qiagen, Hilden, Germany).

To remove overhanging ends, the isolated fragments were then incubated with T4 DNA polymerase in the presence of dNTPs (100 μM each), followed by phenol/chloroform extraction and ethanol precipitation. The fragments were used immediately in ligation reactions or frozen at −20° C. for subsequent use.

Description of the vectors. The vector pMAL4.31 was constructed on a pASK-IBA backbone (Skerra 1994) with the beta-lactamase (bla) gene exchanged with the Kanamycin resistance gene. In addition, the bla gene was cloned into the multiple cloning site. The sequence encoding mature beta-lactamase is preceded by the leader peptide sequence of ompA to allow efficient secretion across the cytoplasmic membrane. Furthermore a sequence encoding the first 12 amino acids (spacer sequence) of mature beta-lactamase follows the ompA leader peptide sequence to avoid fusion of sequences immediately after the leader peptidase cleavage site, since e.g. clusters of positive charged amino acids in this region would decrease or abolish translocation across the cytoplasmic membrane (Kajava et al., 2000). A SmaI restriction site serves for library insertion. An upstream FseI site and a downstream NotI site, which were used for recovery of the selected fragment, flank the SmaI site. The three restriction sites are inserted after the sequence encoding the 12 amino acid spacer sequence in such a way that transcription of the bla gene stops 15 bp after the NotI site. A 3n+1 bp insertion restores the bla ORF so that beta-lactamase protein is produced with a consequent gain of Ampicillin resistance.

The vector pMAL9.1 was constructed by cloning the lamB gene into the multiple cloning site of pEH1 (Etz, H. et al., 2001). Subsequently, a linker containing the restriction sites FseI, SmaI and NotI was inserted in lamB after the sequence coding for serine-155 by PCR mutagenesis. The reading frame for this insertion was constructed in such a way that transfer of frame-selected DNA fragments excised by digestion with FseI and NotI from plasmid pMAL4.31 yields a continuous reading frame of lamB and the respective insert.

The vector pHIE11 was constructed by cloning the fhuA gene into the multiple cloning site of pEH1 (Etz, H. et al., 2001). Subsequently a sequence was inserted in fhuA after the sequence coding for proline-405, containing the restriction site FseI, XbaI and NotI. Vector pHIE14 differs from pHIE11 by an additional sequence of 162 bp cloned into the XbaI site. As described above for pMAL9.1, cloning of a 3n+1 bp fragment into the FseI and NotI sites results in a continuous reading frame of fhuA and the respective insert.

Cloning and evaluation of the library for frame selection. Genomic DNA fragments from Mcat RH4 were ligated into the SmaI site of the vector pMAL4.31. Recombinant DNA was electroporated into DH10B electrocompetent E. coli cells (GIBCO BRL) and transformants plated on LB-agar supplemented with Kanamycin (50 μg/ml) and Ampicillin (50 μg/ml). Plates were incubated over night at 37° C. and colonies collected for large scale DNA extraction. A representative plate was stored and saved for collecting colonies for colony PCR analysis and large-scale sequencing. A simple colony PCR assay was used to initially determine the rough fragment size distribution as well as insertion efficiency. From sequencing data the precise fragment size was evaluated, junction intactness at the insertion site as well as the frame selection accuracy (3n+1 rule).

Cloning and evaluation of the library for bacterial surface display. Genomic DNA fragments were excised with the restriction enzymes FseI and NotI from the pMAL4.31 vector, containing the Moraxella catarrhalis RH4 library. The entire population of fragments was then transferred into plasmids pMAL9.1 (LamB) or pHIE11 (FhuA), which have been digested with FseI and NotI. Using these two restriction enzymes, which recognize an 8 bp GC rich sequence, the reading frame that was selected in the pMAL4.31 vector is maintained in each of the platform vectors. The plasmid library was then transformed into E. coli DH5alpha cells by electroporation. Cells were plated onto large LB-agar plates supplemented with 50 μg/mL Kanamycin and grown over night at 37° C. at a density yielding clearly visible single colonies. Cells were then scraped off the surface of these plates, washed with fresh LB medium and stored in aliquots for library screening at −80° C.

Results

Genome Sequencing of Moraxella catarrhalis RH4

The evaluation of the constructed libraries as well as of the subsequent immuno-screening is facilitated considerably if the genome sequence of the Moraxella catarrhalis strain from which libraries are constructed is known.

Therefore, the genome of M. catarrhalis RH4 was done using second generation sequencing technology. Primary sequence acquisition on a Roche GS20/FLX system yielded 591,043 high quality sequence reads with an average reading length of 224 bp (132 Mb raw data). By de novo assembly 44 contigs were obtained. After comparison with the 41 contigs of the unfinished M. catarrhalis ATCC43617 genome, all gaps could be closed preliminary by PCR methods resulting in one contig of 1.9 Mb.

Bacterial Surface Display Libraries

Ligation and subsequent transformation of approximately 1 μg of pMAL4.31 frame selection plasmid DNA and approximately 50 ng of fragmented and blunt-ended genomic Mcat RH4 DNA yielded 3.6×10⁵ to 1.2×10⁶ clones after frame selection. Based on the frame-selected DNA, two libraries (50/2 and 250) were generated in the pMAL9.1 (LamB) or pHIE11 (FhuA) display vectors, respectively. Frame-selected genomic DNA fragments were excised with FseI and NotI restriction digest, and approximately 5 ng fragments were ligated with 0.1 μg display plasmid DNA (pMAL9.1, LamB display scaffold, or pHIE11, FhuA display scaffold). Transformation into DH5alpha cells resulted in approximately 860,000 clones (50/2 library) and 913,500 clones (250 library). For each library, colonies were scraped off LB plates, pooled and frozen without further amplification.

To assess the size distributions and Mcat genome coverage of the libraries, randomly chosen library clones were sequenced (FIG. 2). The 50/2 and 250 libraries exhibited average insert sizes of 87 and 199 bp, respectively (FIG. 2). The 50/2 library comprised 860,000 colony-forming units, giving a calculated coverage of the Mcat genome of 40-fold. The 250 library comprised 913,500 colony-forming units, giving a calculated coverage of the Mcat genome of 98-fold.

For both libraries, the displayed inserts were distributed equally along the complete genome of Mcat RH4 (FIG. 2). I.e., both libraries provided a non-biased and highly redundant sampling of the complete Mcat RH4 genome.

Example 4

Identification of Highly Immunogenic Peptide Sequences from Moraxella catarrhalis (Mcat) Using Bacterial Surface Display Genomic Libraries and Human Sera

Experimental Procedures

MACS screening. Approximately 1×10⁸ colony-forming units from a given library were grown in 5 mL LB-medium supplemented with 50 μg/mL Kanamycin at 37° C. for 50 min (FhuA library) and 30 min (LamB library), respectively. Expression was induced by the addition of 1 mM IPTG for 40 min (FhuA) and 2 h (LamB), respectively. Cells were washed twice with fresh LB medium and approximately 8×10⁶ cells re-suspended in 100 μL LB medium and transferred to an Eppendorf tube.

An amount of 20 μg of biotinylated, human IgGs was added to the cells and the suspension incubated overnight at 4° C. with gentle shaking. The cells were harvested, washed twice with 1 ml LB-Kan, and resuspended in 100 μl LB-Kan. An amount of 15 μl of streptavidin coupled microbeads (Miltenyi Biotec) were added and the mixture was incubated at 4° C. for 45 min. The MACS microbead cell suspension was washed once, resuspended in 1 ml LB-Kan and loaded onto pre-equilibrated MACS separation columns MS (Miltenyi Biotec) which was fixed to the magnet (the MS columns were equilibrated by washing with 3 ml 70% ethanol and 2×3 ml LB-Kan).

The columns were then washed three times with 3 ml LB-Kan, and elution was performed with 2 ml LB-Kan by removing the magnet. After washing the columns three times with LB-Kan, the eluate was reloaded, washed and eluted as before. Aliquots of the final eluate after three column binding/elution rounds were plated on LB-Kan agar plates and grown over night at 37° C.

Evaluation of selected clones by sequencing and Western blot analysis. Randomly selected clones were grown overnight at 37° C. in 200 μl LB medium supplemented with 50 μg/mL Kanamycin. Bacterial clones on LB/Kan agar were used for sequencing at Agowa (Germany).

For Western blot analysis, library E. coli clones were precultured in LB-Kan and induced with 1 mM IPTG for 2 h (FhuA) or 4 h (LamB). E. coli library clones which were shown by DNA sequence analysis not to display peptides, but did display the FhuA or LamB scaffold proteins as appropriate, served as negative controls in Western. Approximately 10 to 20 μg of total bacterial protein was separated by 10% SDS-PAGE and blotted onto PVDF membranes (Amersham Pharmacia Biotech, England). The reactivity of peptides displayed on the LamB or FhuA fusion proteins were detected using purified human IgG as the primary antibody at a dilution of approximately 1:3,000 to 1:5,000, and HRP-coupled goat anti-human IgG at a dilution of 1:50,000 as secondary antibody. Detection was performed using the Immobilon HRP substrate (Millipore, U.S.). Alternatively, rabbit anti-FhuA or rabbit anti-LamB polyclonal immune sera were used as primary antibodies in combination with HRP-coupled donkey anti-rabbit to verify expression of scaffold proteins for all clones.

Peptide ELISA.

Biotin-labeled peptides (at the N-terminus) were resuspended in DMSO to ˜2 mg/ml concentration, divided into smaller aliquots and stored at −20° C. (for short term storage) or −80° C. (long term storage). Streptavidin-coated microtiter ELISA plates (Nunc, 436014) were washed three times with PBS containing 0.2% Tween-20 detergent (PBST). The peptides were diluted in PBS at concentration 5 μg/ml and used for coating the plates at 4° C. overnight. The streptavidin/peptide-coated plates were blocked with PBS supplemented with 2% BSA for 1 hr at RT and then washed with PBST three times. Sera were diluted 1:1,000 in PBS supplemented with 1% BSA and incubated with the peptides on the plates for 1 hr at room temperature. The bound antibodies were detected with highly specific Horseradish Peroxidase (HRP)-conjugated anti-human IgG secondary antibodies (Pierce, 31412) used at 1:2,000 dilution. Antigen-antibody complexes were quantified by measuring the conversion of the substrate ABTS (Sigma, A3219) to colored product based on OD405 nm readings by automatic ELISA reader (SynergyTM2, Biotek).

Results

For surface display libraries representing the genome of the Mcat strain RH4, nine screens were performed, using two bacterial surface display libraries in LamB and FhuA and four human IgG pools (Table 1). It was observed that the IgG pools IC20 and P39 selected mainly antigens from the already well known immunodominant UspA2H and Hag antigens (Murphy 2005; Mawas et al., 2009; Tan et al., 2007a). Therefore, these IgG pools were preadsorbed with coli library clones displaying immunodominant UspA2H and Hag sequences (Table 1). Library screening with the adsorbed IC20 and P39 IgG pools had the intended effect of identifying less dominant Mcat antigens (Table 1). The IgG pools P36 and P37 did not show predominant selection of already known immunodominant antigens, and were therefore not adsorbed with bacterial library clones displaying immunodominant Mcat antigen fragments (Table 1).

For both libraries and in all 9 screenings, screening with biotinylated human IgG produced between 1.1 and 38-fold more binding clones (relative to input), than did screening without human IgG (Table 1, “specific enrichment”). Thus, the clones selected with the biotinylated human IgG were enriched for Mcat sequences recognized by IgG purified from the immune serum pools.

For each screen, approximately 800 clones were randomly selected and the sequences of the displayed Mcat peptides deduced by DNA sequencing. For Mcat ORFs that were selected more than three times per screen, library clones tested in Western blot analysis using the corresponding IgG pool as used for screening (Table 2). As expected based on the specific enrichment observed during screening (Table 1), a high percent and in most cases the majority of the selected clones were positive in Western blotting with human sera (Table 2).

Following DNA sequence and Western analysis, lead candidate antigens were selected, for which overlapping synthetic peptides were designed. The reactivity of human sera against the peptides was examined by ELISA (an example is given in Table 3).

Multiple peptides from individual antigens from Mcat RH4 were recognized by human sera (an example is given in Table 3). In paired serum samples from the same patient, there was evidence for seroconversion against peptides from MCRH1546 and MCRH1586. As mentioned for the Western blot analysis, the lack of difference between acute and convalescent sera for peptides derived from e.g. MCRH1565 (Table 3) could be due to otitis media being a multifactorial disease, with several viral as well as bacterial pathogens potentially being involved, which could reduce immune responses against Mcat.

The antigenic peptides recognized with the greatest seroconversion rates using the sera obtained from otitis media patients suggested that the antibodies targeting these epitopes played important role in overcoming the infection caused by this pathogen. On the other hand, the antigenic peptides that elicited high antibody titers in healthy population may have been important for overcoming the initial stages of infection (colonization).

Based on the combination of DNA sequence analysis, Western blotting and peptide ELISA, 214 antigens were identified from Moraxella catarrhalis strain RH4, that reacted with human sera (Table 4). The identified epitopes/proteins are immunogenic in humans, suggesting that they are expressed by the pathogen during infection and are capable of inducing a strong immune response (Table 4).

Example 5

Determination of the Conservation of the Immunogenic Mcat Proteins in Diverse Mcat Isolates

An ideal vaccine should be present in all, or the vast majority of strains of the target organism the vaccine is directed against. In order to examine the conservation of the open reading frames encoding the identified Mcat antigens, PCR was performed on 47 Mcat isolates (Table 5) with primers specific for the gene of interest. The conservation of all 214 identified Mcat antigens (Table 4) was thus tested.

Experimental Procedures

Isolation of Genomic DNA:

Moraxella spp. bacteria from a frozen stab were grown overnight at 37° C. on BHI agar. Next day, one well isolated colony was used to inoculate 7 ml BHI medium, followed by incubation with shaking at 37° C. for 18 h.

The 7 ml liquid cultures were harvested by centrifugation at 13,000×g for 2 min, the supernatant was removed by aspiration, and bacterial pellets lysed in 600 μl of Lysis Solution (Wizard Genomic Purification kit, Promega). Bacterial genomic DNA was purified according to the manufacturer's instructions (Wizard Genomic Purification kit, Promega).

The bacterial genomic DNA was dissolved in 60 μl of sterile bi-distilled water. The quality of the isolated genomic DNA was examined by 1% agarose gel electrophoresis and ethidium bromide (EtBr) staining, and the genomic DNA was stored at −80° C.

PCR:

PCR was performed with primers specific for the genes of interest. DNA oligonucleotide primers were designed using the public domain program Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_slow.cgi), or picked by hand. The fragment size was set to 1,000 bp, and regions identified as antigenic were incorporated in the amplified fragment. In cases where the ORF was smaller than 1,000 bp, the entire ORF was used for the analysis. The PCR primers are listed in Table 7.

PCR was performed in 25 μl reaction volumes, using Taq polymerase (1 U, Solis Biodyne, Estonia), 200 μM dNTPs (Fermentas, Germany), 2 mM MgCl₂ (Solis Biodyne, Estonia), 0.4 μMol of each oligonucleotide (MWG, Germany) and 10-20 ng of genomic DNA.

The cycling conditions were:

Heat denature 95° C., 5 min.

30-35 cycles of (95° C. 30 s, anneal 30 s, 72° C. 1 min 20 s)

72° C. 10 min

Soak 4° C.

The annealing temperature was adjusted for primers as required, but was generally 56° C. The PCR reactions were electrophoresed on 1.0% agarose TAE gels, together with 100 bp ladder (Invitrogen, 15628-050, 1 μg/ml). Gels were stained with EtBr and photographed on a standard UV table using an Olympus SP-500 UZ camera, appropriate filters, and AlphaEase FC software (v 5.0.1, Alpha Innotech Corporation).

To avoid false-positive results, negative control PCR reactions with water as template were included in all runs.

To avoid false-negative results, the library isolate RH4 was included in all PCR runs, to serve as positive control. In addition, all PCR reactions negative in the first amplification round were repeated in a second round, generally under the same conditions. In some cases, a third round was done applying changed cycling conditions and a proofreading thermostable DNA polymerase.

For each ORF, the number of isolates, where the ORF could be detected by PCR was first determined, based on the PCR analysis strategy described above. This number was termed “N”. The level of conservation can be calculated for each ORF as: N/47. N is stated in Table 6.

Results

An example of the PCR analysis is given in FIG. 3A. The antigenic open reading frame MCRH3709 was conserved in 47 out of 47 examined Mcat isolates (FIG. 3A and Table 6). The antigenic open reading frame MCRH3844 was conserved in 26 out of 47 examined Mcat isolates (FIG. 3A and Table 6).

