Patent Application
20080160027
The present application relates generally to the fields of immunology, bacteriology and molecular biology. More particularly, the application relates to methods for obtaining and administering vaccines generated from the investigation of expression libraries constructed from a Chlamydia pneumoniae genome. In particular embodiments, the present application concerns methods and compositions for the vaccination of vertebrate animals against Chlamydia pneumoniae infections, wherein the vaccination of the animal is accomplished through a protein or gene derived from the genes or gene fragments validated as vaccines. In particular embodiments, the animal is a human.
Intracellular bacteria of the genus Chlamydia are important pathogens in both man and vertebrate animals causing blindness in man, sexually transmitted disease and community acquired pneumonia, and most likely act as co-factors in atherosclerotic clot formation in human coronary heart disease.
Specifically, Chlamydia pneumoniae is a major agent of community-acquired respiratory infection and pneumonia. In addition, Chlamydia pneumoniae is strongly associated with atherosclerotic coronary heart disease in developed countries, and is thought to be involved in the pathogenesis of asthma. These public health concerns indicate a requirement for control of such infections.
While antibiotics can be successfully used for the treatment of acute pulmonary infection caused by Chlamydia pneumoniae, once infection and pathology are established, antibiotic treatment has little effect on the outcome of chlamydial diseases. For instance, in large-scale field trials, antibiotic treatment did not influence atherosclerosis that had been associated with increased antibody levels against Chlamydia pneumoniae and the presence of the agent in lesions (Hammerschlag 2003).
As an alternative to a whole pathogen vaccine, recent trends in vaccine development have turned to component or subunit vaccine compositions. Such vaccines are far safer and more consistently manufactured, but have often shown reduced efficacy relative to live or inactivated pathogen vaccines. This has been attributed to reduced complexity and inefficient adjuvants; however another consideration is that the best antigens are rarely if ever established for a vaccine. A solution to this antigen discovery problem is expression library immunization. ELI is a recombinant DNA pooling strategy that enables to assay the full repertoire of genome-encoded components of a pathogen for protective antigens using genetic immunization (GI).
Since the original demonstration of ELI by intramuscular injection of genetic vaccine constructs for protection against Mycoplasma pulmonis pneumonia in mice, a number of methods have been used to deliver genes into a host to raise immune responses against the encoded product. The most commonly used ones have been injection into intramuscular (IM) or intradermal (ID) sites and DNA-coated particle delivery into skin epidermis with a gene gun (Barry et al 2004). In an ELI screen, the whole genome of a pathogen is reconstructed as gene fragments (Barry et al 1995). This library of fragments is manipulated into mammalian expression constructs, partitioned into sublibrary pools, and then used as inocula for test animals. Following pathogen exposure, vaccine utility is evaluated by the single criterion of disease protection (Stemke-Hale et al 2005). Another technology has been developed to speed construction and improve the quality of expression libraries. Linear expression elements (LEEs) are recombinant-DNA constructs that are built wholly in vitro. Namely, there is no amplification or propagation step that uses a live system such as bacterial cloning. LEEs are built by generating an open reading frame (ORF) by PCR, gene assembly, or some other in vitro DNA construction method, and then covalently or non-covalently attaching gene control elements such a promoter and terminator (Sykes et al 1999). The desired recombinant expression vector is constructed completely in vitro and ready to deliver directly in vivo.
The complete 1,230 kb genome sequence of the CDC/CWL-029 strain of Chlamydia pneumoniae has been published by Kalman et al (Nat. Genetics 1999). Using bioinformatics approaches, this knowledge allows identification of all putative ORFs for production of LEE vaccine constructs. Thus, all ORFs can be screened for protective candidate antigens for use in a vaccine against Chlamydia pneumoniae. This approach has been used for testing all Chlamydia pneumoniae genes, and highly protective vaccine candidate genes have been located.
Accordingly, the present application relates to antigens and nucleic acids encoding such antigens obtainable by screening a Chlamydia pneumoniae genome. In more specific aspects, the application relates to methods of isolating protective antigens and nucleic acids and to methods of using such isolated antigens for producing immune responses. The ability of an antigen to produce an immune response may be employed in vaccination or antibody preparation techniques.
In some embodiments, the application relates to isolated polynucleotides having a region that comprises a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21 a complement of any of these sequences, or fragments thereof, or sequences closely related to these sequences. In some more specific embodiments, the application relates to such polynucleotides comprising a region having a sequence comprising at least 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 200, or more contiguous nucleotides in common with at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21 or its complement. Of course, such polynucleotides may comprise a region having all nucleotides in common with at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21 or its complement.
In another aspect, the application relates to polypeptides having sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20 or fragments thereof, or sequences closely related to these sequences. The application also relates to methods of producing such polypeptides using recombinant methods, for example, using the polynucleotides described above.
The application relates to antibodies against Chlamydia pneumoniae antigens, including those directed against an antigen having polypeptide sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 or an antigenic fragment thereof, or sequences closely related to these sequences. The antibodies may be polyclonal or monoclonal and produced by methods known in the art.
The present application contemplates vaccines comprising: (a) a pharmaceutically acceptable carrier, and (b) at least one polynucleotide having a Chlamydia pneumoniae sequence. In one embodiment, the at least one polynucleotide may be isolated from a Chlamydia pneumoniae genomic DNA expression library but it need not be. As discussed below, the polynucleotides need not be of natural origin, or to encode an antigen that is precisely a naturally occurring Chlamydia pneumoniae antigen. It is anticipated that polynucleotides and antigens within the scope of this application may be synthetic and/or engineered to mimic, or improve upon, naturally occurring polynucleotides and still be useful in the invention.
In some embodiments, the at least one polynucleotide has a sequence isolated from Chlamydia pneumoniae, for example, a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21, or fragment thereof, or sequences closely related to these sequences. In more specific such embodiments, the at least one polynucleotide has a sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, or SEQ ID NO:19, or fragment thereof, or sequences closely related to these sequences. In even more specific embodiments, the at least one polynucleotide has a sequence of SEQ ID NO:5 or SEQ ID NO:3.
In some embodiments, the polynucleotide encodes an antigen having a sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or antigenic fragment thereof, or sequences closely related to these sequences.
In several embodiments, the polynucleotide is comprised in a genetic immunization vector. Such a vector may, but need not, comprise a gene encoding a mouse ubiquitin fusion polypeptide. The vector, in some preferred embodiments, will comprise a promoter operable in eukaryotic cells, for example, but not limited to a CMV promoter. Such promoters are well known to those of skill in the art. In some embodiments, the polynucleotide is comprised in a viral expression vector, for example, but not limited to, a vector selected from the group consisting of adenovirus, adeno-associated virus, retrovirus and herpes-simplex virus.