For the 214 ORFs encoding antigenic Mcat proteins (Table 4), it was found that the majority of ORFs were conserved amongst a set of 47 reference Mcat isolates comprising worldwide geographical locations (Table 5, Table 6 and FIG. 3). For example, 138 antigenic ORFs were conserved in all 47 Mcat isolates (Table 6 and FIG. 3B), and the majority or 92% of the 214 examined ORFs were conserved in more than 90% of the 47 examined Mcat isolates (FIG. 3C).

Thus, the identified antigenic Mcat proteins (Table 4) were generally highly conserved (Table 6 and FIG. 3), supporting their use as vaccine antigen.

Example 6

Testing Protectivity of M. catarrhalis Candidate Antigen-Induced Immune Response Against M. catarrhalis Infection

Study Rationale:

Although M. catarrhalis is a human pathogen, several animal models have been developed in order to study protective potential of candidate antigens against M. catarrhalis infection and colonization. Pulmonary clearance models involve the direct inoculation or aerosolization of bacteria into the nasal cavity or lungs of experimental animals, including rats and mice. The rate at which the bacteria are cleared from the respiratory tract (nasal cavity, broncho-alveolar space and lungs) is measured following pulmonary challenge by live bacteria. Pulmonary clearance models are being used to evaluate potential vaccine antigens of both H. influenzae and M. catarrhalis (Murphy T F, 2005). Groups of animals are immunized with a putative vaccine antigen and an appropriate adjuvant. The enhancement of clearance by immunization with a putative vaccine antigen compared with controls is interpreted as a positive result, suggesting that the antigen may have a protective effect. The observation that an antigen induces enhanced pulmonary clearance in such animal model represents a line of evidence that the antigen may induce protection in humans.

The protective potential of M. catarrhalis protein antigens was studied in two mouse pulmonary clearance models performed independently by two different research groups. According to the Model #1 mice are immunized by intraperitoneal immunization using incomplete Freund's adjuvant (IFA), bacterial challenge is performed by introducing bacteria (M. catarrhalis strain RH4, 1×10⁶) directly into trachea and the bacterial colony forming units (CFU) are determined in lungs and bronchoalveolar lavage 6 hr after the challenge. Model #2 is performed using the same bacterial strain but a different mouse strain; mice are immunized by mucosal immunization in the presence of Intercell's IC31® adjuvant and bacterial challenge is performed by intranasal inoculation using the a similar CFU load (5×10⁶). The bacterial CFU are measured 12 hr after challenge in lungs and nasopharyngeal lavage. The highly conserved M. catarrhalis proteins that were selected during the library screening using human sera were expressed and purified as recombinant proteins and used for immunization of mice, in order to induce a hyperimmune response and evaluate the rate of bacterial clearance following the challenge.

Experimental Procedures

Cloning and Expression of Recombinant M. catarrhalis Proteins

Cloning of genes/DNA fragments: The gene/DNA fragment of interest was amplified from genomic DNA of M. catarrhalis strain RH4 by PCR using gene specific primers. Apart from the gene specific part, the primers had restriction sites that aided in a directional cloning of the amplified PCR product. The gene annealing (specific) part of the primer ranged between 15-30 bases in length. The PCR products obtained were digested with the appropriate restriction enzymes and cloned into the pET28b (+) vector (Novagen) for His-tagged proteins. The constructs are listed in Table 8. Once the recombinant plasmid was confirmed to contain the gene of interest, E. coli BL21-CodonPlus (DE3)-RIPL cells (Stratagene) that served as expression host were transformed.

TABLE 8
List of genes selected for expression.
						SEQ ID	SEQ ID
Nr. of	Name of			DNA	Protein	NO	NO
construct	construct	Vector	RE	(start/stop)	(start/stop)	(DNA)	(protein)
1	MCRH0357-1	pET28b(+)	NcoI/XhoI	106-1455	36-485	860	887
2	MCRH0391-1	pET28b(+)	NcoI/XhoI	58-570	20-190	861	888
3	MCRH0546-1	pET28b(+)	NcoI-BspHI/XhoI	118-834	40-278	862	889
4	MCRH0759-1	pET28b(+)	NcoI/XhoI	61-480	21-160	863	890
5	MCRH0759-2	pET28b(+)	NcoI/XhoI	460-2739	154-913	864	891
6	MCRH1493-1	pET28b(+)	NcoI/NotI	112-705	38-235	865	892
7	MCRH1546-1	pET28b(+)	NcoI/XhoI	58-333	20-111	866	893
8	MCRH1701-1	pET28b(+)	NcoI/XhoI	58-786	20-262*	867, 868	894, 895
9	MCRH2506-1	pET28b(+)	NcoI/XhoI	61-456	21-152	869	896
10	MCRH2669-1	pET28b(+)	NcoI/NotI	88-723	30-241	870	897
11	MCRH2844-1	pET28b(+)	NcoI/XhoI	70-2037	24-679	871	898
12	MCRH3040-1	pET28b(+)	NcoI/XhoI	91-1815	31-605	872	899
13	MCRH3040-2	pET28b(+)	NcoI/XhoI	1693-2757	565-919	873	900
14	MCRH3709-1	pET28b(+)	NcoI-AflIII/XhoI	79-1158	27-386	874	901
15	MCRH3729-1	pET28b(+)	NcoI/XhoI	88-1125	30-375	875	902
16	MCRH3759-1	pET28b(+)	NcoI/XhoI	79-444	27-148	876	903
17	MCRH3966-1	pET28b(+)	NcoI/XhoI	58-1410	20-470	877	904
18	MCRH4526-1	pET28b(+)	NcoI/XhoI	139-576	47-192	878	905
19	MCRH4526-2	pET28b(+)	NcoI-BspHI/XhoI	544-2883	182-961	879	906
20	MCRH4706-1	pET28b(+)	NcoI-AflIII/XhoI	130-1536	44-512	880	907
21	MCRH4712-1	pET28b(+)	NcoI/XhoI	91-315	31-105	881	908
22	MCRH4730-1	pET28b(+)	NcoI/XhoI	82-1674	28-558	882	909
23	MCRH4740-1	pET28b(+)	NcoI-BspHI/XhoI	76-909	26-303	883	910
24	MCRH5107-1	pET28b(+)	NcoI/XhoI	109-765	37-255	884	911
25	MCRH5471-1	pET28b(+)	NcoI/XhoI	166-909	56-303	885	912
26	MCRH5471-2	pET28b(+)	NcoI-BspHI/XhoI	877-2358	293-786	886	913
The nomenclature of the genes is derived from the project-internal naming of the genome of M. catarrhalis strain RH4 (unpublished data). For a comprehensive study on the recently published genome, supplemented with a revised genomic annotation, please see de Vries S. P., et al., 2010 and the corresponding GenBank accession CP002005.1 (gi: 295919923). Notably the published genomic sequence was fully reversed, then several places repositioned and corrected, thus generally improved.
The restriction sites (RE) used for cloning and the position (start/stop) of the amplicons are indicated for each construct.
*The residue Threonine at position 261 relative to the native protein (“wild-type” fragment DNA/protein: SEQ ID NOs 867, 894) was changed to a Proline according to the sequence confirmation of the expression construct (“mutated” fragment DNA/protein: SEQ ID NOs 868, 895). Mutation could be due to PCR amplification or be a mutation in the particular RH4 strain used for PCR amplification.

Expression and Purification of Proteins:

E. coli BL21-CodonPlus (DE3)-RIPL cells harboring the recombinant plasmid were grown into log phase in the required culture volume. Once an OD_600nm of 0.6 was reached the culture was induced with 0.1 mM IPTG for 3 hours at 37° C. The cells were harvested by centrifugation, lysed by a combination of the freeze-thaw method followed by disruption of cells with High pressure Homogenizer Panda 2K, (Niro Soavi). The lysate was separated by centrifugation into soluble (supernatant) and insoluble (pellet) fractions. Depending on the location of the protein different purification strategies were applied. A) If the His-tagged protein was in the soluble fraction, protein purification was done by binding the supernatant to Ni-Sepharose beads (Ni-Sepharose™ 6 Fast Flow, GE Healthcare). Due to the presence of the hexa Histidine (6×HIS) at the C-terminus or N-terminus of the expressed protein, it bound to the Ni-Sepharose while the other contaminating proteins were washed from the column by wash buffer. The protein was eluted by 250 mM Imidazole in 50 mM Tris, 150 mM NaCl buffer at pH 8.0. The eluate was dialyzed against 50 mM Tris, 150 mM NaCl buffer at pH 8.0 and concentrated if necessary. The protein concentration was assayed by Bradford or BCA and the protein quality and integrity was checked by SDS-PAGE and Western blot.

B) If the protein was present in the insoluble fraction, the pellet was washed several times to purify inclusion bodies. Inclusion bodies were solubilized in suitable buffer containing 8 M Urea and applied onto the Ni-NTA column under denaturing conditions (in buffer containing 8 M Urea) using the same materials and procedure as mentioned above. Contaminating proteins were washed from the column under denaturing conditions. Refolding of the His-tagged protein was performed while the protein was immobilized on the Ni-NTA matrix using buffer without urea. After renaturation, proteins were eluted by the addition of 250 mM Imidazole. The eluate was dialyzed to remove traces of urea and concentrated if the volume was large, checked by SDS-PAGE and measured by the Bradford or BCA method. Alternative, dilution refolding of the His-tagged protein was performed after the protein was eluted under denaturing conditions from the Ni-Sepharose beads by diluting the protein in an appropriate buffer without urea but containing e.g. surfactants. After renaturation, proteins were concentrated checked by SDS-PAGE and protein concentration measured.

Mouse Pulmonary Clearance Study Employing Systemic Immunization:

Male BALB/c mice were used for studies (5 mice per each experimental group). Proteins were emulsified in a 1:1 ratio with Incomplete Freund's adjuvant. Killed bacteria (WKC) were used as a positive control, whereas the negative control was the adjuvant vehicle injected alone. Both control groups were formulated and delivered in the same manner as the tested antigens. OmpCD protein, a Moraxella catarrhalis outer membrane protein which increased bacterial clearance in published studies (Tymothy et al., 1998; Becker et al., 2007), was tested in parallel for the sake of comparison to our candidate antigens. Three immunizations were delivered at weekly intervals (days 0, 7 and 14), with the following protocol timelines:

On Days 0, 7 and 14:

• • the mice received an intraperitoneal injection (i.p.) of 50 μg of protein emulsified in Incomplete Freund's adjuvant.

On Day 21:

0 h Bacterial challenge:

• • live bolus challenge with live Moraxella catarrhalis strain RH4. Bacterial strains and growth conditions:

Moraxella catarrhalis strain RH4 was isolated from the blood of an infected patient (provided by Arne Forsgren, Malmö University Hospital, Sweden). Bacteria were grown on horse blood agar at 37° C./5% CO₂ or in brain heart infusion (BHI, Becton Dickinson and Company) liquid medium at 37° C. with shaking. Bacteria were grown overnight on chocolate agar, harvested and washed three times in PBS. The concentration was assessed by measurement of optical density at 405 nm and adjusted to a challenge dosage of 10⁶ CFU in 20 μl [5×10⁷ CFU/ml]. Mice were sedated by intravenous (tail vein) injection of 0.12-0.15 ml (20 mg alphadone in PBS/kg body weight). The trachea was visualised through the oral cavity, a 22.5 G catheter is inserted and a 20 μl volume of PBS containing the live bacteria is introduced into the lungs with two 0.3 ml volumes of air.

0-6 h Observations on Clinical Status of Mice

6 h Autopsy and Collection of Tissues for Bacteria and White Cell Counts in the Lung:

• • at 6 h post-challenge, mice were euthanized by anesthetic overdose. Blood was collected by heart puncture, allowed to clot and serum collected following centrifugation at 4° C., 450×g, 10 mins. Serum is stored at −80° C.
  • the lungs were lavaged with 0.5 ml PBS (Bronchoalveolar lavage—BAL). Following lavage, the lungs were excised, connective tissue and heart are removed, placed in PBS and homogenised. BAL and lung homogenate were assessed for bacterial counts by plating, on chocolate agar plates, duplicate 20 μl volumes of a 10-fold series titration. Colonies were counted next day and calculated.

Results:

Several vaccine antigen candidates have induced immune response which provided significantly faster clearance of the bacteria compared to IFA (adjuvant vehicle) control (FIG. 4).

The percentage of the bacteria (colony forming units, CFU) recovered in bronchoalveolar lavage calculated in reference to adjuvant control in phosphate based saline (IFA) is shown in (FIG. 4A). The median % value of the bacteria recovered after challenge from the animals immunized with killed whole cells (WKC) was 10%, whereas OmpCD protein did not show any increase in clearance compared to adjuvant control (IFA); therefore the bacterial CFU recovered was 100% of the IFA control group. Candidate antigens that provided ≧80% reduction in bacterial load in bronchoalveolar lavage (between 20% and 40% CFU were recovered compared to adjuvant control and OmpCD) were: MCRH5107-1 and MCRH4712-1. Therefore, these two antigens were almost as efficient as the whole killed cells positive control. Antigens that induced between 40% and 60% bacterial CFU reduction were MCRH0357-1, MCRH0759-1, and MCRH4730-1, whereas antigen MCRH2844-1 provided only 25% CFU reduction compared to adjuvant control in this study model. Antigen MCRH4526-1 provided 40% reduction in bacterial CFU recovered from BAL, compared to adjuvant control.

FIG. 4B shows the bacterial recovery from the lung tissue collected from the mice 6 h after challenge with M. catarrhalis RH4. In the lung homogenates from the animals immunized with whole killed cells, the bacterial recovery was 40% of the median CFU recovered from lungs of adjuvant (IFA) immunized mice, and in the animals immunized with the protein OmpCD there was no reduction in bacterial recovery compared to adjuvant control. The animals immunized with several M. catarrhalis candidate antigens had between 50% and 60% of bacteria recovered from the lung tissue compared to adjuvant control; these antigens were: MCRH0391-1, MCRH1493-1 and MCRH5107-1.

FIG. 4C shows a total % of recovered bacteria compared to adjuvant control group (IFA) when the results from both FIG. 4A (recovery in bronchoalveolar lavage) and FIG. 4B (recovery from lung tissue homogenate) were combined. Several Moraxella antigens still show a faster clearance of the bacteria compared to negative (IFA) control. MCRH5107-1 antigen provided overall 55% reduction in bacterial load, since bacteria recovered from both lungs and BAL represented only 45% of the median bacterial CFU recovered from the animals immunized with adjuvant control (IFA). Additional antigens that provided ˜40% faster bacterial clearance were: MCRH0357-1, MCRH0391-1 and MCRH1493-1.

Therefore, the described in vivo studies support the further use of selected antigens for testing and development of the vaccine against otitis media.

Mouse Pulmonary Clearance Study Employing Mucosal Immunization:

Bacterial Strains and Growth Conditions

Moraxella catarrhalis strain RH4 was isolated from the blood of an infected patient and obtained from Arne Forsgren (Malmö University Hospital, Sweden). Bacteria were grown on horse blood agar at 37° C./5% CO₂ or in brain heart infusion (BHI, Becton Dickinson and Company) liquid medium at 37° C. with shaking.

Mice

Five- to seven-week-old female C57/BL6 bom (wt) mice were kept under specific, pathogen-free conditions. The animals were housed five per cage in a standardized 12 h light/dark cycle and received commercial food and water ad libitum. All the experiments were conducted in conformity with the European Communities Council Directive 86/609/EEC and the Swedish animal protection legislation.

Intranasal Immunization and Blood Sampling of Mice

On day 0, before immunization, 10 μl blood samples from the tail were taken, for preparation of a pre-immunization serum pool.

On days 0, 21 and 42, groups of 10 mice were immunized intranasally with 20 (10 μl to each nostril) of antigen formulated in Intercell's adjuvant IC31®. Since the immunogenicity of the antigens was not known at the beginning of this study, maximum available antigen dose was used for immunization whenever possible, depending on the protein availability (Table 9).

Adjuvantation was done according to a detailed protocol supplied by Intercell AG. Briefly, 17.5 μl protein solution was mixed with 2.5 μl IC31® (2000 nmol/ml of KLK(L)₅KLK and 80 nmol/ml of Oligo(dIdC)₁₃), and incubated 30 min at room temperature. The adjuvanted protein was used to immunize mice within 1 hour of preparation. PBS control mice received PBS alone. Adjuvant control mice received 17.5 μA 50 mM Tris/HCl pH8 mixed with 2.5 μl IC31®.

For the intranasal immunization the mice were anaesthetized by light inhalation of isoflurane (Forene; Abbott) (4% isoflurane, flow 500-700 ml/min).

Mouse Challenge

Mice were anaesthetized by light inhalation of isoflurane (Forene; Abbott) (4% isoflurane, flow 500-700 ml/min) and subsequently inoculated intranasally with 40 μl (20 μl to each nostril) live M. catarrhalis strain RH4˜5×10⁶ cfu.