The vaccines of the application may comprise multiple polynucleotide sequences. In some embodiments, the vaccine will comprise at least a first polynucleotide having a Chlamydia pneumoniae sequence and a second polynucleotide having a Chlamydia pneumoniae sequence, wherein the first polynucleotide and the second polynucleotide have different sequences. In some more specific embodiments, the first polynucleotide may have a sequence of SEQ ID NO:4, or SEQ ID NO:6.
The present application also involves vaccines comprising: (a) a pharmaceutically acceptable carrier; and (b) at least one Chlamydia pneumoniae antigen. In several embodiments, the at least one Chlamydia pneumoniae antigen has a sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID 5 NO:22 or antigenic fragment thereof, or sequences closely related to these sequences. In some specific embodiments, the at least one Chlamydia pneumoniae antigen has a sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:20, or an antigenic fragment thereof, or sequences closely related to these sequences. In even more specific embodiments, the at least one Chlamydia pneumoniae antigen has a sequence of SEQ ID NO:4, or SEQ ID NO:6.
The present application also relates to methods of immunizing an animal comprising providing to the animal at least one Chlamydia pneumoniae antigen, or antigenic fragment thereof, in an amount effective to induce an immune response. In further embodiments, the Chlamydia pneumoniae antigens are comprised of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:20. As discussed above, and described in detail below, the Chlamydia pneumoniae antigens useful in the invention need not be native antigens. Rather, these antigens may have sequences that have been modified in any number of ways known to those of skill in the art, so long as they result in or aid in an antigenic response. In one embodiment, the animal is a human.
In some embodiments of the present application, the provision of the at least one Chlamydia pneumoniae antigen comprises: (a) preparing a cloned expression library from fragmented genomic DNA, cDNA or sequenced genes of Chlamydia pneumoniae; (b) screening the cloned expression library to identify highly protective genes; (c) administering at least one clone of the identified highly protective genes in a pharmaceutically acceptable carrier into an animal; and (d) expressing at least one Chlamydia pneumoniae antigen in the animal. The highly protective genes may comprise at least one or more polynucleotides having a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, OR SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or fragment thereof, or sequences closely related to these sequences. The expression library may be cloned in a genetic immunization vector, such vectors and other suitable vectors being well known in the art. The vector may comprise a gene encoding a mouse ubiquitin fusion polypeptide designed to link the expression library polynucleotides to the ubiquitin gene. The vector may comprise a promoter operable in eukaryotic cells, for example a CMV promoter, or any other suitable promoter. An acceptable vector is described in
In some embodiments, the provision of the Chlamydia pneumoniae antigen(s) may comprise: (a) preparing a pharmaceutical composition comprising at least one polynucleotide encoding a Chlamydia pneumoniae antigen or fragment thereof; (b) administering one or more prepared antigen or antigen fragment in a pharmaceutically acceptable carrier into an animal; and (c) expressing one or more Chlamydia pneumoniae antigens in the animal. The one or more polynucleotides can be comprised in one or more expression vectors, as described above and elsewhere in this specification.
Alternatively, the provision of the Chlamydia pneumoniae antigen(s) may comprise: (a) preparing a pharmaceutical composition of at least one Chlamydia pneumoniae antigen or an antigenic fragment thereof; and (b) administering the at least one antigen or fragment into an animal. The antigen(s) may be administered by a first intramuscular injection, intravenous injection, parenteral injection, epidermal injection, inhalation or oral route.
In the embodiments of the application, the animal is a mammal. In some cases the mammal is a bovine, in others, the mammal is a human.
In some embodiments, these methods may induce an immune response against Chlamydia pneumoniae. Alternatively, these methods may be practiced in order to induce an immune response against a Chlamydia species other than Chlamydia pneumoniae, for example, but not limited to, Chlamydia psittaci. Chlamydia trachomatis, and/or Chlamydia pecorum. In some embodiments, these methods may be employed to induce an immune response against a non-Chlamydia infection or other disease.
Thus, the present application is, in one embodiment, directed to a method of immunizing comprising the step of administering a Chlamydia pneumoniae antigen to an animal in an amount effective to induce an immune response against Chlamydia pneumoniae, wherein the antigen comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In one embodiment, the method of immunizing also comprises administering a second Chlamydia pneumoniae antigen in an amount effective to induce an immune response against Chlamydia pneumoniae, wherein the second antigen is distinct from the first antigen and comprises an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In one embodiment, the antigen is administered in a pharmaceutically acceptable carrier. In one embodiment, the animal is a human.
This specification discusses methods of obtaining polynucleotide sequences effective for generating an immune response against Chlamydia pneumonia by: (a) preparing a cloned expression library from fragmented genomic DNA of the genus Chlamydia; (b) administering one or more clones of the library in a pharmaceutically acceptable carrier into the animal in an amount effective to induce an immune response; and (c) selecting from the library the polynucleotide sequences that induce an immune response, wherein the immune response in the animal is protective against Chlamydia pneumoniae infection. Such methods may further comprise testing the animal for immune resistance against a Chlamydia pneumoniae bacterial infection by challenging the animal with Chlamydia pneumoniae. In some cases, the genomic DNA has been fragmented physically or by restriction enzymes, for example, but not limited to, fragments that average, about 200-1000 base pairs in length. In some cases, each clone in the library may comprise a gene encoding a mouse ubiquitin fusion polypeptide designed to link the expression library polynucleotides to the ubiquitin gene, but this is not required in all cases. In some cases, the library may comprise about 1×103 to about 1×106 clones; in more specific cases, the library could have 1×105 clones. In some preferred methods, about 0.01 μg to about 200 μg of DNA, from the clones is administered into the animal. In some situations the genomic DNA, cDNA or sequenced gene is introduced by intramuscular injection or epidermal injection. In some versions of these protocols, the cloned expression library further comprises a promoter operably linked to the DNA that permits expression in a vertebrate animal cell.
The application also discloses methods of preparing antigens that confer protection against infection in an animal comprising the steps of: (a) preparing a cloned expression library from fragmented genomic DNA of the Chlamydia pneumoniae genome; (b) administering one or more clones of the library in a pharmaceutically acceptable carrier into the animal in an amount effective to induce an immune response; (c) selecting from the library the polynucleotide sequences that induce an immune response and expressing the polynucleotide sequences in cell culture; and (d) purifying the polypeptide(s) expressed in the cell culture. Often, these methods further comprise testing “the animal for immune resistance against infection by challenging the animal with Chlamydia pneumoniae or other pathogens.