For mouse inoculation, RH4 was grown in BHI medium to an OD_620nm of 0.4. Bacteria were pelleted, and resuspended in PBS. Mice were held in a head-up vertical position during the inoculation and kept in that position for at least 10 seconds after the inoculation.

Euthanasia, Tissue Collection and Bacterial Culture

Mice were killed at 6 hours post-infection using an overdose of isoflurane and neck dislocation. Blood samples to quantitate the antibody response following the intranasal immunizations of mice with the purified recombinant proteins were obtained from each mouse and put on ice to clot. After centrifugation individual serum samples were frozen at −20° C.

Tracheal-nasopharyngeal lavage samples were obtained by inserting a needle in the trachea pointing cranially, and flushing with PBS, collecting the first 100 μl of liquid coming out of the nares.

Both lungs were removed, placed in 1 ml PBS plus protease inhibitor (Roche), homogenized using cell strainers (100 μm, Becton Dickinson and Company) and used for serial plating to quantify viable bacteria.

The experiments were repeated at least over two independent replicates. Data on the number of colony-forming units (cfu) in the nasopharynx and lungs of the mice were corrected for the infectious dose to 5×10⁶ cfu bacteria and analyzed with nonparametric Kruskal-Wallis tests and Dunn's posttesting.

TABLE 9
List of antigens and the immunization dose.
			Dose/
		Conc.	mouse
Group	Antigen	[μg/ml]	[μg]	Batch number
1st antigen*	MCRH2506-1	2.97	51.97	CS718/151841
2nd antigen*	MCRH2844-1	5.9	103.25	JF672/143356
3rd antigen*	MCRH0759-1	2.33	40.77	JF672/143335
4th antigen*	MCRH3709-1	1.03	18.03	CS718/151869
5th antigen*	MCRH0357-1	0.94	16.45	CS718/151980
6th antigen*	MCRH3729-1	6.46	113.05	MS708/145598d
7th antigen*	MCRH3759-1	1.68	29.40	CS718/151880b
8th antigen*	MCRH4730-1	5.43	95.03	JF672/143337b
positive control*	OmpCD	2.50	43.75	CS718/151880a
negative control,
IC31 ® alonex
negative control,
PBS alone
*17.5 μl antigen plus 2.5 μl IC31 ® adjuvant
x17.5 μl buffer (50 mM Tris/HCl pH 8) plus 2.5 μl IC31 ® adjuvant

Results

Enhancement of Pulmonary Clearance

All groups of mice immunized with recombinant proteins showed a greater clearance of bacteria from lungs compared to the positive control protein OmpCD (FIG. 5). The effect was significant for MCRH2506-1 with one log reduced bacterial recovery compared to the mice immunized with adjuvants alone (IC31®) (p<0.01). There was also a significant reduction for MCRH2844-1 (p<0.05) and MCRH0759-1 (p<0.05) compared to IC31® alone, if sterile lung cultures were removed from the analysis. Based on the observation that sterile lung cultures appeared randomly in the PBS group, they were likely a technical artifact rather than biology involved in elimination of bacteria, hence introducing variability of intranasal inoculation. The number of sterile cultures detected per each group are indicated in Table 10.

Immunization with recombinant proteins showed a reduced bacterial load in the nasopharyngeal lavage for the candidates MCRH2506-1, MCRH0759-1, MCRH3759-1 and MCRH4730-1. The reduction compared to the IC31® control group was, however, not statistically significant for the analysis with or without sterile NAL cultures (FIG. 6). Nevertheless, since the same antigens which provided faster clearance in both broncho-alveolar lavage in the Model #1, MCRH3759-1 and MCRH4730-1, showed also effects in the naso-pharyngeal lavage in this study, the observed effects are considered worth further investigation.

TABLE 10
Summary of sterile cultures identified per each experimental group.
			Total instances	Total instances
		Number of	of sterile lung	of sterile NAL
		mice/	cultures	cultures
Group	Antigen	experiments	(sterile/tested)	(sterile/tested)
1st antigen*	MCRH2506-1	30/3	7/30	7/29
2nd antigen*	MCRH2844-1	20/2	0/20	3/18
3rd antigen*	MCRH0759-1	30/3	5/30	10/30
4th antigen*	MCRH3709-1	20/2	1/20	8/19
5th antigen*	MCRH0357-1	20/2	4/20	4/17
6th antigen*	MCRH3729-1	20/2	5/20	3/20
7th antigen*	MCRH3759-1	30/3	5/30	9/30
8th antigen*	MCRH4730-1	20/2	0/20	6/20
positive control*	OmpCD	50/5	14/50	8/47
negative control,		50/5	11/50	8/47
IC31 ® alonex
negative control,		50/5	7/50	13/49
PBS alone
*17.5 μl antigen plus 2.5 μl IC31 ® adjuvant
x17.5 μl buffer (50 mM Tris/HCl pH 8) plus 2.5 μl IC31 ® adjuvant

CONCLUSIONS

There is significant correlation of the data from two independently performed studies using two different mouse pulmonary clearance models, except in the case of antigen MCRH2506-1, which showed a significant effect only in the second model which employed mucosal immunization route and adjuvant. Compared to adjuvant control in both nasopharyngeal lavage (NAL) and lung tissue MCRH2506-1 induced >1 log reduction in bacterial CFU; similar protective effects of this antigen were reported using mouse pulmonary clearance model which employed different mouse strain and challenge by inhalation of bacteria in the presence of anesthetic (Ruckdeschel E A et al., 2009). Considering the fact that Moraxella catarrhalis is not mouse pathogen and is easily cleared by mouse immune system, slight differences in the mouse physiology, mode of challenge, immunization route and the time window during which tissue samples are removed for measuring bacterial load are all expected to add to variation in the data obtained in different models. Interestingly, the antigens MCRH3759-1 and MCRH4730-1 which showed effects only in nasopharyngeal lavage (NAL) in the mucosal immunization model, also showed effects only in bronchoalveolar lavage (BAL) in the systemic immunization pulmonary clearance model. This may suggest that these antigens may be effective in preventing bacterial adherence to the respiratory epithelium, but are less effective in clearing already established infection within deeper layers of lung tissue. Additional antigens which provided more effective bacterial clearance in both models include: MCRH2844-1 and MCRH0759-1. Therefore, these data additionally confirm the selection of candidate antigens for testing in the future studies.

REFERENCES

The following references which have been recited in the present specification in a truncated version are incorporated herein by reference in their entirety.

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The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.

Claims

1. An isolated nucleic acid molecule encoding an antigen or a fragment thereof, comprising a nucleic acid sequence, which is selected from the group consisting of: a) a nucleic acid molecule having at least 70% sequence identity to a nucleic acid molecule having a nucleotide sequence selected from the group comprising SEQ ID NOs 860 to 886 and 1 to 214,b) a nucleic acid molecule which is complementary to the nucleic acid molecule of a),c) a nucleic acid molecule comprising at least 15 sequential bases of the nucleic acid molecule of a) or b),d) a nucleic acid molecule which anneals under stringent hybridisation conditions to the nucleic acid molecule of a), b), or c),e) a nucleic acid molecule which, but for the degeneracy of the genetic code, would hybridize to the nucleic acid molecule defined in a), b), c), or d).

2.-7. (canceled)

8. A vector comprising a nucleic acid molecule according to claim 1.

9. (canceled)

10. A host cell comprising the vector as defined in claim 8.

11. An antigen that is immunologically reactive with sera from a human having a Moraxella infection, or from an uninfected healthy human who was previously infected with Moraxella, wherein the antigen comprises or consists of an isolated polypeptide or an active fragment or an active variant thereof from Moraxella, most preferably from Moraxella catarrhalis.

12. An antigen comprising or consisting of an isolated polypeptide selected from the group consisting of SEQ ID NOs 887 to 913 and 215 to 428, or an active fragment or an active variant thereof.

13. An antigen that is immunologically reactive with sera from a human having a Moraxella infection, or from an uninfected healthy human who was previously infected with Moraxella, wherein the antigen comprises or consists of an isolated polypeptide or an active fragment or an active variant thereof from Moraxella, most preferably from Moraxella catarrhalis, wherein the polypeptide is encoded by a nucleic acid molecule as defined in claim 1.

14. The antigen according to claim 11, wherein the active fragment thereof consists of at least 50%, especially at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, 96%, 97% or 98%, most preferably 99% of said polypeptide, especially of a polypeptide as defined by any of the SEQ ID NOs 887 to 913 or 215 to 428.

15. The antigen according to claim 11, wherein the active variant thereof has at least 50%, especially at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, 96%, 97% or 98%, most preferably 99% sequence identity to said polypeptide, especially to a polypeptide as defined by any of the SEQ ID NOs 887 to 913 or 215 to 428.

16. The antigen according to claim 11, whereby the antigen further comprises or consists of at least one amino acid residue heterologous to the antigen, preferably a marker protein.

17. The antigen according to claim 16, wherein the additional amino acid residue(s) is/are flanking the antigen N-terminally, C-terminally or N- and C-terminally.

18. The antigen according to claim 11, whereby the antigen further comprises or consists of either a leader or a secretory sequence, a sequence employed for purification, or a proprotein sequence.

19. An antigen comprising at least one core amino acid sequence as indicated in column “Predicted immunogenic aa” or “Predicted class II-restricted T cell epitope/regions” or “Location of identified immunogenic region (aa)” of Table 4, whereby more preferably the core amino acid sequence is selected from the group consisting of: amino acids 4-10, 12-40, 46-52, 61-76, 78-94, 96-103, 107-114, 126-133, 135-150, 153-162, 167-174, 182-195, 199-208, 210-218, 226-244, 247-255, 262-276, 285-292, 9-99, 108-128, 132-169, 179-286, and 17-72 of SEQ ID NO 215; amino acids 7-17, 25-32, 36-53, 66-76, 83-91, 100-113, 124-131, 151-158, 168-179, 184-198, 213-225, 227-237, 247-256, 261-270, 275-281, 284-293, 295-305, 315-329, 345-358, 388-415, 436-443, 449-456, 471-483, 1-168, 180-204, 212-253, 261-313, 326-467, 472-486, 10-60, 92-130, and 207-280 of SEQ ID NO 216; amino acids 45-53, 58-65, 70-78, 81-87, 94-100, 163-179, 184-192, 201-207, 212-222, 224-230, 254-260, 263-268, 18-158, 163-232, and 211-260 of SEQ ID NO 217; amino acids 4-12, 25-33, 40-50, 62-69, 74-85, 97-109, 127-138, 141-149, 153-161, 197-203, 206-215, 217-223, 258-265, 272-286, 308-316, 325-335, 343-358, 366-371, 373-381, 400-406, 417-426, 444-458, 466-486, 498-514, 522-539, 544-555, 561-572, 603-619, 621-638, 658-672, 687-695, 710-723, 731-744, 747-752, 764-769, 789-805, 808-823, 830-840, 873-885, 888-900, 902-920, 930-943, 964-970, 992-1003, 1005-1016, 1033-1038, 1045-1057, 1059-1068, 1089-1096, 1104-1110, 1115-1142, 1150-1156, 1158-1169, 1188-1198, 1200-1205, 1-80, 88-153, 167-222, 231-249, 256-289, 304-337, 340-488, 491-679, 681-780, 803-878, 880-942, 951-986, 988-1014, 1030-1106, 1109-1133, 1136-1179, 1183-1203, 128-158, 161-232, 277-314, 590-627, 712-848, 957-963, 1007-1033, and 1074-1093 of SEQ ID NO 218; amino acids 4-29, 45-56, 68-78, 84-97, 100-109, 127-141, 145-151, 173-179, 189-197, 202-209, 214-220, 238-263, 266-279, 288-297, 307-315, 330-336, 343-351, 358-367, 373-382, 388-403, 412-421, 423-429, 435-441, 449-459, 462-468, 488-501, 503-522, 536-542, 559-572, 603-611, 623-630, 675-681, 684-690, 705-719, 1-33, 45-204, 210-414, 431-480, 486-532, 536-582, 597-691, 704-724, 496-512, and 542-618 of SEQ ID NO 219; amino acids 14-24, 50-70, 76-92, 100-128, 130-143, 150-155, 178-187, 215-221, 233-251, 261-267, 275-288, 295-301, 319-324, 339-364, 368-384, 404-453, 461-472, 493-510, 522-532, 534-550, 570-577, 598-633, 641-655, 666-672, 676-682, 762-772, 793-808, 819-826, 836-858, 860-867, 872-896, 900-907, 909-918, 929-948, 961-974, 990-1003, 1005-1015, 1018-1025, 1066-1076, 1081-1090, 1100-1113, 1124-1131, 1146-1152, 1191-1197, 1202-1208, 1214-1231, 1237-1254, 1282-1310, 1313-1325, 1333-1353, 1365-1378, 6-26, 35-55, 58-163, 166-184, 190-252, 258-300, 305-493, 495-559, 567-587, 591-654, 672-750, 753-813, 827-866, 870-889, 898-983, 993-1011, 1038-1107, 1139-1162, 1168-1191, 1199-1256, 1261-1355, 1365-1387, 199-293, 420-438, 643-694, 719-771, 823-881, 1050-1144, and 1267-1339 of SEQ ID NO 220; amino acids 14-42, 50-65, 69-81, 96-109, 122-129, 146-161, 170-184, 194-200, 206-215, 221-234, 241-248, 264-270, 273-281, 288-299, 303-316, 320-338, 345-352, 365-372, 426-443, 475-484, 499-515, 518-524, 537-546, 555-563, 565-572, 582-588, 601-608, 612-629, 642-647, 662-683, 698-703, 706-711, 718-731, 739-746, 758-766, 776-782, 801-807, 825-835, 840-852, 878-886, 889-897, 908-915, 920-933, 935-946, 972-979, 982-992, 1003-1008, 1013-1023, 1030-1042, 1065-1081, 1095-1103, 1116-1126, 1132-1138, 1155-1173, 1176-1184, 1186-1199, 1203-1209, 1216-1224, 1242-1262, 1270-1282, 1295-1306, 1308-1316, 1342-1348, 1352-1357, 1-74, 84-228, 233-258, 264-383, 391-449, 451-490, 496-547, 559-610, 616-635, 640-731, 752-778, 799-862, 874-990, 1064-1110, 1114-1138, 1144-1167, 1183-1203, 1209-1230, 1241-1281, 1289-1363, 443-561, 644-713, 767-806, and 990-996 of SEQ ID NO 221; amino acids 4-19, 25-33, 36-51, 53-67, 105-110, 1-72, 74-108, and 55-108 of SEQ ID NO 222; amino acids 15-36, 42-96, 101-112, 3-115, and 4-23 of SEQ ID NO 223; amino acids 4-10, 12-22, 25-32, 35-48, 54-59, 65-70, 100-111, 115-126, 132-149, 156-164, 166-188, 197-207, 220-235, 243-265, 272-278, 309-318, 322-329, 340-353, 355-370, 378-388, 393-398, 410-417, 441-447, 449-455, 1-227, 248-378, 395-427, 445-459, and 310-399 of SEQ ID NO 224; amino acids 5-39, 1-40, and 3-52 of SEQ ID NO 225; amino acids 10-16, 20-40, 58-80, 83-89, 98-119, 126-132, 138-149, 173-179, 193-210, 229-235, 242-248, 266-272, 293-301, 303-315, 335-350, 355-362, 368-377, 379-390, 402-408, 420-431, 438-443, 451-464, 471-476, 486-501, 504-525, 547-568, 606-612, 637-644, 649-656, 685-701, 728-740, 774-780, 784-791, 793-800, 806-816, 818-834, 853-860, 878-888, 890-895, 926-937, 969-976, 983-997, 1-48, 54-82, 101-122, 126-184, 186-235, 237-325, 350-443, 448-512, 523-545, 553-583, 595-623, 634-657, 660-729, 732-786, 804-841, 845-975, 980-1000, 23-130, 227-257, 269-346, 680-775, 817-894, and 922-983 of SEQ ID NO 226; amino acids 4-31, 34-55, 98-105, 127-137, 157-175, 187-194, 228-238, 255-262, 268-273, 281-296, 302-307, 314-327, 329-339, 346-352, 394-401, 407-416, 452-458, 466-477, 534-550, 561-571, 573-580, 597-605, 610-622, 624-630, 639-644, 650-656, 666-672, 689-696, 706-712, 728-735, 743-748, 785-790, 803-812, 820-825, 827-833, 843-848, 861-868, 4-51, 95-116, 125-207, 217-244, 254-363, 375-426, 440-469, 474-506, 527-574, 576-641, 653-682, 750-800, 803-835, 29-69, 204-337, 400-502, and 578-765 of SEQ ID NO 227; amino acids 8-16, 19-52, 61-68, 77-86, 88-98, 106-116, 126-131, 143-153, 163-178, 183-191, 211-217, 6-56, 85-99, 106-127, 140-222, and 101-185 of SEQ ID NO 228; amino acids 15-26, 46-53, 59-68, 75-86, 93-104, 116-122, 126-140, 154-165, 175-181, 206-216, 230-243, 251-257, 294-301, 305-311, 324-336, 364-372, 374-381, 394-400, 419-427, 435-447, 451-459, 1-108, 114-192, 208-285, 310-348, 375-483, 32-98, 122-144, 157-323, 336-351, 369-389, and 433-440 of SEQ ID NO 229; amino acids 4-10, 12-23, 27-40, 42-49, 59-67, 94-99, 1-41, 47-100, and 7-58 of SEQ ID NO 230; amino acids 4-22, 38-52, 110-117, 122-132, 144-154, 167-175, 1-29, 33-61, 71-128, 138-189, 24-38, 46-67, and 128-177 of SEQ ID NO 231; amino acids 4-9, 13-29, 39-45, 50-64, 67-77, 85-94, 103-120, 127-146, 148-156, 163-178, 180-192, 204-211, 213-238, 241-256, 266-295, 1-21, 42-140, 145-261, 263-315, and 135-227 of SEQ ID NO 232; amino acids 4-15, 33-58, 67-120, 129-139, 144-171, 1-178, and 7-61 of SEQ ID NO 233; amino acids 18-24, 26-34, 40-47, 53-70, 74-88, 103-125, 128-137, 150-159, 164-174, 181-188, 201-209, 220-230, 232-238, 249-272, 278-287, 290-303, 318-332, 346-353, 379-387, 394-419, 421-430, 433-441, 443-450, 470-488, 498-507, 509-524, 532-539, 543-550, 557-579, 602-608, 611-617, 624-632, 641-657, 671-676, 694-701, 720-730, 753-762, 764-785, 792-797, 801-807, 817-840, 853-859, 868-874, 878-894, 899-913, 915-921, 929-935, 1-43, 49-70, 100-164, 173-192, 198-258, 265-306, 315-371, 373-433, 438-461, 465-523, 525-741, 761-785, 787-808, 810-925, 937-953, 288-408, 577-608, 622-764, 826-899, and 923-948 of SEQ ID NO 234; amino acids 10-23, 36-44, 46-63, 70-79, 89-106, 118-128, 140-149, 161-170, 197-207, 233-245, 1-185, 190-234, 240-254, and 182-251 of SEQ ID NO 235; amino acids 6-20, 26-32, 41-47, 50-72, 75-83, 102-112, 114-140, 156-172, 178-187, 197-218, 228-269, 275-297, 307-336, 352-368, 370-377, 387-397, 1-399, and 303-363 of SEQ ID NO 236; amino acids 8-38, 40-46, 53-70, 88-94, 127-136, 140-145, 154-174, 186-191, 198-226, 236-243, 249-256, 260-266, 269-280, 320-333, 342-349, 357-363, 365-381, 385-430, 433-449, 456-465, 467-475, 488-493, 498-503, 510-520, 557-609, 618-624, 631-644, 646-673, 679-685, 690-696, 698-734, 780-797, 802-808, 810-829, 831-870, 879-890, 910-916, 926-942, 944-980, 1-76, 82-103, 105-156, 158-289, 295-474, 506-557, 565-803, 806-857, 867-898, 914-988, and 888-947 of SEQ ID NO 237; amino acids 16-23, 25-33, 41-53, 56-63, 70-79, 87-98, 100-111, 120-128, 131-149, 160-171, 214-230, 232-242, 247-255, 259-266, 273-282, 284-292, 308-347, 355-364, 370-376, 379-386, 554-563, 10-182, 212-232, 236-358, 364-409, 461-475, 478-498, 542-577, 140-160, 232-300, 321-394, and 514-522 of SEQ ID NO 238; amino acids 9-35, 39-51, 54-67, 71-78, 93-102, 105-116, 122-129, 138-147, 155-162, 168-192, 194-231, 253-261, 264-276, 281-293, 8-241, 244-265, 269-302, and 152-279 of SEQ ID NO 239; amino acids 35-56, 61-70, 74-101, 120-129, 152-165, 171-183, 192-224, 241-271, 278-285, 291-301, 303-319, 334-340, 367-391, 401-426, 437-442, 446-469, 475-488, 495-520, 523-529, 532-541, 543-550, 554-564, 570-598, 624-630, 633-638, 643-655, 663-685, 687-694, 698-725, 735-751, 758-765, 778-795, 1-22, 36-70, 72-128, 141-223, 231-267, 270-334, 339-605, 613-652, 655-778, 115-232, 312-332, 539-555, and 558-620 of SEQ ID NO 240; amino acids 15-39, 54-61, 65-71, 84-89, 98-111, 126-144, 154-160, 189-196, 199-206, 218-224, 259-275, 1-170, 175-230, 244-278, and 162-217 of SEQ ID NO 241; amino acids 22-37, 45-57, 59-65, 82-93, 102-112, 160-172, 200-207, 229-236, 244-268, 275-322, 327-344, 346-369, 376-414, 428-451, 459-490, 495-527, 531-555, 561-582, 600-612, 616-622, 625-635, 638-669, 683-709, 726-732, 735-763, 11-57, 72-88, 99-122, 139-221, 236-274, 279-328, 332-766, 60-145, and 187-223 of SEQ ID NO 242; amino acids 4-12, 32-78, 81-89, 91-100, 105-111, 125-132, 170-176, 179-189, 194-204, 223-229, 235-245, 259-276, 282-289, 303-313, 318-326, 334-345, 350-356, 364-370, 387-405, 409-422, 424-430, 442-457, 459-465, 490-516, 521-532, 534-541, 566-571, 1-208, 230-310, 315-438, 440-456, 467-580, 186-238, and 398-572 of SEQ ID NO 243; amino acids 14-20, 28-35, 41-47, 52-61, 73-86, 92-103, 124-141, 143-158, 172-180, 185-191, 193-221, 239-250, 262-273, 280-290, 304-324, 328-357, 367-376, 378-384, 394-401, 420-428, 439-458, 460-469, 474-492, 22-125, 130-150, 157-177, 179-368, 403-495, 59-74, 94-141, 143-203, and 297-362 of SEQ ID NO 244; amino acids 8-17, 25-39, 41-67, 69-85, 93-115, 123-143, 145-157, 166-174, 178-185, 188-199, 204-210, 221-238, 245-252, 257-268, 284-290, 294-306, 312-320, 1-170, 175-247, 261-323, 17-28, 61-99, and 221-240 of SEQ ID NO 245; amino acids 5-14, 16-26, 28-45, 54-60, 72-82, 98-104, 106-114, 121-132, 137-164, 169-175, 188-194, 214-220, 233-239, 283-296, 302-310, 336-347, 357-364, 400-407, 419-425, 433-439, 443-452, 464-470, 492-499, 511-521, 528-534, 536-548, 564-571, 577-595, 597-611, 615-623, 637-649, 652-670, 681-700, 716-724, 726-738, 766-792, 806-833, 839-851, 853-872, 880-899, 905-912, 919-927, 935-956, 958-970, 980-988, 990-995, 1004-1013, 1017-1031, 1033-1039, 1043-1055, 1059-1068, 1087-1093, 1105-1114, 1116-1122, 1128-1134, 1149-1158, 1164-1172, 1197-1204, 1208-1215, 1230-1244, 1284-1290, 8-53, 68-252, 265-283, 287-320, 329-353, 357-383, 389-470, 475-495, 507-527, 534-749, 773-870, 876-898, 906-928, 938-1150, 1153-1255, 1267-1287, 1291-1314, 291-408, 411-425, 500-552, 719-741, 975-1034, 1089-1156, 1203-1234, and 1273-1301 of SEQ ID NO 246; amino acids 9-20, 25-40, 48-61, 70-84, 89-113, 116-145, 161-181, 183-193, 196-212, 217-229, 235-246, 251-276, 284-290, 300-325, 338-349, 365-376, 387-393, 401-421, 429-438, 448-458, 6-382, 385-465, 204-286, and 310-463 of SEQ ID NO 247; amino acids 9-47, 52-61, 63-76, 82-88, 95-106, 112-126, 132-138, 145-154, 162-176, 178-223, 228-234, 236-247, 253-290, 300-307, 311-325, 327-347, 353-361, 379-408, 426-436, 454-460, 490-504, 506-517, 526-532, 539-545, 550-574, 580-591, 598-612, 616-622, 625-634, 652-668, 670-679, 682-688, 1-463, 466-510, 519-641, 647-702, 7-54, 198-314, 333-381, 404-452, and 523-549 of SEQ ID NO 248; amino acids 4-20, 24-38, 40-64, 66-85, 8-88, and 15-72 of SEQ ID NO 249; amino acids 6-15, 28-33, 35-46, 54-65, 74-108, 141-146, 172-178, 204-217, 221-231, 242-248, 251-257, 276-294, 298-304, 316-332, 341-353, 356-363, 369-382, 3-44, 64-172, 175-191, 202-348, 56-110, 143-224, and 252-308 of SEQ ID NO 250; amino acids 4-28, 49-60, 66-71, 158-164, 215-221, 254-275, 288-294, 318-326, 338-344, 366-380, 389-395, 400-421, 423-430, 450-458, 465-470, 474-491, 495-504, 512-517, 543-563, 577-582, 587-594, 617-623, 631-637, 644-652, 677-691, 694-700, 705-733, 748-783, 791-819, 833-840, 847-854, 876-886, 1-39, 46-64, 80-100, 105-130, 142-162, 166-233, 249-342, 352-467, 470-511, 539-591, 596-687, 689-859, and 609-738 of SEQ ID NO 251; amino acids 7-31, 42-54, 56-64, 94-110, 112-122, 128-136, 172-195, 198-224, 256-263, 281-287, 298-310, 340-346, 356-372, 1-147, 155-276, 278-319, 329-387, 391-405, and 87-164 of SEQ ID NO 252; amino acids 4-20, 33-41, 58-65, 78-86, 93-100, 102-108, 114-161, 194-200, 202-212, 218-225, 230-235, 243-250, 269-278, 291-301, 312-317, 329-340, 352-375, 416-422, 427-434, 443-453, 468-476, 481-491, 510-532, 537-554, 560-566, 574-588, 612-623, 653-663, 678-702, 721-728, 742-748, 770-778, 796-805, 807-817, 824-832, 856-864, 871-878, 885-900, 1-20, 27-172, 175-195, 218-270, 276-308, 334-388, 409-449, 451-712, 741-824, 828-913, 16-32, 133-197, 277-385, 556-573, and 798-874 of SEQ ID NO 253; amino acids 7-14, 17-30, 35-40, 62-69, 80-86, 88-99, 109-118, 120-133, 154-179, 203-209, 216-226, 237-245, 253-260, 265-273, 286-291, 315-339, 353-371, 390-398, 412-430, 435-448, 456-464, 476-487, 492-503, 522-533, 537-549, 579-590, 606-622, 648-654, 664-679, 681-692, 700-711, 718-730, 743-752, 763-798, 808-815, 823-838, 846-864, 891-925, 927-933, 935-948, 25-79, 83-126, 151-284, 292-408, 415-511, 532-577, 594-627, 634-735, 740-809, 815-866, 885-916, 53-163, 191-246, 457-515, 520-528, 620-642, 759-800, and 894-943 of SEQ ID NO 254; amino acids 21-31, 48-58, 60-66, 83-89, 102-115, 144-154, 159-168, 175-186, 195-200, 206-218, 244-250, 259-272, 293-299, 301-307, 324-330, 339-348, 358-369, 380-391, 404-421, 429-437, 456-467, 481-486, 495-502, 509-533, 536-550, 553-571, 575-580, 599-620, 1-93, 98-116, 118-149, 174-199, 202-302, 319-393, 410-468, 493-578, 582-623, 251-272, 290-363, and 424-502 of SEQ ID NO 255; amino acids 12-19, 30-48, 55-61, 66-74, 103-115, 117-131, 176-183, 192-197, 200-222, 8-52, 80-171, 173-201, 208-237, 240-258, 15-64, 77-168, and 190-252 of SEQ ID NO 256; amino acids 4-19, 31-58, 61-70, 84-96, 104-112, 131-138, 143-165, 168-178, 197-208, 213-220, 255-268, 273-281, 313-320, 334-354, 359-367, 369-376, 382-399, 406-420, 429-439, 441-461, 463-507, 513-544, 550-557, 569-580, 583-589, 598-618, 640-650, 675-680, 683-711, 713-739, 797-805, 1-55, 59-164, 168-185, 193-238, 244-333, 343-375, 378-517, 521-583, 602-656, 670-761, 788-808, 124-151, 326-341, and 567-720 of SEQ ID NO 257; amino acids 5-12, 22-37, 47-75, 78-94, 102-107, 110-118, 124-130, 140-154, 161-166, 185-193, 205-213, 218-237, 244-257, 264-269, 276-283, 289-315, 317-323, 334-341, 1-105, 121-344, 141-168, and 256-313 of SEQ ID NO 258; amino acids 33-48, 56-63, 65-79, 84-101, 109-122, 127-160, 211-216, 224-229, 239-256, 1-108, 110-265, 4-75, and 108-199 of SEQ ID NO 259; amino acids 6-14, 24-40, 47-60, 79-98, 102-108, 114-120, 122-132, 154-160, 168-179, 187-206, 221-226, 231-253, 255-268, 296-314, 316-337, 341-347, 357-378, 383-407, 418-425, 443-452, 456-462, 485-495, 503-510, 512-523, 533-543, 550-555, 1-101, 115-152, 154-203, 210-314, 321-368, 375-412, 414-434, 438-494, 497-529, 544-558, 35-136, and 348-355 of SEQ ID NO 260; amino acids 4-11, 14-22, 24-29, 33-40, 42-52, 54-70, 87-111, 121-128, 132-143, 149-168, 180-190, 197-215, 223-232, 244-251, 296-319, 328-349, 363-368, 378-399, 414-431, 434-440, 451-457, 1-27, 29-48, 59-199, 206-257, 264-378, 398-459, and 322-374 of SEQ ID NO 261; amino acids 4-20, 28-34, 36-42, 56-72, 80-86, 97-110, 118-127, 132-138, 145-150, 173-179, 181-195, 232-244, 250-263, 270-276, 308-314, 324-331, 336-343, 355-364, 398-403, 1-37, 52-81, 93-152, 173-252, 266-406, 29-90, and 125-197 