The application relates to methods of preparing antibodies against a Chlamydia pneumoniae antigen comprising the steps of (a) selecting a Chlamydia pneumoniae antigen that confers immune resistance against Chlamydia pneumoniae infection when challenged with Chlamydia pneumoniae; (b) generating an immune response in a vertebrate animal with the antigen identified in step (a); and (c) obtaining antibodies produced in the animal.
The application also relates to methods of assaying for the presence of Chlamydia pneumoniae infection in a vertebrate animal comprising: (a) obtaining an antibody directed against a Chlamydia pneumoniae antigen; (b) obtaining a sample from the animal; (c) admixing the antibody with the sample; and (d) assaying the sample for antigen-antibody binding, wherein the antigen-antibody binding indicates Chlamydia pneumoniae infection in the animal. In some cases, the antibody directed against the antigen is further defined as a polyclonal antibody. In others, the antibody directed against the antigen is further defined as a monoclonal antibody. In some embodiments, the Chlamydia pneumoniae antigen has a sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, or fragment thereof, or sequences closely related to these sequences. The assaying the sample for antigen-antibody binding may be by precipitation reaction, radioimmunoassay, ELISA, Western blot, immunofluorescence, or any other method known to those of skill in the art.
The application also relates to kits for assaying a Chlamydia pneumoniae infection comprising, in a suitable container: (a) a pharmaceutically acceptable carrier; and (b) an antibody directed against a Chlamydia pneumoniae antigen, wherein the antibody binds to a Chlamydia pneumoniae antigen having the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38.
The application further relates to methods of assaying for the presence of a Chlamydia pneumoniae infection in an animal comprising: (a) obtaining an oligonucleotide probe comprising a sequence comprised within one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, or a complement thereof; and (b) employing the probe in a PCR or other detection protocol.
As used herein in the specification, “a” or “an” may mean one or more. As used herein, when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.
As used herein, “plurality” means more than one. In certain specific aspects, a plurality may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300, 400, 500, 750, 1,000, 2,000, 3,000, 4,000, 5,000, 7,500, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 125,000, 150,000, 200,000 or more, and any integer derivable therein, and any range derivable therein.
As used herein, “any integer derivable therein” means a integer between the numbers described in the specification, and “any range derivable therein” means any range selected from such numbers or integers.
As used herein, a “fragment” refers to a sequence having or having at least 5, 10, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous residues of the recited SEQ ID NOS, but less than the full-length of the SEQ, ID NOS. It is contemplated that the definition of “fragment” can be applied to amino acid and nucleic acid fragments.
As used herein, an “antigenic fragment” refers to a fragment, as defined above, that can elicit an immune response in an animal. The term “animal” may refer to any animal, including vertebrate animals and particularly including humans.
Reference to a sequence in an organism, such as a “Chlamydia sequence” refers to a segment of contiguous residues that is unique to that organism or that constitutes a fragment (or full-length region(s)) found in that organism (either amino acid or nucleic acid).
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present application. The application may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Chlamydia pneumoniae is a species of chlamydiae bacteria that infects humans and is a major cause of pneumonia. Chlamydia pneumoniae has a complex life cycle and must infect another cell in order to reproduce and thus is classified as an obligate intracellular pathogen. In addition to its role in pneumonia, there is evidence associating Chlamydia pneumoniae with atherosclerosis and with asthma.
Chlamydia pneumoniae is a common cause of pneumonia around the world. Chlamydia pneumoniae is typically acquired by otherwise healthy people and is a form of community-acquired pneumonia. Because treatment and diagnosis are different from historically recognized causes such as Streptococcus pneumoniae, pneumonia caused by Chlamydia pneumoniae is categorized as an “atypical pneumonia.”
Typically, treatment for pneumonia is begun before the causative microorganism is identified. This empiric therapy includes an antibiotic active against the bacteria. The most common type of antibiotic used is a macrolide such as azithromycin or clarithromycin. If testing reveals that Chlamydia pneumoniae is the causative agent, therapy may be switched to doxycycline, which may be slightly more effective against the bacteria. Sometimes a quinolone antibiotic such as levofloxacin may be started empirically. This group is not as effective against Chlamydia pneumoniae. Treatment is typically continued for ten to fourteen days for known infections.
The present application is directed to compositions and methods for the immunization of vertebrate animals, including humans, against infections using nucleic acid sequences and polypeptides elucidated by screening Chlamydia pneumoniae. These compositions and methods will be useful for immunization against Chlamydia pneumoniae infections and other infections and disease states. In particular embodiments, a vaccine composition directed against Chlamydia pneumoniae infections is provided. The vaccine according to the present application comprises Chlamydia pneumoniae genes and polynucleotides identified by the inventors, that confer protective resistance in vertebrate animals to Chlamydia pneumoniae bacterial infections, and other infections. In other embodiments, the application provides methods for immunizing an animal against Chlamydia pneumoniae infections and methods for screening and identifying Chlamydia pneumoniae genes that confer protection against infection.
Referring to
The preparation of an expression library is performed using the techniques and methods familiar one of skill in the art. The pathogen's genome, may or may not be known or possibly may even have been cloned. Thus one obtains DNA (or cDNA), representing substantially the entire genome or all open reading frames of the pathogen (e.g., Chlamydia pneumoniae) The DNA is broken up, by physical fragmentation or restriction endonuclease, into segments of some length so as to provide a library of about 105 (approximately 18× the genome size) members. Alternatively, LEEs of all PCR-amplified open reading frames are constructed. The library is then tested by inoculating a subject with purified DNA of the library or sub-library and the subject challenged with a pathogen, wherein immune protection of the subject from pathogen challenge indicates a clone that confers a protective immune response against infection.
The present application discloses Chlamydia pneumoniae polynucleotide compositions and methods that induce a protective immune response in vertebrate animals challenged with a Chlamydia pneumoniae bacterial infection. The preparation and purification of antigenic Chlamydia polypeptides, or fragments thereof and antibody preparations directed against Chlamydia antigens, or fragments thereof are described below.
Thus, in certain embodiments, genes or polynucleotides encoding Chlamydia pneumoniae polypeptides or fragments thereof are provided. It is contemplated that in other embodiments, a polynucleotide encoding a Chlamydia pneumoniae polypeptide or polypeptide fragment will be expressed in prokaryotic or eukaryotic cells and the polypeptides purified for use as anti-Chlamydia pneumoniae antigens in the vaccination of vertebrate animals or in generating antibodies immunoreactive with Chlamydia pneumoniae polypeptides (i.e., antigens).
The present application, therefore, discloses polynucleotides encoding antigenic Chlamydia pneumoniae polypeptides capable of inducing a protective immune response in vertebrate animals and for use as an antigen to generate anti-Chlamydia pneumoniae or other pathogen antibodies. In certain instances, it may be desirable to express Chlamydia pneumoniae polynucleotides encoding a particular antigenic Chlamydia pneumoniae polypeptide domain or as a sequence to be used as a vaccine or in generating anti-Chlamydia pneumoniae or other pathogen antibodies. Nucleic acids according to the present application may encode an entire Chlamydia pneumoniae gene, or any other fragment of the Chlamydia pneumoniae sequences set forth herein. Experiments have been conducted to demonstrate the efficiency of both fragments and full length genes in providing a protective immune response. The nucleic acid may be derived from genomic DNA, i.e., cloned or PCR-amplified directly from the genome of a particular organism. In other embodiments, however, the nucleic acid may comprise complementary DNA (cDNA). A protein may be derived from the designated sequences for use in a vaccine or to isolate useful antibodies.
The term “cDNA” is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression.
It also is contemplated that a given Chlamydia pneumoniae polynucleotide from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same polypeptide (see Table 1 below). In addition, it is contemplated that a given Chlamydia polypeptide from a species may be generated using alternate codons that result in a different nucleic acid sequence but encodes the same polypeptide.
As used in this application, the term “a nucleic acid encoding a Chlamydia pneumoniae polynucleotide” refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine (Table 1, below), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of given Chlamydia pneumoniae gene or polynucleotide. Sequences that are essentially the same as those set forth in a Chlamydia pneumoniae gene or polynucleotide may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of a Chlamydia pneumoniae polynucleotide under standard conditions.
Thus, modifications and changes may be made in the structure of a gene and a functional molecule that encodes a protein or polypeptide with desirable characteristics may be obtained. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine *−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
The DNA segments of the present application include those encoding biologically functional equivalent Chlamydia pneumoniae proteins and peptides, as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
Referring now to
Naturally, the present application also encompasses nucleotide segments that are complementary, or essentially complementary to identified sequences of a Chlamydia pneumoniae polynucleotide. Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules and are well known in the art. As used herein, the term “complementary sequences” means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of a Chlamydia pneumoniae polynucleotide under relatively stringent conditions well known in the art, for example, using site-specific mutagenesis. Such sequences may encode the entire Chlamydia pneumoniae polypeptide or functional or non-functional fragments thereof.
For the purposes of the present application, a Chlamydia pneumoniae polypeptide used as an antigen may be a naturally-occurring Chlamydia pneumoniae polypeptide that has been extracted using protein extraction techniques well known to those of skill in the art, such as ELI, and prepared in a pharmaceutically acceptable carrier for the vaccination of an animal against Chlamydia pneumoniae infection. In alternative embodiments, the Chlamydia pneumoniae polypeptide or antigen may be a synthetic peptide. In still other embodiments, the peptide may be a recombinant peptide produced through molecular engineering techniques.
Chlamydia pneumoniae genes or their corresponding cDNA identified in the present application can be inserted into an appropriate cloning vehicle for the production of Chlamydia pneumoniae polypeptides as antigens. The transcription of a polypeptide sequence from a polynucleotide sequence is well known in the art.
In addition, sequence variants of the polypeptide can be prepared. The variants may, for instance, be minor sequence variants of the polypeptide that arise due to natural variation within the population, or they may be homologues found in other species. They also may be sequences that do not occur naturally, but that are sufficiently similar that they function similarly and/or elicit an immune response that cross-reacts with natural forms of the polypeptide. Sequence variants can be prepared by standard methods of site-directed mutagenesis well known in the art.
Another synthetic or recombinant variation of a Chlamydia-antigen is a polyepitopic moiety comprising repeats of epitopic determinants found naturally on Chlamydia pneumoniae proteins. Such synthetic polyepitopic proteins can be made up of several homomeric repeats of anyone Chlamydia pneumoniae protein epitope; or can comprise of two or more heteromeric epitopes expressed on one or several Chlamydia pneumoniae protein epitopes.
Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
Insertional variants include fusion proteins such as those used to allow rapid purification of the polypeptide and also can include hybrid proteins containing sequences from other proteins and polypeptides which are homologues of the polypeptide. For example, an insertional variant could include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species, such as Chlamydia psittaci or Chlamydia trachomatis. Other insertional variants can include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, into a protease cleavage site.
In one embodiment, major antigenic determinants of the polypeptide may be identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response. For example, the polymerase chain reaction (PCR) can be used to prepare a range of cDNAs encoding peptides lacking successively longer fragments of the C-terminus of the protein. The immunogenic activity of each of these peptides then identifies those fragments or domains of the polypeptide that are essential for this activity. Further experiments in which only a small number of amino acids are removed or added at each iteration then allows the location of other antigenic determinants of the polypeptide. Thus, the polymerase chain reaction, a technique for amplifying a specific segment of DNA via multiple cycles of denaturation-renaturation, using a thermostable DNA polymerase, deoxyribonucleotides and primer sequences is contemplated.
Another embodiment for the preparation of the polypeptides according to the application is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. Because many proteins exert their biological activity via relatively small regions of their folded surfaces, their actions can be reproduced by much smaller designer (mimetic) molecules that retain the bioactive surfaces and have potentially improved pharmacokinetic/dynamic properties.
The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. However, unlike proteins, peptides often lack well defined three dimensional structure in aqueous solution and tend to be conformationally mobile. Progress has been made with the use of molecular constraints to stabilize the bioactive conformations. By affixing or incorporating templates that fix secondary and tertiary structures of small peptides, synthetic molecules (protein surface mimetics) can be devised to mimic the localized elements of protein structure that constitute bioactive surfaces. Methods for predicting, preparing, modifying, and screening mimetic peptides are described in U.S. Pat. No. 5,933,819 and U.S. Pat. No. 5,869,451 (each specifically incorporated herein by reference). It is contemplated in the present application, that peptide mimetics will be useful in screening modulators of an immune response.
In certain embodiments, the synthesis of a Chlamydia pneumoniae peptide fragment is considered. The peptides of the application can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with well known protocols. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the application is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
The present application contemplates the purification, and in particular embodiments, the substantial purification, of Chlamydia pneumoniae polypeptides. The term “purified protein or peptide” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
Generally, “purified” will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50% or more of the proteins in the composition.
Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a “− fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater-fold purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE. It will, therefore, be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain and adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
The present application provides antibody compositions that are immunoreactive with a Chlamydia pneumoniae polypeptide of the present application, or any portion thereof.
An antibody can be a polyclonal or a monoclonal antibody. An antibody may also be monovalent or bivalent. A prototype antibody is an immunoglobulin composed by four polypeptide chains, two heavy and two light chains, held together by disulfide bonds. Each pair of heavy and light chains forms an antigen binding site, also defined as complementarity-determining region (CDR). Therefore, the prototype antibody has two CDRs, can bind two antigens, and because of this feature is defined bivalent. The prototype antibody can be split by a variety of biological or chemical means. Each half of the antibody can only bind one antigen and, therefore, is defined monovalent. Means for preparing and characterizing antibodies are well known in the art.