of SEQ ID NO 262; amino acids 15-23, 31-38, 51-66, 89-105, 123-130, 135-140, 149-154, 198-208, 212-220, 228-239, 249-257, 261-267, 275-281, 300-310, 322-343, 354-365, 372-384, 394-404, 409-420, 422-429, 446-455, 459-468, 1-87, 104-194, 199-392, 404-425, 443-466, 82-164, 236-325, and 392-453 of SEQ ID NO 263; amino acids 4-10, 17-46, 51-80, 87-92, 99-121, 135-150, 170-176, 200-212, 243-252, 277-282, 310-331, 340-349, 1-160, 168-188, 200-263, 276-355, and 126-294 of SEQ ID NO 264; amino acids 4-18, 35-48, 52-67, 73-80, 90-96, 108-147, 156-171, 188-207, 227-234, 239-248, 263-286, 301-330, 347-363, 365-395, 409-419, 421-431, 442-447, 454-468, 471-482, 484-510, 517-525, 545-555, 1-62, 84-178, 182-264, 295-396, 403-452, 461-502, 520-568, 53-62, 112-187, 233-265, and 375-402 of SEQ ID NO 265; amino acids 13-31, 36-48, 54-64, 67-83, 93-104, 106-126, 128-137, 144-154, 161-169, 179-196, 205-221, 232-255, 257-263, 265-305, 307-324, 326-340, 354-367, 372-378, 383-402, 14-409, 23-124, and 134-172 of SEQ ID NO 266; amino acids 5-21, 26-35, 37-44, 57-69, 71-78, 84-93, 95-101, 1-78, 81-112, and 41-103 of SEQ ID NO 267; amino acids 13-19, 21-42, 47-55, 57-66, 68-74, 77-82, 84-90, 92-101, 106-112, 147-169, 171-183, 185-200, 221-230, 240-252, 269-275, 284-296, 299-308, 311-322, 332-342, 344-358, 374-382, 386-391, 403-412, 445-451, 462-477, 484-494, 557-563, 565-574, 1-47, 50-65, 80-108, 121-135, 145-234, 236-322, 326-379, 396-488, 502-522, 564-589, 14-77, 217-269, and 380-542 of SEQ ID NO 268; amino acids 4-34, 36-44, 51-57, 63-68, 70-80, 91-130, 136-173, 186-193, 195-201, 207-213, 226-234, 242-266, 268-284, 290-308, 315-332, 368-378, 386-402, 413-437, 1-87, 89-172, 175-193, 222-244, 247-366, 381-409, 416-437, 191-197, and 350-438 of SEQ ID NO 269; amino acids 11-24, 29-51, 61-91, 98-116, 119-129, 132-142, 10-66, 69-102, 111-145, and 2-98 of SEQ ID NO 270; amino acids 16-49, 55-74, 78-88, 91-112, 119-127, 140-145, 156-165, 167-173, 199-209, 234-249, 251-263, 265-272, 282-290, 299-305, 322-330, 1-333, 32-89, 108-141, and 246-333 of SEQ ID NO 271; amino acids 19-43, 49-55, 78-92, 1-65, 68-113, and 45-108 of SEQ ID NO 272; amino acids 15-23, 52-84, 97-103, 110-133, 149-154, 160-183, 203-227, 233-241, 243-250, 313-330, 338-351, 373-382, 384-396, 401-407, 425-452, 469-474, 476-482, 490-513, 519-528, 533-545, 591-613, 619-649, 23-105, 115-152, 154-220, 226-263, 276-296, 302-368, 378-402, 414-477, 481-536, 539-581, 588-652, 158-222, 327-423, and 427-526 of SEQ ID NO 273; amino acids 4-11, 19-29, 43-52, 82-96, 101-110, 138-144, 157-165, 258-268, 326-334, 336-344, 346-358, 377-383, 386-394, 397-405, 413-427, 429-434, 437-443, 449-458, 486-495, 514-521, 535-546, 554-560, 566-578, 589-596, 603-612, 618-625, 644-659, 662-671, 675-680, 707-720, 722-735, 737-752, 754-771, 788-802, 816-830, 836-844, 851-870, 884-909, 39-105, 132-176, 183-247, 258-292, 300-324, 328-398, 405-506, 512-532, 544-653, 659-838, 840-885, 78-130, 215-292, 655-713, and 818-880 of SEQ ID NO 274; amino acids 5-14, 21-27, 29-37, 46-72, 83-96, 113-121, 129-134, 1-63, 81-141, and 58-141 of SEQ ID NO 275; amino acids 4-9, 20-34, 36-54, 88-94, 124-148, 155-161, 182-202, 204-211, 220-232, 1-53, 75-116, 126-174, 177-235, and 92-234 of SEQ ID NO 276; amino acids 4-22, 31-38, 49-55, 82-91, 1-24, 28-49, and 7-65 of SEQ ID NO 277; amino acids 4-28, 30-83, 85-92, 94-123, 125-157, 163-196, 216-234, 240-258, 260-289, 307-313, 315-323, 328-378, 392-424, 428-440, 458-472, 474-482, 491-506, 508-530, 539-564, 1-298, 301-566, 14-31, and 282-347 of SEQ ID NO 278; amino acids 7-17, 20-28, 30-37, 39-47, 49-61, 77-96, 114-128, 149-154, 166-180, 182-200, 240-248, 256-271, 1-158, 163-275, 60-112, and 149-246 of SEQ ID NO 279; amino acids 6-25, 31-37, 43-57, 63-72, 82-90, 96-107, 109-118, 123-140, 144-149, 155-162, 168-182, 184-190, 213-219, 237-244, 247-256, 262-268, 271-284, 295-301, 307-320, 325-337, 346-364, 1-46, 48-196, 208-231, 234-278, 290-374, and 184-277 of SEQ ID NO 280; amino acids 27-46, 53-77, 103-123, 130-142, 153-159, 171-179, 182-209, 215-222, 224-233, 237-243, 271-276, 2-210, 215-271, and 12-138 of SEQ ID NO 281; amino acids 17-24, 38-45, 49-67, 80-86, 94-112, 119-141, 154-159, 165-171, 183-200, 218-226, 234-252, 263-291, 328-336, 338-349, 357-385, 397-405, 423-440, 445-453, 457-466, 470-477, 487-493, 8-134, 141-167, 175-224, 231-473, 479-496, 284-393, and 462-469 of SEQ ID NO 282; amino acids 4-9, 16-23, 56-64, 70-82, 86-93, 1-20, 39-103, and 11-36 of SEQ ID NO 283; amino acids 5-38, 69-77, 83-99, 143-151, 154-160, 176-186, 198-210, 226-268, 270-288, 293-303, 305-324, 327-342, 344-353, 388-394, 432-438, 1-49, 54-69, 96-110, 130-248, 251-270, 312-368, 382-450, 65-116, 179-231, and 328-395 of SEQ ID NO 284; amino acids 8-23, 58-70, 74-80, 85-91, 95-103, 110-127, 143-150, 153-160, 164-171, 189-201, 218-224, 228-233, 236-242, 248-255, 1-27, 30-46, 53-71, 77-171, 181-264, and 3-91 of SEQ ID NO 285; amino acids 4-19, 30-49, 57-83, 88-100, 111-119, 122-129, 154-159, 168-173, 189-200, 222-229, 237-243, 251-259, 1-22, 33-51, 55-99, 114-139, 163-204, 220-250, and 108-262 of SEQ ID NO 286; amino acids 4-19, 26-51, 53-62, 68-79, 83-96, 99-105, 114-122, 128-135, 138-146, 155-164, 180-199, 201-218, 226-232, 237-249, 257-264, 1-174, 176-277, and 47-117 of SEQ ID NO 287; amino acids 12-17, 23-31, 42-62, 81-88, 96-103, 109-118, 122-128, 136-146, 151-158, 188-203, 210-224, 229-238, 270-287, 296-308, 310-333, 341-354, 361-368, 383-395, 404-420, 423-435, 443-456, 460-503, 510-517, 521-526, 541-548, 565-570, 575-586, 606-617, 625-637, 648-655, 683-696, 702-713, 722-728, 740-748, 779-786, 789-801, 807-812, 820-829, 849-854, 862-868, 9-61, 75-124, 132-288, 299-547, 592-608, 612-660, 666-695, 733-753, 776-813, 818-854, 858-875, 59-133, 260-272, 382-410, 440-515, and 711-872 of SEQ ID NO 288; amino acids 15-24, 28-56, 63-87, 98-104, 112-121, 125-133, 142-159, 166-176, 189-198, 200-207, 216-223, 225-231, 236-243, 245-252, 262-287, 295-304, 315-323, 326-332, 340-356, 361-368, 370-376, 387-398, 401-410, 412-420, 422-434, 442-455, 469-476, 486-496, 507-514, 519-527, 547-556, 559-564, 569-575, 588-594, 604-609, 611-622, 634-662, 676-686, 712-718, 723-738, 752-758, 779-790, 793-822, 833-850, 866-891, 894-908, 935-950, 1-155, 162-181, 186-321, 346-600, 608-760, 778-897, 910-953, 700-781, and 800-903 of SEQ ID NO 289; amino acids 25-31, 41-54, 79-86, 88-96, 111-125, 135-141, 145-152, 159-182, 188-195, 208-214, 257-267, 275-297, 306-312, 326-333, 342-358, 370-377, 399-414, 420-428, 434-439, 446-492, 499-507, 509-521, 527-533, 2-33, 36-217, 231-251, 263-363, 367-427, 430-527, 2-122, and 192-306 of SEQ ID NO 290; amino acids 6-13, 18-26, 28-46, 49-56, 62-69, 74-84, 88-110, 116-132, 135-149, 156-178, 181-191, 202-223, 247-260, 274-281, 295-303, 336-344, 368-375, 377-390, 395-404, 409-433, 450-458, 470-506, 1-83, 100-280, 295-370, 374-509, 63-151, 220-291, and 321-388 of SEQ ID NO 291; amino acids 4-11, 16-27, 31-41, 62-81, 101-108, 116-144, 169-185, 203-218, 228-234, 236-245, 255-273, 283-294, 298-304, 306-313, 315-340, 342-350, 357-371, 378-390, 402-416, 421-436, 444-456, 458-464, 481-487, 496-508, 510-531, 536-541, 544-551, 556-569, 576-590, 600-614, 627-636, 644-651, 664-670, 675-688, 700-720, 727-770, 775-797, 802-818, 828-835, 864-884, 887-892, 899-907, 918-928, 940-958, 965-980, 7-194, 202-235, 261-383, 401-505, 524-617, 624-727, 735-749, 761-879, 898-973, 975-989, 96-113, 634-656, and 658-734 of SEQ ID NO 292; amino acids 4-12, 14-23, 26-35, 51-78, 83-94, 100-106, 109-119, 121-135, 140-149, 153-177, 179-202, 212-224, 229-239, 241-252, 260-312, 330-336, 342-352, 368-383, 393-403, 1-100, 105-343, 365-406, 76-101, and 295-353 of SEQ ID NO 293; amino acids 4-28, 31-38, 65-81, 1-84, and 9-69 of SEQ ID NO 294; amino acids 4-11, 26-35, 55-74, 78-90, 109-115, 125-141, 164-169, 173-181, 193-203, 212-231, 253-266, 289-295, 297-313, 350-356, 380-388, 410-416, 425-433, 449-462, 466-473, 478-497, 562-584, 586-606, 614-619, 718-724, 727-738, 745-752, 782-789, 812-821, 827-834, 844-854, 864-874, 902-908, 920-927, 952-957, 966-973, 982-988, 1010-1019, 1027-1034, 1050-1057, 1082-1088, 1111-1125, 1129-1135, 1150-1163, 1-104, 108-152, 161-190, 195-323, 337-366, 369-410, 415-449, 453-495, 532-552, 559-624, 677-697, 724-751, 784-802, 804-824, 834-931, 949-977, 979-999, 1060-1149, 378-625, and 795-908 of SEQ ID NO 295; amino acids 7-31, 36-49, 59-66, 78-85, 89-95, 101-120, 127-151, 174-183, 185-194, 207-217, 221-230, 238-247, 258-268, 290-297, 309-315, 1-42, 58-233, 236-279, 290-315, and 181-234 of SEQ ID NO 296; amino acids 17-25, 60-72, 80-90, 97-109, 111-117, 1-72, 74-120, and 22-110 of SEQ ID NO 297; amino acids 4-31, 44-53, 55-70, 76-99, 109-115, 123-130, 136-142, 147-155, 158-191, 199-220, 223-235, 243-257, 264-281, 286-299, 313-323, 350-365, 373-391, 397-441, 455-466, 1-35, 45-218, 225-323, 347-386, 388-403, 405-446, 448-469, 105-193, and 327-355 of SEQ ID NO 298; amino acids 4-19, 47-53, 69-75, 81-87, 109-122, 125-140, 142-156, 191-199, 219-238, 246-271, 279-290, 296-302, 305-313, 321-327, 331-345, 1-32, 75-89, 107-127, 133-171, 188-310, 325-349, 43-142, and 300-314 of SEQ ID NO 299; amino acids 4-43, 49-56, 66-74, 80-94, 97-110, 116-136, 139-145, 151-163, 165-178, 182-210, 213-220, 222-231, 241-252, 256-271, 277-293, 300-314, 330-336, 340-363, 369-375, 381-404, 419-426, 439-445, 447-453, 465-501, 1-38, 43-62, 77-137, 140-173, 179-291, 328-376, 386-400, 406-504, 19-76, 195-221, and 271-292 of SEQ ID NO 300; amino acids 4-19, 25-31, 40-63, 71-80, 89-98, 101-119, 125-151, 159-164, 173-201, 219-227, 247-258, 263-284, 308-319, 321-326, 10-99, 101-185, 200-294, 297-347, and 74-161 of SEQ ID NO 301; amino acids 12-18, 25-31, 37-43, 76-94, 121-135, 146-176, 191-199, 205-212, 259-290, 300-313, 340-347, 364-373, 375-385, 413-420, 424-430, 446-462, 465-473, 475-486, 505-522, 537-544, 557-563, 573-582, 588-602, 604-613, 620-630, 638-675, 677-683, 1-28, 33-134, 137-179, 181-221, 255-323, 333-439, 443-532, 537-692, 25-59, 156-270, 272-335, and 557-565 of SEQ ID NO 302; amino acids 10-22, 24-54, 63-70, 77-86, 94-110, 112-133, 1-60, 88-136, and 17-135 of SEQ ID NO 303; amino acids 7-24, 28-47, 76-86, 92-103, 122-129, 135-141, 152-157, 162-173, 192-205, 210-225, 230-236, 270-286, 289-297, 299-314, 316-328, 334-341, 349-356, 376-382, 384-395, 4-47, 50-248, 261-393, 61-67, 168-195, and 257-320 of SEQ ID NO 304; amino acids 4-9, 20-39, 42-48, 67-101, 129-135, 156-162, 170-176, 178-189, 192-197, 228-235, 260-266, 272-278, 291-301, 330-342, 347-360, 367-373, 383-390, 397-409, 432-443, 466-487, 506-517, 520-545, 551-561, 588-598, 602-608, 622-630, 15-126, 129-166, 171-217, 227-307, 309-361, 363-431, 461-496, 501-637, 172-215, 218-291, 417-440, and 498-574 of SEQ ID NO 305; amino acids 8-28, 34-67, 1-70, and 2-32 of SEQ ID NO 306; amino acids 9-26, 33-40, 79-93, 98-105, 112-121, 128-137, 144-156, 160-167, 183-190, 198-204, 210-224, 250-275, 283-289, 296-302, 314-321, 336-344, 346-353, 355-362, 367-380, 382-391, 393-403, 407-413, 416-423, 434-443, 469-492, 499-509, 550-559, 596-603, 613-633, 635-642, 648-659, 1-30, 33-99, 106-126, 129-331, 340-511, 522-565, 578-599, 608-667, 59-118, and 160-215 of SEQ ID NO 307; amino acids 7-20, 25-33, 36-64, 74-83, 117-124, 126-137, 146-159, 164-173, 181-186, 195-231, 240-255, 258-266, 268-276, 283-290, 5-90, 113-250, 260-293, 9-47, 145-198, and 227-239 of SEQ ID NO 308; amino acids 4-24, 31-41, 43-57, 61-68, 72-78, 83-88, 115-121, 123-135, 190-200, 214-224, 239-248, 291-299, 305-312, 328-333, 351-362, 1-101, 103-140, 202-263, 286-362, and 189-242 of SEQ ID NO 309; amino acids 4-21, 110-126, 128-134, 139-149, 1-29, 31-62, 65-94, 99-149, and 7-80 of SEQ ID NO 310; amino acids 4-9, 23-29, 37-52, 57-73, 80-98, 101-119, 146-163, 175-181, 198-208, 222-228, 243-249, 258-263, 269-278, 283-298, 309-324, 329-339, 342-353, 356-363, 365-374, 389-395, 397-404, 408-415, 424-429, 432-450, 457-490, 503-524, 526-556, 568-575, 584-592, 596-619, 621-646, 653-673, 675-694, 699-706, 1-104, 121-135, 143-176, 182-274, 281-301, 306-385, 393-502, 505-648, 654-709, 162-186, 309-397, 435-463, and 503-565 of SEQ ID NO 311; amino acids 5-21, 24-49, 51-63, 65-76, 111-117, 133-145, 149-156, 189-197, 217-230, 240-245, 288-295, 297-316, 321-335, 339-346, 369-382, 401-421, 1-89, 106-125, 129-207, 210-230, 255-357, 360-453, 166-220, and 369-473 of SEQ ID NO 312; amino acids 4-11, 22-37, 39-54, 62-70, 78-84, 94-100, 106-111, 1-51, 59-102, and 41-100 of SEQ ID NO 313; amino acids 16-26, 29-46, 48-54, 94-101, 103-110, 117-125, 134-148, 156-180, 190-196, 221-239, 1-35, 61-81, 88-116, 139-205, 207-227, and 43-75 of SEQ ID NO 314; amino acids 5-19, 21-27, 30-35, 40-57, 67-75, 104-114, 121-130, 152-161, 167-175, 187-205, 219-227, 244-250, 1-76, 83-155, 158-191, 218-253, and 15-137 of SEQ ID NO 315; amino acids 15-38, 40-49, 57-66, 68-76, 80-88, 91-109, 117-122, 148-154, 161-169, 195-214, 220-230, 233-241, 263-269, 285-293, 301-312, 324-340, 349-367, 372-386, 398-407, 426-434, 436-443, 446-452, 456-476, 478-498, 510-517, 523-550, 558-565, 589-604, 610-619, 634-639, 659-668, 680-688, 698-704, 732-739, 741-754, 758-767, 783-796, 800-812, 816-826, 842-860, 1-83, 85-115, 136-179, 192-223, 257-360, 375-486, 501-527, 530-618, 626-644, 653-867, 598-632, 690-700, and 772-833 of SEQ ID NO 316; amino acids 4-12, 41-49, 61-66, 111-118, 123-128, 139-144, 159-166, 178-185, 206-214, 236-248, 262-268, 275-293, 297-314, 348-360, 368-399, 1-68, 71-150, 193-226, 230-296, 305-406, 37-125, and 272-341 of SEQ ID NO 317; amino acids 8-15, 18-54, 61-80, 109-114, 143-149, 153-160, 172-185, 188-194, 196-207, 221-230, 238-244, 273-279, 291-297, 305-319, 325-333, 342-362, 17-110, 118-139, 149-238, 247-365, 62-107, and 123-182 of SEQ ID NO 318; amino acids 15-28, 34-59, 105-135, 137-144, 155-184, 187-212, 221-233, 240-260, 267-278, 286-291, 299-315, 319-334, 1-32, 39-55, 76-122, 124-279, 282-337, 50-100, 116-176, and 261-288 of SEQ ID NO 319; amino acids 4-22, 43-56, 71-108, 119-125, 127-138, 146-155, 164-171, 173-181, 190-199, 214-234, 1-36, 57-112, 121-181, 190-241, and 4-108 of SEQ ID NO 320; amino acids 11-19, 24-31, 37-42, 61-71, 85-91, 94-100, 105-115, 123-138, 140-148, 153-161, 164-173, 185-192, 194-202, 212-222, 227-233, 244-250, 276-287, 298-304, 309-315, 319-330, 342-353, 367-374, 387-394, 403-425, 439-445, 514-520, 528-543, 545-553, 569-584, 586-595, 603-621, 8-273, 283-299, 309-346, 367-490, 494-537, 558-632, 17-74, 155-189, 220-302, 418-540, and 550-612 of SEQ ID NO 321; amino acids 4-20, 41-47, 49-56, 68-86, 95-101, 108-121, 123-137, 145-150, 158-178, 187-198, 218-226, 231-237, 240-250, 252-262, 269-282, 287-317, 1-62, 70-209, 217-319, 140-149, 174-233, and 243-300 of SEQ ID NO 322; amino acids 16-21, 29-36, 43-61, 68-84, 89-136, 159-197, 206-216, 224-240, 244-251, 270-286, 292-307, 313-323, 327-332, 339-364, 388-395, 402-409, 423-440, 448-461, 464-476, 1-15, 19-260, 292-312, 314-451, 465-483, 242-272, and 435-457 of SEQ ID NO 323; amino acids 8-28, 30-35, 55-64, 69-79, 87-95, 132-143, 161-177, 186-198, 222-227, 247-258, 266-288, 340-352, 1-40, 44-96, 103-154, 158-177, 181-202, 208-268, 272-355, 158-168, and 286-334 of SEQ ID NO 324; amino acids 5-24, 39-46, 50-64, 79-89, 103-109, 116-133, 137-150, 159-167, 169-177, 184-202, 209-229, 245-254, 272-282, 294-301, 340-356, 368-378, 388-395, 429-444, 450-463, 484-490, 536-545, 547-557, 565-601, 615-627, 648-655, 1-33, 35-108, 118-236, 245-260, 264-324, 329-358, 360-397, 448-485, 492-536, 541-599, 607-669, 162-181, 331-422, 481-534, and 591-669 of SEQ ID NO 325; amino acids 26-55, 58-73, 75-81, 87-96, 131-140, 150-158, 6-151, and 15-96 of SEQ ID NO 326; amino acids 4-9, 43-52, 63-71, 97-104, 117-125, 130-136, 143-161, 167-179, 185-191, 194-201, 205-211, 219-225, 240-245, 247-256, 261-279, 286-301, 323-338, 340-349, 359-370, 390-397, 399-414, 439-473, 485-496, 505-521, 534-561, 566-571, 575-588, 598-606, 623-629, 653-665, 675-680, 683-690, 692-700, 730-755, 769-798, 802-815, 818-826, 852-859, 862-875, 27-88, 90-173, 176-283, 303-404, 421-443, 473-487, 507-540, 544-642, 645-673, 677-761, 767-840, 843-878, 24-123, 243-316, 430-472, and 512-571 of SEQ ID NO 327; amino acids 40-55, 59-119, 145-180, 182-210, 215-230, 264-274, 293-305, 308-314, 316-328, 339-346, 348-354, 388-393, 395-414, 416-425, 431-436, 442-451, 467-473, 477-487, 491-498, 505-513, 519-542, 548-565, 1-336, 344-379, 395-438, 440-457, 476-571, 230-331, and 454-460 of SEQ ID NO 328; amino acids 5-24, 32-46, 72-77, 83-98, 100-106, 113-135, 140-150, 154-170, 176-199, 218-240, 259-278, 282-301, 307-323, 379-386, 389-430, 449-457, 465-484, 504-520, 537-561, 568-581, 1-321, 323-344, 353-371, 374-588, 341-374, and 406-416 of SEQ ID NO 329; amino acids 11-20, 27-37, 39-54, 67-73, 79-89, 92-102, 112-120, 125-138, 145-152, 172-178, 181-191, 193-204, 213-220, 222-235, 241-257, 281-288, 298-306, 331-359, 361-403, 420-426, 434-456, 462-470, 477-484, 497-504, 520-527, 535-548, 566-575, 584-592, 606-613, 624-629, 635-651, 654-660, 683-694, 697-703, 714-725, 728-757, 767-779, 787-794, 801-814, 834-843, 858-867, 869-875, 888-899, 912-933, 936-946, 3-31, 36-63, 66-80, 103-208, 231-299, 307-399, 445-517, 528-586, 597-618, 632-652, 661-742, 745-781, 785-852, 855-893, 909-947, 293-326, 691-787, and 896-950 of SEQ ID NO 330; amino acids 4-21, 29-46, 61-77, 81-88, 1-88, and 24-78 of SEQ ID NO 331; amino acids 8-41, 48-53, 73-78, 81-98, 115-140, 143-149, 154-162, 182-196, 207-215, 221-226, 230-239, 257-264, 268-276, 287-303, 1-49, 69-149, 161-227, 230-306, 39-67, and 77-105 of SEQ ID NO 332; amino acids 4-11, 16-23, 31-39, 64-79, 82-89, 98-105, 111-117, 136-146, 162-168, 186-193, 196-205, 219-236, 268-287, 289-300, 308-314, 323-339, 349-365, 368-374, 1-51, 93-245, 259-377, 3-9, 107-183, and 210-318 of SEQ ID NO 333; amino acids 4-28, 35-48, 56-64, 91-125, 135-143, 150-159, 1-26, 54-136, 144-167, and 14-88 of SEQ ID NO 334; amino acids 4-16, 21-39, 53-83, 88-93, 99-133, 152-164, 167-187, 192-209, 211-231, 245-273, 278-284, 289-304, 317-333, 335-341, 346-353, 359-366, 378-389, 394-402, 424-438, 447-460, 473-480, 482-492, 494-500, 506-520, 523-541, 558-574, 577-588, 598-605, 611-619, 622-629, 633-643, 649-669, 685-701, 707-727, 735-749, 768-780, 783-789, 798-810, 1-153, 178-240, 254-282, 284-349, 366-446, 450-784, 794-813, 79-169, 194-251, and 451-509 of SEQ ID NO 335; amino acids 4-30, 38-46, 56-70, 80-90, 101-110, 117-123, 149-157, 162-172, 178-191, 211-218, 230-256, 261-283, 292-310, 335-344, 348-354, 361-369, 382-396, 403-414, 425-431, 452-458, 460-472, 484-504, 508-514, 524-541, 562-580, 597-603, 627-643, 645-657, 688-696, 707-713, 719-736, 746-759, 769-775, 791-797, 802-812, 824-830, 838-847, 856-864, 880-901, 909-916, 1-52, 64-78, 81-202, 221-297, 335-381, 384-454, 464-552, 559-590, 598-620, 622-644, 646-668, 672-821, 833-898, 25-108, and 505-560 of SEQ ID NO 336; amino acids 16-24, 37-46, 60-73, 79-90, 97-116, 130-141, 143-150, 155-161, 179-186, 193-206, 212-225, 230-253, 258-267, 290-296, 306-344, 366-381, 383-390, 398-405, 418-425, 438-449, 465-482, 489-499, 503-509, 530-543, 545-567, 7-44, 51-79, 81-114, 116-263, 285-307, 311-351, 379-416, 427-482, 491-509, 535-570, 126-230, 248-286, 364-508, and 510-517 of SEQ ID NO 337; amino acids 4-10, 13-21, 27-40, 90-95, 121-128, 197-203, 239-246, 253-259, 305-311, 322-336, 349-356, 358-364, 372-379, 393-408, 411-417, 427-434, 457-467, 481-490, 492-497, 504-513, 533-542, 561-571, 592-600, 641-646, 661-667, 671-682, 711-720, 722-734, 763-771, 779-796, 833-838, 855-865, 873-879, 886-896, 901-908, 913-924, 931-939, 1-19, 24-55, 88-108, 118-153, 172-190, 198-281, 299-361, 369-392, 430-578, 661-689, 698-738, 795-850, 861-901, 904-924, 927-946, 37-262, 272-351, 453-474, 499-518, 527-547, 569-643, and 897-954 of SEQ ID NO 338; amino acids 22-28, 39-49, 4-20, 26-44, and 5-65 of SEQ ID NO 339; amino acids 4-39, 52-63, 70-88, 97-112, 1-88, 94-125, and 56-113 of SEQ ID NO 340; amino acids 10-29, 35-40, 46-62, 77-84, 94-129, 146-153, 158-164, 166-174, 196-202, 209-215, 225-240, 250-258, 272-287, 305-313, 318-335, 339-353, 362-381, 384-390, 395-424, 439-452, 454-463, 467-476, 482-496, 504-529, 535-562, 564-573, 576-588, 596-602, 604-619, 625-658, 664-675, 685-696, 714-727, 746-758, 770-777, 780-789, 793-799, 845-861, 863-873, 890-901, 905-910, 921-934, 1-30, 35-106, 117-348, 357-381, 390-551, 553-800, 804-835, 844-882, 893-937, 231-286, 394-429, and 686-758 of SEQ ID NO 341; amino acids 8-31, 35-70, 83-89, 91-102, 108-116, 130-156, 169-191, 203-217, 238-256, 270-292, 298-311, 315-331, 335-345, 364-370, 376-382, 384-401, 405-419, 421-430, 434-441, 445-452, 467-481, 503-511, 516-522, 524-531, 534-539, 547-560, 570-582, 5-104, 122-260, 271-340, 353-564, 566-603, 106-129, 215-224, and 454-522 of SEQ ID NO 342; amino acids 8-27, 29-37, 42-49, 63-69, 74-80, 95-118, 140-148, 156-170, 189-195, 197-209, 218-223, 227-235, 247-255, 268-275, 294-299, 305-313, 316-333, 347-359, 369-376, 381-386, 399-410, 423-432, 436-455, 462-475, 524-537, 578-588, 597-605, 616-622, 624-634, 636-644, 648-655, 657-665, 693-698, 706-723, 726-732, 736-778, 782-805, 820-832, 835-846, 848-870, 882-909, 927-933, 951-957, 962-971, 976-982, 986-1001, 1016-1021, 1042-1050, 1054-1061, 1066-1075, 1-68, 84-100, 152-182, 198-344, 348-413, 416-552, 558-621, 624-788, 800-892, 958-1060, 1064-1078, 155-209, 427-501, 658-688, 809-841, 862-871, and 927-1010 of SEQ ID NO 343; amino acids 15-36, 55-67, 70-76, 81-88, 110-125, 128-134, 136-188, 197-214, 216-223, 244-250, 257-263, 265-280, 300-306, 311-319, 2-36, 52-72, 82-152, 156-188, 191-225, 232-322, 21-78, 90-154, and 168-215 of SEQ ID NO 344; amino acids 4-25, 32-41, 51-58, 71-78, 81-89, 101-123, 130-138, 140-151, 165-171, 176-193, 202-214, 244-259, 266-282, 285-293, 295-301, 316-325, 329-334, 344-350, 381-390, 405-415, 423-431, 1-120, 126-160, 173-257, 266-313, 322-342, 354-434, 8-56, and 181-269 of SEQ ID NO 345; amino acids 4-46, 53-60, 74-86, 97-104, 121-128, 149-160, 206-217, 252-258, 274-280, 287-294, 311-327, 356-370, 378-383, 394-403, 425-432, 434-447, 473-484, 491-502, 543-556, 599-608, 610-623, 641-657, 671-684, 698-707, 719-733, 748-758, 772-779, 798-808, 814-820, 830-843, 854-866, 872-884, 891-899, 901-907, 920-932, 1-91, 114-128, 130-149, 156-190, 207-306, 308-330, 342-429, 436-450, 454-662, 666-937, 103-238, 247-273, 451-470, 687-734, and 753-796 of SEQ ID NO 346; amino acids 35-41, 52-58, 66-74, 78-87, 92-99, 101-109, 120-132, 153-164, 176-182, 186-194, 208-238, 246-262, 272-284, 291-311, 317-322, 328-343, 4-104, 112-131, 150-214, 216-345, 36-58, and 282-336 of SEQ ID NO 347; amino acids 5-27, 29-42, 57-71, 73-80, 92-99, 101-107, 126-136, 142-151, 155-163, 173-179, 214-232, 235-254, 262-274, 297-309, 311-323, 330-358, 362-379, 383-394, 396-416, 432-457, 464-472, 474-485, 487-498, 511-519, 527-536, 1-35, 53-118, 123-280, 283-440, 442-542, and 22-120 of SEQ ID NO 348; amino acids 4-15, 27-61, 64-77, 84-97, 102-122, 144-215, 223-239, 242-252, 257-263, 271-286, 295-302, 312-324, 328-348, 380-400, 403-411, 417-428, 434-440, 444-451, 453-467, 485-496, 522-538, 540-545, 552-560, 566-577, 582-588, 1-149, 151-408, 442-594, and 324-391 of SEQ ID NO 349; amino acids 21-41, 43-123, 125-137, 139-157, 171-190, 193-203, 205-226, 242-256, 264-274, 289-299, 301-311, 323-368, 375-396, 400-408, 418-434, 1-437, 84-149, 163-176, and 260-321 of SEQ ID NO 350; amino acids 4-11, 19-34, 44-65, 70-115, 130-140, 142-168, 170-176, 185-206, 221-227, 1-65, 81-124, 127-206, 215-232, and 157-215 of SEQ ID NO 351; amino acids 4-19, 44-51, 66-104, 122-129, 136-141, 143-150, 154-159, 166-173, 181-205, 207-218, 1-15, 21-119, 123-213, and 77-182 of SEQ ID NO 352; amino acids 4-15, 17-34, 40-51, 61-71, 84-89, 137-146, 153-159, 163-170, 175-191, 203-210, 216-228, 242-250, 259-271, 279-284, 291-329, 333-372, 376-394, 399-404, 406-412, 416-424, 446-451, 1-76, 82-168, 179-209, 227-249, 253-473, and 234-319 of SEQ ID NO 353; amino acids 8-15, 17-26, 30-45, 47-74, 80-92, 94-111, 114-120, 124-133, 148-173, 175-181, 183-196, 198-208, 212-231, 238-244, 248-262, 266-285, 290-302, 308-317, 327-332, 334-341, 346-355, 357-365, 373-380, 1-47, 68-99, 104-174, 178-378, and 135-280 of SEQ ID NO 354; amino acids 12-28, 32-56, 61-72, 74-89, 95-128, 130-144, 150-163, 177-189, 196-214, 218-282, 294-305, 313-382, 384-398, 404-420, 426-454, 456-461, 1-224, 229-283, 296-464, and 350-406 of SEQ ID NO 355; amino acids 6-17, 22-31, 35-63, 67-85, 89-101, 128-147, 149-155, 161-170, 172-179, 187-198, 228-234, 240-247, 284-291, 298-308, 326-337, 344-350, 357-366, 1-28, 30-82, 86-267, 271-307, 310-376, 5-136, and 172-260 of SEQ ID NO 356; amino acids 9-32, 36-52, 72-90, 149-157, 165-177, 188-196, 223-233, 237-245, 264-273, 280-293, 298-307, 309-348, 352-362, 366-374, 381-396, 405-413, 416-421, 428-435, 438-444, 449-457, 464-476, 491-555, 579-597, 602-628, 646-652, 663-670, 674-692, 739-747, 762-771, 779-789, 809-816, 1-40, 49-69, 72-92, 97-265, 271-369, 373-452, 456-476, 496-527, 535-555, 567-637, 652-819, 297-327, 539-627, and 712-776 of SEQ ID NO 357; amino acids 5-16, 21-27, 39-46, 60-77, 92-104, 112-118, 129-136, 141-164, 167-176, 178-214, 220-228, 234-251, 284-301, 303-310, 315-328, 336-353, 362-375, 380-390, 393-409, 411-417, 430-438, 448-459, 461-468, 1-15, 53-86, 89-225, 237-320, 331-398, 406-467, 107-184, and 256-311 of SEQ ID NO 358; amino acids 13-34, 36-42, 49-62, 86-92, 124-130, 132-145, 1-38, 41-140, and 4-89 of SEQ ID NO 359; amino acids 16-22, 45-51, 80-85, 88-108, 115-127, 139-157, 160-176, 184-189, 198-204, 227-234, 249-270, 277-295, 314-325, 331-337, 344-367, 397-424, 432-500, 512-521, 524-530, 555-563, 569-583, 586-617, 622-653, 661-673, 679-696, 710-738, 754-759, 764-786, 793-808, 813-820, 831-837, 844-850, 864-889, 891-899, 912-926, 961-970, 975-982, 1016-1024, 1027-1034, 1051-1059, 1096-1102, 1134-1147, 1158-1177, 1179-1185, 1188-1215, 1235-1250, 1271-1284, 1308-1315, 1-34, 79-113, 129-325, 327-360, 363-383, 385-514, 530-565, 577-701, 704-941, 944-981, 985-1005, 1012-1032, 1049-1088, 1097-1183, 1188-1320, 108-194, 370-410, 411-442, 956-1046, and 1055-1236 of SEQ ID NO 360; amino acids 20-32, 67-73, 84-91, 102-108, 148-163, 198-209, 232-237, 7-94, 98-116, 121-196, 205-224, 240-254, 113-132, and 167-245 of SEQ ID NO 361; amino acids 7-16, 36-43, 69-77, 101-114, 136-150, 155-161, 183-195, 197-206, 210-218, 225-237, 1-77, 97-156, 172-215, 221-240, and 92-164 of SEQ ID NO 362; amino acids 17-23, 52-62, 75-87, 107-112, 120-135, 140-146, 170-176, 188-206, 210-220, 228-255, 273-282, 304-314, 324-331, 344-355, 363-370, 2-35, 81-179, 181-264, 267-288, 292-376, 2-126, and 245-300 of SEQ ID NO 363; amino acids 4-9, 11-17, 24-85, 89-103, 114-119, 126-135, 151-158, 169-175, 191-203, 226-237, 247-256, 261-266, 278-283, 306-316, 321-331, 333-340, 350-358, 361-392, 397-408, 412-419, 1-113, 117-183, 186-275, 294-362, 370-448, 468-485, 63-136, 248-305, and 458-466 of SEQ ID NO 364; amino acids 4-23, 41-47, 52-59, 62-75, 86-92, 98-104, 106-112, 126-143, 152-157, 160-166, 183-192, 196-201, 207-214, 227-235, 238-245, 248-265, 276-285, 304-314, 324-342, 350-356, 360-375, 390-403, 411-435, 437-446, 453-461, 1-39, 43-79, 88-160, 165-236, 241-426, 434-470, 132-199, and 214-286 of SEQ ID NO 365; amino acids 8-31, 35-48, 75-98, 100-106, 128-134, 151-166, 168-177, 202-223, 252-257, 265-277, 288-302, 306-316, 371-377, 385-396, 408-413, 429-445, 472-480, 1-331, 352-386, 404-488, 80-156, and 237-303 of SEQ ID NO 366; amino acids 4-10, 22-32, 39-46, 52-69, 90-96, 104-117, 126-142, 148-161, 163-174, 178-195, 200-210, 216-222, 225-236, 238-246, 248-265, 279-285, 313-322, 324-336, 1-203, 205-339, 31-111, and 205-228 of SEQ ID NO 367; amino acids 6-26, 33-63, 66-74, 125-148, 153-185, 225-230, 234-250, 253-278, 286-299, 312-319, 335-346, 348-361, 383-401, 414-419, 423-430, 432-440, 447-454, 470-478, 488-508, 519-533, 537-554, 16-83, 126-172, 175-210, 245-325, 329-448, 469-557, 81-97, 197-371, and 470-500 of SEQ ID NO 368; amino acids 4-9, 11-32, 44-56, 68-74, 82-98, 104-109, 131-140, 144-164, 176-182, 188-194, 206-212, 219-225, 228-235, 237-244, 254-271, 277-287, 294-302, 312-318, 324-331, 341-350, 352-358, 399-405, 411-419, 438-447, 452-459, 465-470, 475-484, 486-506, 514-523, 525-532, 537-543, 1-73, 112-209, 231-294, 296-314, 326-381, 388-414, 423-457, 462-484, 495-514, 522-544, 547-568, and 495-559 of SEQ ID NO 369; amino acids 4-26, 29-36, 45-52, 57-63, 68-75, 77-88, 93-108, 114-120, 138-143, 148-155, 161-179, 198-211, 214-224, 229-242, 244-253, 268-274, 302-311, 319-331, 335-349, 364-374, 377-384, 388-394, 423-436, 441-446, 452-471, 478-501, 506-514, 522-546, 558-579, 589-604, 623-631, 1-36, 40-114, 125-255, 266-395, 413-511, 530-557, 564-583, 605-640, 54-144, and 543-627 of SEQ ID NO 370; amino acids 5-10, 24-30, 37-53, 62-94, 101-111, 118-125, 145-151, 156-162, 164-186, 191-198, 204-212, 224-233, 244-253, 263-269, 277-297, 304-322, 334-341, 355-372, 379-384, 393-406, 412-425, 427-435, 437-449, 461-471, 483-506, 515-527, 535-543, 546-559, 562-570, 576-584, 588-601, 640-647, 651-673, 681-689, 712-734, 1-63, 73-369, 371-428, 432-454, 457-567, 573-635, 655-698, 707-744, 600-685, and 741-753 of SEQ ID NO 371; amino acids 13-19, 27-38, 78-87, 105-129, 134-142, 151-179, 184-197, 206-214, 225-235, 246-265, 274-314, 322-330, 334-343, 1-47, 49-66, 70-96, 102-285, 287-322, 330-346, 14-57, 76-167, and 191-245 of SEQ ID NO 372; amino acids 15-21, 26-45, 49-60, 62-69, 77-87, 96-104, 108-114, 117-130, 135-144, 149-160, 174-181, 186-193, 203-218, 228-234, 236-242, 257-264, 285-326, 332-342, 344-349, 353-370, 373-425, 428-437, 447-457, 472-483, 490-505, 516-526, 546-554, 556-570, 572-585, 601-609, 613-624, 626-640, 647-663, 665-671, 678-689, 691-697, 707-719, 721-751, 769-778, 1-64, 73-192, 209-327, 329-418, 421-529, 534-586, 599-646, 650-682, 703-793, 86-169, 318-384, 502-520, and 529-602 of SEQ ID NO 373; amino acids 4-10, 19-26, 28-34, 41-60, 62-81, 89-97, 99-115, 143-156, 162-181, 186-193, 197-207, 225-231, 233-240, 254-266, 283-296, 309-323, 325-338, 341-352, 363-373, 388-413, 420-426, 1-37, 41-119, 133-251, 255-277, 291-355, 359-411, 93-182, and 360-391 of SEQ ID NO 374; amino acids 5-37, 48-77, 82-89, 91-102, 119-127, 129-138, 145-179, 186-194, 197-203, 205-212, 219-235, 243-250, 273-289, 294-304, 309-349, 352-363, 377-388, 391-399, 407-415, 418-443, 449-463, 471-477, 479-494, 496-503, 2-44, 85-108, 127-242, 245-259, 271-288, 291-329, 339-433, 443-457, 468-517, 34-88, 104-164, 168-227, 235-259, 279-301, and 345-377 of SEQ ID NO 375; amino acids 4-24, 32-39, 44-51, 60-70, 86-96, 98-105, 108-117, 122-138, 142-156, 167-181, 192-212, 214-223, 231-240, 254-278, 15-131, 139-285, 9-96, and 181-194 of SEQ ID NO 376; amino acids 12-31, 41-47, 50-73, 86-99, 103-109, 141-147, 152-167, 180-195, 201-214, 216-237, 240-246, 251-265, 275-281, 289-294, 301-314, 320-333, 342-349, 351-361, 367-389, 402-408, 426-435, 459-469, 9-27, 31-92, 94-134, 147-199, 202-268, 271-291, 303-333, 336-465, 480-498, and 72-139 of SEQ ID NO 377; amino acids 23-34, 41-52, 74-86, 92-99, 107-121, 123-130, 154-159, 162-169, 193-227, 229-243, 249-255, 260-269, 17-154, 183-272, and 137-270 of SEQ ID NO 378; amino acids 4-29, 81-88, 106-128, 177-200, 207-214, 221-234, 240-250, 255-277, 279-285, 291-324, 326-347, 349-362, 376-392, 400-412, 415-423, 425-440, 444-449, 453-460, 1-52, 60-99, 102-161, 166-189, 203-294, 312-429, 436-468, 487-512, and 419-508 of SEQ ID NO 379; amino acids 4-11, 42-49, 