Peptides corresponding to one or more antigenic determinants of a Chlamydia polypeptide of the present application also can be prepared. Such peptides should generally be at least five or six amino acid residues in length, will preferably be about 10, 15, 20, 25 or about 30 amino acid residues in length, and may contain up to about 35-50 residues or so. Synthetic peptides will generally be about 35 residues long, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, Calif.). Longer peptides also may be prepared, e.g., by recombinant means.
The identification and preparation of epitopes from primary amino acid sequences on the basis of hydrophilicity is taught in U.S. Pat. No. 4,554,101 (Hopp), incorporated herein by reference. Through the methods disclosed in Hopp, one of skill in the art would be able to identify epitopes from within an amino acid sequence such as a Chlamydia pneumoniae polypeptide sequence. Predictable computer simulations and software well known in the art may be used to supplement and assist in predicting antigenic regions, such as PEPPLOT® available from the University of Wisconsin Biotechnology Center in Madison, Wis. or MACVECTOR available from IBI of New Haven, Conn.
In further embodiments, major antigenic determinants of a Chlamydia pneumoniae polypeptide may be identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response. For example, PCR can be used to prepare a range of peptides lacking successively longer fragments of the C-terminus of the protein. The immunoactivity of each of these peptides is determined to identify those fragments or domains of the polypeptide that are immunodominant. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinants of the polypeptide to be more precisely determined.
Another method for determining the major antigenic determinants of a polypeptide is the SPOTS system (Genosys Biotechnologies, Inc., The Woodlands, Tex.). In this method, overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody. The antigenic determinants of the peptides which are initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive peptide.
Once one or more such analyses are completed, polypeptides are prepared that contain at least the essential features of one or more antigenic determinants. The peptides are then employed in the generation of antisera against the polypeptide. Minigenes or gene fusions encoding these determinants also can be constructed and inserted into expression vectors by standard methods, for example, using peR cloning methodology.
The use of such small peptides for antibody generation or vaccination typically requires conjugation of the peptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine serum albumin. Methods for performing this conjugation are well known in the art.
The present application provides monoclonal antibody compositions that are immunoreactive with a Chlamydia pneumoniae polypeptide. As detailed above, in addition to antibodies generated against a full length Chlamydia polypeptide, antibodies also may be generated in response to smaller constructs comprising epitopic core regions, including wild-type and mutant epitopes. In other embodiments of the application, the use of anti-Chlamydia pneumoniae single chain antibodies, chimeric antibodies, diabodies and the like are contemplated.
As used herein, the term “antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
Monoclonal antibodies (mAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred.
However, “humanized” Chlamydia pneumoniae antibodies also are contemplated, as are chimeric antibodies from mouse, rat, goat or other species, fusion proteins, single chain antibodies, diabodies, bispecific antibodies, and other engineered antibodies and fragments thereof. As defined herein, a “humanized” antibody comprises constant regions from a human antibody gene and variable regions from a non-human antibody gene. A “chimeric antibody, comprises constant and variable regions from two genetically distinct individuals. An anti-Chlamydia pneumoniae humanized or chimeric antibody can be genetically engineered to comprise a Chlamydia pneumoniae antigen binding site of a given of molecular weight and biological lifetime, as long as the antibody retains its Chlamydia pneumoniae antigen binding site.
The term “antibody” is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab′, Fab, F(ab′h, single domain antibodies (DABs), Fv, scFv (single chain Fv), chimeras and the like. Methods and techniques of producing the above antibody-based constructs and fragments are well known in the art (U.S. Pat. No. 5,889,157; U.S. Pat. No. 5,821,333; U.S. Pat. No. 5,888,773, each specifically incorporated herein by reference).
U.S. Pat. No. 5,889,157 describes a humanized B3 scFv antibody preparation. The B3 scFv is encoded from a recombinant, fused DNA molecule, that comprises a DNA sequence encoding humanized Fv heavy and light chain regions of a B3 antibody and a DNA sequence that encodes an effector molecule. The effector molecule can be any agent having a particular biological activity which is to be directed to a particular target cell or molecule. Described in U.S. Pat. No. 5,888,773, is the preparation of scFv antibodies produced in eukaryotic cells, wherein the scFv antibodies are secreted from the eukaryotic cells into the cell culture medium and retain their biological activity. It is contemplated that similar methods for preparing multi-functional anti-Chlamydia pneumoniae fusion proteins, as described above, may be utilized in the present application.
Means for preparing and characterizing antibodies also are well known in the art. The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic Chlamydia pneumoniae composition in accordance with the present application and collecting antisera from that immunized animal.
A wide range of animal species can be used for the production of antisera. Typically, the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary, therefore, to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhold limpet hemocyanin (KLH) and bovine serium albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable molecule adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.
Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, Quil-A, a plant saponin, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated. MHC antigens may even be used. Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (SmithKline Beecham, Pa.); low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson & Johnson, Mead, N.J.), cytokines such as y-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.
A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified Chlamydia pneumoniae polypeptide, peptide or domain, be it a wild-type or mutant composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.
The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rate are contemplated in some embodiments; however, the use of rabbit, sheep or frog cells also is possible.
The animals are injected with antigen, generally as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary. The antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster injections with the same antigen would occur at approximately two-week intervals, or the gene encoding the protein of interest can be directly injected.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
Often, a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×107 to 2×108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art. For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
Fusion procedures usually produce viable hybrids at low frequencies, about 1×10−6 to 1×10−8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. HAT medium, a growth medium containing hypoxanthine, aminopterin and thymidine, is well known in the art as a medium for selection of hybrid cells. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas then would be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. First, a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. Second, the individual cell lines could be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the application can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present application can be synthesized using an automated peptide synthesizer.
It also is contemplated that a molecular cloning approach may be used to generate monoclonals. For this, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.
Alternatively, monoclonal antibody fragments encompassed by the present application can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in, for example, E. coli.
Compositions of the present application comprise an effective amount of a purified Chlamydia pneumoniae polynucleotide and/or a purified Chlamydia pneumoniae a protein, polypeptide, peptide, epitopic core region, and the like, dissolved and/or dispersed in a pharmaceutically acceptable carrier and/or aqueous medium. Aqueous compositions of gene therapy vectors expressing any of the foregoing are also contemplated.
The phrases “pharmaceutically and/or pharmacologically acceptable” refer to molecular entities and/or compositions that do not produce an adverse, allergic and/or other untoward reaction when administered to an animal.