51-68, 91-97, 111-122, 130-142, 156-178, 186-230, 247-252, 278-297, 303-331, 337-344, 355-360, 383-391, 415-421, 439-444, 452-463, 468-481, 516-524, 537-542, 558-570, 576-582, 590-597, 621-633, 655-661, 667-673, 680-688, 728-734, 764-771, 776-789, 803-810, 825-841, 844-853, 877-891, 893-907, 924-933, 947-960, 966-974, 984-989, 998-1011, 1018-1024, 1028-1034, 1044-1053, 1084-1093, 1104-1110, 1114-1121, 1146-1154, 1166-1173, 1189-1196, 1202-1210, 1212-1218, 1220-1228, 1243-1248, 2-25, 30-82, 95-287, 304-370, 444-491, 507-662, 677-698, 704-746, 763-811, 818-920, 926-970, 989-1127, 1130-1198, 1203-1239, 200-260, 562-610, 815-858, 892-903, 961-1060, and 1065-1073 of SEQ ID NO 380; amino acids 25-41, 50-56, 58-70, 101-111, 118-126, 138-147, 161-168, 242-252, 271-291, 320-329, 336-342, 353-364, 369-375, 383-389, 418-432, 473-484, 499-514, 520-532, 537-545, 548-560, 567-575, 588-606, 612-622, 628-634, 647-658, 666-673, 686-693, 700-706, 714-720, 734-744, 754-759, 786-798, 820-827, 852-859, 864-885, 892-899, 908-915, 921-933, 941-946, 950-957, 1-52, 55-157, 189-250, 269-311, 358-576, 582-774, 783-837, 855-924, 930-961, 75-155, 197-242, 257-412, 457-547, 715-788, and 874-936 of SEQ ID NO 381; amino acids 29-36, 49-55, 92-100, 109-116, 127-135, 140-148, 153-158, 169-176, 179-191, 214-221, 227-237, 240-246, 1-43, 55-73, 108-155, 164-249, and 20-87 of SEQ ID NO 382; amino acids 5-19, 30-41, 44-49, 64-70, 72-80, 82-88, 95-100, 115-120, 141-163, 1-39, 57-119, 131-166, and 15-83 of SEQ ID NO 383; amino acids 18-53, 64-75, 91-98, 103-109, 112-119, 154-167, 173-181, 200-208, 226-241, 261-268, 283-289, 294-309, 344-371, 374-382, 388-398, 416-423, 425-439, 442-453, 456-472, 491-497, 502-513, 515-524, 527-542, 547-562, 599-608, 610-620, 623-632, 636-656, 666-675, 683-692, 697-703, 709-716, 747-769, 778-785, 792-805, 829-836, 863-870, 877-905, 913-922, 937-950, 952-985, 991-999, 1-110, 114-160, 169-189, 216-238, 244-364, 378-409, 422-446, 453-475, 487-570, 590-640, 643-713, 722-809, 816-952, 969-1017, 155-173, 239-280, 501-575, 689-727, 766-789, and 826-880 of SEQ ID NO 384; amino acids 29-35, 40-64, 66-75, 115-122, 141-173, 179-187, 199-206, 209-215, 234-240, 244-251, 259-272, 297-303, 319-325, 332-348, 358-368, 372-382, 388-397, 399-405, 431-439, 446-454, 43-130, 136-211, 242-297, 314-333, 338-387, 396-416, 422-459, 19-144, 219-359, and 438-452 of SEQ ID NO 385; amino acids 4-23, 30-35, 77-84, 92-97, 105-113, 115-120, 131-137, 140-151, 200-211, 226-239, 241-247, 255-261, 271-278, 304-309, 395-400, 429-435, 443-453, 478-493, 521-530, 535-553, 563-568, 571-581, 623-628, 660-667, 693-700, 1-28, 74-140, 151-185, 194-218, 225-298, 300-355, 358-443, 445-463, 468-553, 567-595, 597-621, 631-676, 681-699, 16-72, 526-619, and 625-694 of SEQ ID NO 386; amino acids 4-18, 26-42, 58-79, 83-89, 99-108, 110-118, 125-140, 143-156, 168-175, 177-189, 211-227, 262-273, 281-291, 356-363, 383-402, 464-472, 1-49, 59-247, 251-321, 338-404, 410-430, 438-493, and 244-462 of SEQ ID NO 387; amino acids 6-28, 57-65, 72-81, 87-99, 1-43, 46-68, 83-104, and 13-105 of SEQ ID NO 388; amino acids 4-12, 32-55, 67-81, 92-100, 119-126, 143-151, 153-163, 182-188, 201-210, 238-244, 250-256, 278-283, 290-296, 303-308, 312-317, 345-354, 356-367, 378-388, 423-434, 442-449, 458-465, 476-488, 494-501, 510-520, 530-538, 551-563, 570-577, 582-601, 628-635, 700-714, 719-725, 744-752, 761-769, 778-786, 790-796, 821-839, 851-867, 872-878, 920-945, 957-975, 1022-1031, 1038-1047, 1084-1090, 1-91, 102-123, 130-170, 173-290, 307-327, 334-354, 359-396, 416-484, 507-563, 567-590, 600-618, 623-648, 661-686, 692-813, 822-887, 905-984, 994-1076, 1079-1093, 219-278, 280-417, 599-696, and 750-759 of SEQ ID NO 389; amino acids 6-39, 46-61, 67-72, 75-93, 107-116, 129-164, 169-176, 181-189, 226-243, 245-255, 267-274, 277-305, 319-326, 342-363, 375-385, 390-404, 419-428, 439-446, 448-465, 478-488, 507-516, 524-530, 546-555, 1-33, 35-63, 71-209, 217-237, 246-260, 283-313, 317-397, 399-431, 445-476, 479-558, and 151-220 of SEQ ID NO 390; amino acids 5-14, 25-50, 84-91, 99-111, 119-125, 129-144, 188-197, 200-208, 228-234, 243-250, 262-269, 275-282, 294-299, 9-175, 183-302, 104-209, and 229-292 of SEQ ID NO 391; amino acids 8-27, 29-42, 45-62, 1-71, and 8-61 of SEQ ID NO 392; amino acids 13-39, 41-47, 56-75, 78-87, 100-107, 121-127, 146-157, 164-171, 173-183, 201-207, 210-216, 221-231, 244-252, 255-263, 281-289, 291-309, 348-374, 390-401, 424-433, 435-445, 458-474, 509-530, 534-545, 548-565, 582-591, 597-602, 605-620, 622-628, 663-672, 686-691, 699-711, 728-742, 745-758, 773-786, 3-118, 120-171, 183-290, 293-313, 316-343, 345-367, 373-555, 561-651, 655-730, 737-773, 261-330, 337-365, and 587-709 of SEQ ID NO 393; amino acids 4-25, 41-54, 92-98, 104-111, 122-131, 144-151, 154-160, 200-207, 215-233, 243-253, 259-270, 278-297, 299-310, 328-337, 345-352, 373-389, 398-405, 419-425, 438-447, 472-479, 483-489, 492-501, 505-519, 531-546, 550-555, 568-574, 581-589, 595-600, 622-631, 683-689, 714-725, 733-738, 747-759, 761-766, 772-779, 795-801, 814-821, 856-868, 899-907, 923-929, 931-937, 1-30, 37-57, 64-86, 93-191, 193-217, 231-277, 296-361, 365-394, 398-529, 534-735, 742-785, 788-912, 506-517, and 532-590 of SEQ ID NO 394; amino acids 4-10, 16-25, 31-59, 66-75, 90-99, 102-107, 136-149, 151-159, 185-202, 204-215, 224-237, 240-248, 254-262, 272-278, 310-316, 337-349, 390-396, 400-409, 420-429, 478-484, 502-517, 533-539, 570-584, 614-621, 625-634, 645-654, 662-672, 677-686, 719-733, 747-753, 762-772, 782-792, 802-811, 820-826, 832-841, 856-862, 912-918, 931-937, 964-970, 989-994, 1006-1011, 1017-1023, 1036-1043, 1053-1064, 1071-1077, 1082-1090, 1103-1109, 1128-1135, 1137-1143, 1156-1165, 1169-1174, 1197-1204, 1213-1222, 1255-1273, 1305-1311, 1315-1322, 1340-1346, 1352-1359, 1386-1393, 1403-1412, 1441-1447, 1459-1468, 1470-1479, 1493-1500, 1511-1520, 1525-1537, 1544-1557, 1564-1569, 1580-1585, 1591-1596, 1627-1633, 1642-1656, 1690-1697, 1707-1713, 1715-1721, 1751-1761, 1773-1781, 1791-1796, 1804-1810, 1846-1851, 1860-1870, 1876-1882, 1910-1917, 1947-1953, 1962-1970, 2008-2019, 2026-2036, 2043-2051, 2085-2090, 2105-2115, 2132-2142, 1-30, 32-88, 143-330, 337-359, 379-434, 480-583, 604-715, 719-736, 744-761, 768-800, 805-850, 874-889, 898-948, 961-1053, 1058-1110, 1135-1150, 1157-1183, 1189-1230, 1235-1255, 1259-1277, 1308-1377, 1383-1422, 1428-1450, 1453-1472, 1474-1501, 1507-1591, 1596-1660, 1669-1703, 1709-1767, 1788-1848, 1861-1876, 1922-1941, 1944-1979, 1998-2061, 2069-2102, 2115-2136, 60-298, 308-571, 616-842, 971-1120, 1126-1252, 1359-1652, 1688-1980, and 2051-2106 of SEQ ID NO 395; amino acids 17-25, 29-38, 41-60, 97-105, 111-117, 124-159, 172-181, 186-192, 200-207, 212-229, 241-257, 267-298, 305-314, 316-322, 335-341, 345-355, 357-364, 367-375, 377-382, 387-400, 407-413, 417-428, 437-443, 448-454, 456-463, 469-477, 483-489, 495-508, 513-524, 540-551, 585-602, 1-98, 108-194, 205-473, 490-510, 517-557, 559-615, and 16-287 of SEQ ID NO 396; amino acids 10-33, 35-41, 50-55, 60-73, 81-90, 106-114, 121-136, 147-158, 179-191, 212-219, 229-236, 266-275, 285-307, 312-318, 334-345, 351-359, 361-366, 373-390, 398-419, 450-464, 475-483, 495-506, 524-530, 534-546, 555-565, 572-583, 596-603, 623-640, 658-664, 675-685, 690-696, 702-711, 714-723, 742-748, 765-772, 783-790, 811-823, 835-842, 848-854, 874-881, 1-31, 33-74, 85-118, 138-158, 165-184, 204-359, 375-402, 408-478, 491-511, 531-640, 653-745, 756-861, 871-889, 34-89, 124-209, 227-245, 271-338, 402-453, and 496-596 of SEQ ID NO 397; amino acids 19-31, 39-48, 50-61, 70-76, 85-96, 98-106, 114-127, 129-159, 166-184, 195-210, 215-224, 234-240, 247-254, 259-265, 267-284, 286-295, 297-307, 310-318, 320-330, 340-347, 1-32, 34-74, 93-115, 134-190, 192-255, 266-339, 119-185, and 241-349 of SEQ ID NO 398; amino acids 16-31, 45-52, 62-68, 89-149, 154-164, 175-185, 187-193, 195-207, 222-230, 253-279, 287-295, 299-323, 1-62, 85-154, 172-222, 224-248, 252-328, 188-255, and 319-334 of SEQ ID NO 399; amino acids 7-15, 38-65, 67-76, 80-103, 123-139, 146-152, 181-188, 196-206, 223-229, 234-245, 259-267, 285-296, 323-333, 340-358, 387-393, 401-409, 417-427, 431-439, 441-450, 472-477, 483-496, 508-524, 591-598, 602-610, 616-624, 627-641, 655-664, 666-679, 687-694, 696-709, 714-726, 759-766, 768-778, 782-793, 804-810, 1-105, 115-293, 300-342, 344-418, 432-562, 567-598, 613-656, 670-713, 757-803, and 23-98 of SEQ ID NO 400; amino acids 9-18, 35-57, 65-74, 82-87, 92-98, 105-117, 128-136, 147-154, 157-165, 181-187, 189-218, 224-232, 2-174, 176-241, and 26-112 of SEQ ID NO 401; amino acids 4-13, 24-35, 49-56, 71-77, 81-98, 103-109, 134-150, 158-169, 176-186, 188-195, 222-229, 249-262, 273-282, 335-341, 358-366, 375-395, 415-423, 431-448, 468-477, 486-492, 500-517, 526-539, 553-558, 563-571, 573-579, 581-603, 606-641, 649-657, 659-669, 1-40, 46-117, 127-171, 186-201, 203-318, 332-352, 356-390, 411-543, 560-669, 13-84, and 395-470 of SEQ ID NO 402; amino acids 43-56, 95-108, 139-145, 158-178, 186-199, 204-216, 224-231, 234-247, 251-275, 282-290, 303-309, 312-329, 331-341, 349-355, 379-405, 410-416, 429-436, 452-457, 463-474, 476-483, 485-491, 507-531, 546-563, 573-585, 628-635, 663-669, 687-700, 718-725, 731-742, 758-769, 788-795, 800-811, 824-831, 841-852, 859-865, 869-881, 885-891, 896-904, 941-951, 955-972, 1-91, 93-195, 199-284, 324-357, 385-405, 409-499, 506-604, 621-678, 688-749, 753-806, 815-882, 887-927, 933-953, 30-60, 107-144, 235-255, 439-492, 620-673, 767-814, and 864-969 of SEQ ID NO 403; amino acids 6-31, 59-73, 76-87, 98-104, 107-118, 126-145, 156-165, 168-177, 185-193, 206-214, 217-237, 245-252, 256-276, 288-304, 314-331, 333-340, 366-378, 387-406, 409-423, 428-437, 457-463, 480-488, 509-519, 536-542, 550-563, 571-593, 606-612, 6-51, 54-151, 161-194, 203-357, 361-474, 477-497, 501-538, 541-615, and 319-382 of SEQ ID NO 404; amino acids 6-12, 17-31, 33-48, 1-53, and 2-60 of SEQ ID NO 405; amino acids 54-61, 64-86, 91-109, 111-120, 127-133, 135-143, 147-152, 166-176, 179-186, 189-207, 212-220, 226-231, 235-242, 250-267, 271-279, 294-308, 316-336, 347-353, 362-368, 380-389, 391-398, 406-418, 441-463, 471-500, 503-522, 524-560, 562-570, 572-582, 584-590, 634-639, 643-652, 657-663, 666-673, 697-707, 714-721, 727-734, 743-751, 758-776, 796-806, 852-867, 870-896, 898-905, 923-945, 948-955, 957-962, 964-969, 982-988, 1000-1006, 1008-1014, 1030-1042, 1056-1068, 1075-1086, 2-97, 111-196, 209-321, 327-349, 353-367, 377-408, 418-491, 500-664, 691-772, 788-1019, 1024-1049, 1054-1077, 1079-1095, 29-54, 261-267, and 635-691 of SEQ ID NO 406; amino acids 4-16, 18-41, 47-54, 106-113, 120-131, 144-150, 165-171, 192-202, 220-227, 229-239, 245-259, 314-327, 358-365, 377-384, 396-402, 414-430, 436-442, 457-466, 474-480, 498-519, 524-531, 535-541, 564-569, 581-597, 622-628, 653-676, 681-686, 719-727, 1-139, 145-180, 186-261, 271-291, 308-328, 332-365, 372-394, 400-440, 453-530, 533-585, 588-631, 639-687, 695-746, 118-193, 209-355, and 505-657 of SEQ ID NO 407; amino acids 6-27, 50-56, 62-72, 80-86, 98-111, 118-127, 138-168, 176-184, 189-201, 205-245, 256-263, 1-44, 46-115, 132-192, 222-242, 246-264, 161-212, and 218-247 of SEQ ID NO 408; amino acids 6-28, 32-41, 46-66, 74-84, 92-108, 110-117, 137-146, 152-170, 173-200, 226-264, 266-275, 284-301, 308-313, 316-335, 355-364, 374-388, 394-404, 406-416, 424-431, 437-452, 456-477, 1-49, 51-166, 170-203, 208-368, 382-486, 489-507, 5-82, 101-118, and 260-376 of SEQ ID NO 409; amino acids 4-13, 16-26, 28-34, 39-45, 47-63, 68-121, 130-145, 150-162, 165-171, 176-203, 215-240, 242-249, 251-261, 282-290, 293-309, 316-322, 334-352, 360-367, 397-410, 412-422, 430-439, 1-67, 73-274, 288-429, 434-465, 119-189, 200-246, and 399-436 of SEQ ID NO 410; amino acids 4-12, 15-22, 25-34, 36-47, 54-79, 92-99, 106-114, 129-136, 150-160, 168-184, 186-215, 222-229, 239-249, 286-293, 299-314, 322-327, 333-358, 371-378, 387-406, 413-422, 435-443, 448-455, 461-471, 474-480, 489-495, 1-42, 57-82, 87-123, 127-498, and 30-110 of SEQ ID NO 411; amino acids 4-29, 42-68, 70-79, 1-52, 57-80, and 15-86 of SEQ ID NO 412; amino acids 8-51, 78-86, 94-99, 107-113, 140-148, 156-162, 173-181, 189-197, 213-229, 264-270, 274-281, 283-293, 308-321, 347-360, 399-411, 425-436, 442-449, 466-482, 486-498, 516-521, 527-534, 557-565, 586-594, 604-617, 622-630, 652-674, 720-731, 742-754, 773-783, 1-129, 131-247, 263-335, 343-364, 370-639, 652-697, 712-779, 187-241, 319-394, 411-485, and 657-679 of SEQ ID NO 413; amino acids 7-14, 36-46, 48-57, 74-81, 85-108, 113-131, 141-154, 168-179, 16-67, 69-91, 96-134, 137-177, and 48-105 of SEQ ID NO 414; amino acids 4-20, 28-33, 39-55, 57-65, 68-82, 84-92, 117-123, 1-85, 92-148, and 15-142 of SEQ ID NO 415; amino acids 4-9, 17-23, 37-53, 71-89, 104-116, 123-128, 133-138, 145-170, 173-205, 213-232, 234-252, 260-268, 275-280, 287-311, 316-345, 357-364, 1-18, 37-57, 68-278, 306-400, 7-100, and 152-243 of SEQ ID NO 416; amino acids 5-19, 38-45, 77-83, 89-99, 108-119, 125-132, 144-153, 161-171, 180-191, 221-227, 233-243, 274-286, 293-299, 1-27, 44-147, 159-255, 262-280, 288-302, 22-73, and 224-290 of SEQ ID NO 417; amino acids 8-15, 17-39, 48-55, 57-77, 87-95, 99-109, 119-132, 146-152, 171-176, 196-203, 220-229, 231-245, 253-259, 273-285, 293-300, 318-324, 331-344, 367-374, 395-401, 409-414, 434-471, 483-490, 509-517, 519-534, 537-544, 554-574, 584-590, 599-609, 615-625, 632-639, 665-672, 676-686, 688-697, 702-708, 714-720, 730-742, 744-752, 780-786, 790-800, 812-836, 2-17, 28-146, 152-170, 198-214, 235-270, 279-326, 333-368, 381-407, 435-608, 624-650, 656-673, 711-779, 782-799, 809-872, 157-185, and 341-483 of SEQ ID NO 418; amino acids 4-57, 60-67, 80-96, 98-118, 5-114, and 38-112 of SEQ ID NO 419; amino acids 11-34, 43-51, 67-108, 116-138, 151-165, 9-30, 40-168, and 13-78 of SEQ ID NO 420; amino acids 18-24, 40-47, 51-62, 67-74, 87-94, 102-109, 112-121, 141-147, 152-164, 175-198, 200-224, 229-235, 245-260, 262-293, 310-323, 326-349, 390-414, 423-431, 440-447, 456-462, 466-476, 481-493, 497-504, 512-538, 545-550, 560-573, 584-591, 596-602, 607-613, 7-67, 71-131, 134-231, 233-260, 276-340, 342-385, 388-443, 447-475, 509-616, and 504-552 of SEQ ID NO 421; amino acids 11-17, 38-49, 52-58, 67-79, 98-104, 7-106, and 16-107 of SEQ ID NO 422; amino acids 5-17, 32-46, 54-60, 89-102, 112-127, 152-161, 169-178, 186-193, 228-240, 247-256, 259-266, 274-296, 3-38, 55-69, 72-135, 165-301, 22-96, 124-131, 143-172, and 209-275 of SEQ ID NO 423; amino acids 17-26, 32-44, 1-43, and 28-49 of SEQ ID NO 424; amino acids 11-17, 26-32, 72-77, 162-168, 204-211, 218-224, 234-243, 270-276, 287-301, 314-333, 339-345, 360-375, 392-398, 403-409, 419-434, 438-444, 454-468, 473-479, 489-503, 508-514, 524-539, 543-549, 559-574, 578-584, 607-616, 618-628, 664-669, 710-715, 718-730, 734-740, 747-763, 766-772, 774-785, 787-796, 73-125, 133-152, 163-246, 264-330, 337-376, 594-635, 660-680, 693-711, 715-816, 6-428, and 493-608 of SEQ ID NO 425; amino acids 14-29, 32-58, 63-76, 78-86, 89-100, 102-110, 131-140, 148-154, 188-201, 210-219, 230-243, 245-254, 262-281, 298-307, 316-324, 340-352, 373-396, 407-414, 440-453, 456-477, 480-487, 495-508, 515-529, 537-544, 552-560, 564-572, 587-592, 605-619, 625-632, 638-644, 660-674, 684-690, 697-719, 732-738, 1-58, 67-95, 123-168, 176-240, 242-284, 287-317, 325-390, 398-418, 423-499, 510-582, 598-631, 635-683, 692-734, 127-190, 537-604, and 697-727 of SEQ ID NO 426; amino acids 13-27, 38-44, 48-55, 57-69, 72-81, 97-108, 126-141, 152-160, 165-177, 180-186, 197-241, 289-296, 309-328, 334-339, 341-353, 1-106, 125-251, 257-306, 310-356, 42-51, and 97-267 of SEQ ID NO 427; amino acids 4-10, 12-21, 29-35, 44-56, 59-67, 74-86, 91-98, 103-114, 1-124, and 9-86 of SEQ ID NO 428.