As used herein, “pharmaceutically acceptable carrier” includes any and/or all solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and/or absorption delaying agents and the like. The use of such media and/or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media and/or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For animal and more particularly human administration, preparations should meet sterility, pyrogenicity, general safety and/or purity standards as required by FDA Office of Biologics standards.
The biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. The active compounds may generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and/or even intraperitoneal routes, or formulated for oral or inhaled delivery. The preparation of an aqueous composition that contains an effective amount of purified Chlamydia pneumoniae polynucleotide or polypeptide agent as an active component and/or ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions and/or suspensions; solid forms suitable for using to prepare solutions and/or suspensions upon the addition of a liquid prior to injection can also be prepared; and/or the preparations can also be emulsified.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions and/or dispersions; formulations including sesame oil, peanut oil and/or aqueous propylene glycol; and/or sterile powders for the extemporaneous preparation of sterile injectable solutions and/or dispersions. In all cases the form must be sterile and/or must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and/or storage and/or must be preserved against the contaminating action of microorganisms, such as bacteria and/or fungi.
Solutions of the active compounds as free base and/or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and/or in oils. Under ordinary conditions of storage and/or use, these preparations contain a preservative to prevent the growth of microorganisms.
A Chlamydia pneumoniae polynucleotide or polypeptide of the present application can be formulated into a composition in a neutral and/or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and/or which are formed with inorganic acids such as, for example, hydrochloric and/or phosphoric acids, and/or such organic acids as acetic, oxalic, tartaric, mandelic, and/or the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, and/or ferric hydroxides, and/or such organic bases as isopropylamine, trimethylamine, histidine, procaine and/or the like. In terms of using peptide therapeutics as active ingredients, the technology of U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and/or 4,578,770, each incorporated herein by reference, may be used.
The carrier can also be a solvent and/or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and/or liquid polyethylene glycol, and/or the like), suitable mixtures thereof, and/or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and/or the like. In many cases, it will be preferable to include isotonic agents, for example, sugars and/or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and/or gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and/or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, and/or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and/or in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and/or the like can also be employed.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and/or the liquid diluent first rendered isotonic with sufficient saline and/or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and/or intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and/or either added to 1000 ml of hypodermoclysis fluid and/or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
A Chlamydia polynucleotide or protein-derived peptides and/or agents may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, and/or about 0.001 to 0.1 milligrams, and/or about 0.1 to 1.0 and/or even about 10 milligrams per dose and/or so. Multiple doses can also be administered.
In addition to the compounds formulated for parenteral administration, such as intravenous and/or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets and/or other solids for oral administration; liposomal formulations; time release capsules; and/or any other form currently used, including cremes.
One may also use nasal solutions and/or sprays, aerosols and/or inhalants in the present application. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops and/or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, the aqueous nasal solutions usually are isotonic and/or slightly buffered to maintain a pH of 5.5 to 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and/or appropriate drug stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known and/or include, for example, antibiotics and/or antihistamines and/or are used for asthma prophylaxis.
Additional formulations which are suitable for other modes of administration include vaginal suppositories and/or pessaries. A rectal pessary and/or suppository may also be used. Suppositories are solid dosage forms of various weights and/or shapes, usually medicated, for insertion into the rectum, vagina and/or the urethra. After insertion, suppositories soften, melt and/or dissolve in the cavity fluids. In general, for suppositories, traditional binders and/or carriers may include, for example, polyalkylene glycols and/or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and/or the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations and/or powders. In certain defined embodiments, oral pharmaceutical compositions will comprise an inert diluent and/or assimilable edible carrier, and/or they may be enclosed in hard and/or soft shell gelatin capsule, and/or they may be compressed into tablets, and/or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and/or used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and/or the like. Such compositions and/or preparations should contain at least 0.1% of active compound. The percentage of the compositions and/or preparations may, of course, be varied and/or may conveniently be between about 2 to about 75% of the weight of the unit, and/or preferably between 25-60%. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and/or the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, and/or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and/or the like; a lubricant, such as magnesium stearate; and/or a sweetening agent, such as sucrose, lactose and/or saccharin may be added and/or a flavoring agent, such as peppermint, oil of wintergreen, and/or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings and/or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, and/or capsules may be coated with shellac, sugar and/or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and/or propylparabens as preservatives, a dye and/or flavoring, such as cherry and/or orange flavor.
Therapeutic kits of the present application are kits comprising a Chlamydia pneumoniae polynucleotide or polypeptide. Such kits will generally contain, in a suitable container, a pharmaceutically acceptable formulation of a Chlamydia pneumoniae polynucleotide or polypeptide or vector expressing any of the foregoing in a pharmaceutically acceptable formulation. The kit may have a single container, and/or it may have a distinct container for each compound.
When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The Chlamydia pneumoniae polynucleotide or polypeptide compositions may also be formulated into a syringeable composition. In which case, the container may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.
The container will generally include at least one vial, test tube, flask, bottle, syringe and/or other container, into which the Chlamydia pneumoniae polynucleotide or polypeptide formulation are placed, preferably, suitably allocated. The kits may also comprise a second container for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
The kits of the present application will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blowmolded plastic containers into which the desired vials are retained.
Irrespective of the number and/or type of containers, the kits of the application may also comprise, and/or be packaged with, an instrument for assisting with the injection/administration and/or placement of the ultimate Chlamydia pneumoniae polynucleotide or polypeptide within the body of an animal. Such an instrument may be a syringe, pipette, forceps, and/or any such medically approved delivery vehicle.
The present application discloses several polynucleotide and polypeptide sequences that code for proteins that provide a protective response against Chlamydia pneumoniae infection.
One of ordinary skill in the art will understand that Table 2 demonstrates first a polynucleotide (e.g., DNA) sequence for a gene or gene fragment and then the corresponding polypeptide (e.g., amino acid) sequence for the same gene or gene fragment. Thus, for example, SEQ ID NO:1 is a polynucleotide sequence corresponding to the polypeptide sequence of SEQ ID NO:2. Both SEQ ID NO:1 and SEQ ID NO:2 code for the same final protein. One of ordinary skill in the art will also understand that fragments of a gene, e.g., cutE_a, is contained within the full length gene, e.g., cutE. Thus, for example, SEQ ID NO:3 is contained in SEQ ID NO:1, and SEQ ID NO:4 is contained in SEQ ID NO:2.
Since the identified sequences demonstrate protective qualities in animal models, as demonstrated in the following examples, these identified sequences, when expressed as antigens, will be efficacious as a vaccine in animals and particularly in humans. Administration of at least one of the identified antigens is effective to induce an immune response in animals, particularly humans. In one embodiment, the antigen comprises the amino acid sequence set forth as SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20 or SEQ ID NO:22. In other embodiments, the antigen comprises the amino acid sequence set forth as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22. In some embodiments at least two different antigens are administered to an animal, in an amount effective to induce an immune response. In some of these embodiments, the two different antigens are antigens encoded by SEQ ID NO:4 and SEQ ID NO:6. In other embodiments at least three different antigens are administered to an animal, in an amount effective to induce an immune response. In some of these embodiments, two of the different antigens are antigens encoded by SEQ ID NO: 4 and SEQ ID NO: 6, and a third different antigen is selected from the group: SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22. In other embodiments, the two different antigens are selected from the group: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22, while the third different antigen is selected from the group: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38.
The following examples are included to demonstrate preferred embodiments of the application. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
MATERIALS AND METHODS. Chlamydia pneumoniae. Chlamydia pneumoniae strain CDC/CWL-029 (ATCC VR-1310) was grown, purified and quantified as described by Vaglenov et al 2005. Briefly, Buffalo Green Monkey Kidney cells (Diagnostic Hybrids, Inc. Athens, Ohio) were used as host cells for propagation of chlamydiae. For purification, embroid bodies in supernatant culture medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Sediments of purified infectious EBs were suspended in sucrose-phosphate-glutamate (SPG) buffer and stored at −80° C.
Animals. Inbred A/J and C57BL/6 female mice were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) at 5 weeks of age. Udel “shoebox” type cages with spun fiber filter top were maintained in static air or ventilated cage racks. Five animals were housed per cage in a temperature-controlled room with a 12-hour light/dark cycle, with ad libitum access to water and one of two diets. Mice were fed a 19% protein/1.33% L-arginine standard rodent maintenance diet. Beginning two weeks before challenge infection and during challenge infection, mice were fed a custom 24% protein/1.8% L-arginine diet (Harlan Teklad, Madison, Wis.). All components except protein/L-arginine were similar to the standard rodent maintenance diet. The custom diet was used because it was associated in preliminary experiments with enhanced immune responses and lower variance than the standard diet composed of non-chemically defined nutrient components. All animal protocols followed NIH guidelines and were approved by the Auburn University Institutional Animal Care and Use Committee (IACUC).
Negative and positive controls. In all experiments, unvaccinated (naïve) but challenged animals served as negative protection controls, and mice immunized with 5×106 genomes of viable Chlamydia pneumoniae one month prior to the vaccine challenge served as positive protection controls (immune). Groups were scored for protection by calculating the percent lung weight increase over that of age-matched unchallenged female A/J mice (138.4 mg), and by calculating the mean logarithm of total Chlamydia pneumoniae per lung. These values were then converted to a relative protection score by normalizing them to the lung weight increase or logarithm of total lung Chlamydia pneumoniae load that was calibrated by control immune (protection score 1=100% protection) and naïve (protection score 0=0% protection) groups. A CMVi-UB LEE construct encoding the luciferase gene (LUC) served as a control for LEE-based immunizations, and a plasmid construct pCMVi-UB carrying the same LUC insert was used as the control for plasmid-based immunizations.
Chlamydia pneumoniae lung challenge infection. Mouse intranasal inoculation was performed as described by Huang et al (1999), and optimal doses for live-immunization and challenge inocula were determined in preliminary experiments. For intranasal inoculation, mice received a light isoflurane inhalation anesthesia. Vaccine protection control mice were inoculated with a low dose of 5×106 Chlamydia pneumoniae elementary bodies in 30 μl SPG buffer. In rounds 1 and 2, higher-dose challenge infection was performed 4 weeks after the last gene gun genetic vaccination or low dose inoculation of live Chlamydia pneumoniae, by intranasal inoculation of 1×108 Chlamydia pneumoniae elementary bodies in 30 μl SPG buffer. In round 3, mice were challenged by an LD50 dose of 5×108 Chlamydia pneumoniae elementary bodies in 30 μl SPG buffer. Mice were sacrificed by CO2 inhalation 2 hours, 3 days, 10 days, or 15 days after inoculation, and lungs and spleen were weighed, snap frozen in liquid nitrogen, and stored at −80° C. until further processing. In all screening experiments, mice were sacrificed 10 days after inoculation. From selected animals, terminal blood was collected in heparinized microcentrifuge tubes by axillary incision under isoflurane anesthesia. Plasma was obtained by centrifugation at 5,000×g for 20 min in a microcentrifuge. Percent lung weight increase was based on naïve lung weights of 138.4 mg for adult A/J mice and 133 mg for adult C57BL/6 mice.
Mouse lung nucleic acid extraction. Mouse lungs were homogenized in guanidinium isothiocyanate Triton X-100-based RNA/DNA stabilization reagent in disposable tissue grinders (Fisher Scientific, Atlanta, Ga.) to create a 10% (wt/vol) tissue suspension. This suspension was used for total nucleic acid extraction by the High Pure® PCR template preparation kit (Roche Applied Science, Indianapolis, Ind.) and for mRNA extraction using oligo (dT)20 silica beads.
For mRNA extraction, a suspension of oligo (dT)20-coated silica beads (25 mg/ml in dH2O; 1 μm particle size, Kisker GbR, Steinfurt, Germany) was used. First, 100 μl of 10% lung suspension was mixed with 10 μl oligo (dT)20 silica bead suspension diluted in 230 μl dilution buffer (0.1 M Tris-HCl, pH 7.5, 0.2 M LiCl, 20 mM EDTA). For mRNA binding, samples were incubated at 72° C. for 3 minutes followed by room temperature for 10 minutes. The silica beads were sedimented by centrifugation at 13,000×g for 2 minutes, supernatants removed by decanting, the beads resuspended in 100 μl DNase buffer (20 mM Tris-HCl, pH 7.0, 1 M NaCl, 10 mM MnCl2) containing 100 U of RNase-free bovine pancreatic DNase I (Roche Applied Science, Indianapolis, Ind.) and incubated for 15 minutes at room temperature. Subsequently, beads were washed three times with wash buffer (10 mM Tris-HCl, pH 7.5, 0.2 M LiCl, 1 mM EDTA) by vigorous vortexing for 2 minutes followed by sedimentation at 13,000×g, and mRNA was eluted by resuspension of the beads in 200 μl DEPC-treated ddH2O followed by incubation at 72° C. for 7 minutes, centrifugal sedimentation, and removal of the supernatant mRNA. The purified nucleic acids samples were stored at −80° C. until used for real-time PCR assays.