20. The antigen according to claim 19, whereby the antigen further consists of a) 1 to 50 additional amino acid residue(s), preferably 1 to 40, more preferably 1 to 30, even more preferably at most 1 to 25, still more preferably at most 1 to 10, most preferably 1, 2, 3, 4 or 5 additional amino acid residue(s); and/orb) at least one additional amino acid residue heterologous to the core amino acid sequence.

21. The antigen according to claim 20, wherein the additional amino acid residue(s) is/are flanking the core amino acid sequence N-terminally, C-terminally or N- and C-terminally.

22. The antigen according to claim 19, wherein the antigen comprises at least 2, at least 3, at least 4, at least 5 or at least 6 core amino acid sequences as defined in claim 19.

23. A process for producing an antigen, or an active fragment or an active variant thereof, comprising expressing the nucleic acid molecule as defined in claim 1.

24. (canceled)

25. (canceled)

26. A pharmaceutical composition, preferably a vaccine, comprising an antigen, or an active fragment or an active variant thereof, as defined in claim 11.

27.-30. (canceled)

31. An antigen, or an active fragment or an active variant thereof, as defined in claim 11 for the treatment or prevention of an infection with Moraxella, most preferably Moraxella catarrhalis.

32. (canceled)

33. An antibody, or at least an effective part thereof, which binds to at least a selective part of an antigen or a fragment thereof, preferably an active fragment thereof, or a variant thereof, preferably an active variant thereof, as defined in claim 11.

34.-38. (canceled)

39. A hybridoma cell line, which produces an antibody as defined in claim 33.

40. A method for producing an antibody, characterized by the following steps: a) initiating an immune response in a non-human animal by administering an antigen, or an active fragment or an active variant thereof, as defined in claim 11, to said animal,b) removing an antibody containing body fluid from said animal, andc) producing the antibody by subjecting said antibody containing body fluid to further purification steps.

41. (canceled)

42. A pharmaceutical composition comprising an antibody according to claim 33.

43.-47. (canceled)

48. A method for diagnosing an infection with a Moraxella organism comprising the steps of: a) contacting a sample obtained from a subject with an antigen, or an active fragment or an active variant thereof, as defined in claim 11; andb) detecting the presence of an antibody against said Moraxella organism in the sample.

49.-55. (canceled)

56. A method for the treatment of a Moraxella infection in an animal or human preferably in need thereof, comprising the step of administering to said animal or human a therapeutically effective amount of an antigen, or an active fragment or an active variant thereof, as defined in claim 11.

57.-59. (canceled)