Analysis of lung nucleic acids by real-time PCR. The primers and probes used in all PCR assays were custom synthesized by Operon, Alameda, Calif. The copy number of Chlamydia pneumoniae genomes per lung was determined by Chlamydia genus-specific 23S rRNA FRET (fluorescence resonance energy transfer) qPCR. One-step duplex RT-qPCR for analysis of lung transcript concentrations was performed in a Lightcycler as described by Wang et al (2004). RT reaction and PCR amplification for the analyte transcripts and an internal reference housekeeping gene transcript (porphobilinogen deaminase, PBGD) were performed in the same tube. All analyte transcript concentrations are expressed as copies per 1000 PBGD reference transcripts. Tim 3 is a CD4 Th1 cell-specific surface protein (GenBank #AF450241), GATA-3 is a CD4 Th2 cell-specific GATA sequence transcription factor (GenBank #X55123), CD45RO is a memory T cell surface protein (GenBank #NM—0112100).
Data analysis. All analyses were performed with the Statistica 7.0 software package (StatSoft, Tulsa, Okla.). Data of Chlamydia pneumoniae genome copies, RT-PCR gene transcripts, and anti-Chlamydophila IgG1 and IgG2a antibody relative light unit values were logarithmically transformed. Normal distribution of data was confirmed by the Shapiro-Wilk's W test, and homogeneity of variances by Levene's test. Data were evaluated by mean plots ±95% confidence intervals, and analyzed by analysis of variance (ANOVA). Post-hoc comparisons of means were performed under the assumption of no a priori hypothesis by the Tukey honest significant difference (HSD) test, or by Dunnett's test for determination of the significant differences between a single control group mean and the remaining treatment group means. Survival data were analyzed by one-sided Fisher's Exact test.
Experimental Outline: Round 1—ELI screen of the complete Chlamydia pneumoniae genome. The genome sequence of Chlamydia pneumoniae isolate CDC/CWL-029 (ATCC strain VR-1310) was extracted from Genbank (AE001363, 1,230,230 bp). The 1,052 annotated genes of Chlamydia pneumoniae were imported into a gene-splitting and primer prediction program; primer pairs to amplify 1,263 open reading frames (ORFs) of 1.5 kb or less were exported. A 1.5 kb maximum ORF length was chosen to ensure sufficient polymerase chain reaction (PCR) quality and yields, and this generated additional fragments. The sequence-specific primers each carried at the 5′-end a common 15 base stretch in which deoxy-uracil bases were interspersed every third position. This design rendered the 5′-ends of all PCR products susceptible to uracil-DNA-glycosylase (UDG) cleavage. Genomic DNA was isolated from purified Chlamydia pneumoniae stock as described by Sykes et al (1996), and used as a template. All products were PCR-amplified from Chlamydia pneumoniae genomic DNA.
First-pass PCR conditions were: 20 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 2 minutes followed by 25 cycles of 94° C. for 1 minute, 50° C. for 1 minute, 72° C. for 2 minutes and lastly 72° C. for 7 minutes. This amplified all but 364 ORFs. Failed PCR reactions were repeated at different annealing temperatures, and all but 38 were amplified. New primers were synthesized, but not re-designed and all but 16 ORFs were amplified. These ORFs were amplified with new, re-designed primers. ORF PCR products were purified by gel-filtration with Sephadex G-50. The purified PCR products were vacuum-concentrated in a Speedvac centrifuge as needed to keep the volume below 200 μl. The pooled PCR products were phenol: chloroform extracted, chloroform extracted and ethanol precipitated.
The products were arrayed into microtiter wells for pooling. The ORFs were combined into 90 pools of approximately 42 ORFs. Each ORF was a member of three unique pools, and the complete genomic set of ORFs is represented in three different sets of 30 pools. This pooling strategy can be conceptualized as a 3-dimensional grid. The purpose is to enable multiplex analyses of the subsequent ELI results and thereby facilitate the selection power of the screen.
To enable non-covalent linkage of expression elements, the ORF pools were exposed to UDG. These samples were combined with 3 expression elements also produced by PCR: the CMV promoter linked to a ubiquitin sequence, the CMV promoter linked to a secretory leader sequence, and a terminator sequence. These were also designed with UDG sensitive ends and prepared for ORF linkage by enzymatically exposing 3′ single stranded ends complementary to the ORFs.
A/J mice were used in all vaccine screening experiments. For Helios gene gun immunization (Bio-Rad Laboratories, Hercules, Calif.), mice received an isoflurane inhalation anesthesia, and were immunized on the outside of each ear. Three gene gun immunizations were performed in one month intervals with 5 mice per vaccine pool. The individual vaccine dose per ORF LEE was approximately 50 ng DNA/mouse (1/42 dose), resulting in a total DNA dose of approximately 2 μg DNA/mouse per pool, split into two immunization doses per mouse.
The numbers of Chlamydia pneumoniae genomes per lung were logarithmically transformed, and the means of all immunization pools determined. The protective capacity of each pool of ˜42 ORF immunization constructs was determined as protection score in a linear equation in which the Log10 of the lung Chlamydia pneumoniae genomes of the low-dose immunized positive protection controls equaled 100% protection, and that of the naïve controls 0% protection. Groups that had higher Chlamydia pneumoniae lung loads than the naïve controls had negative protection scores. The protective potential of the Chlamydia pneumoniae ORFs was matrix analyzed in two ways: 1) by ranking in descending order the sum of the protection scores of the X, Y, and Z pools in which any one ORF was a member, and 2) by residency of an ORF in 3 protective groups (1 each from the x, y, and z sets), which represents an intersection of planes X, Y, and Z. Using both analyses of inferred protection, 46 candidates were identified.
Round 2—Initial Chlamydia pneumoniae vaccine candidate screen. After the total Chlamydia pneumoniae genome screen, the 46 highest scoring inferred candidates were tested individually. Subsequent steps were identical to those described above for Round 1. The inocula per gene gun-dose were comprised of 200 ng of the candidate ORF and 800 ng of pUC118 filler DNA. Each mouse received 2 doses and each group had 10 mice. All other gene gun vaccination parameters were identical to the round 1 experiment.
Round 3—Confirmatory Chlamydia pneumoniae vaccine candidate screen. After the round 2 screen of 46 candidates, the highest ranked 12 candidates were cloned as full genes, excluding ide and Cpn0095 for which only the identified fragments ide_b, ide_ab, and Cpn0095_a were tested. The candidates were tested individually in a high-dose Chlamydia pneumoniae challenge using an inoculum that in titration experiments killed 50% of inoculated naïve mice within 10 days. This experiment was designed as a rigorous challenge of the protective efficacy of the final candidate genes, with a multiple readout evaluating protection from disease by survival of mice and determination of lung weight increase, as well as elimination of Chlamydia pneumoniae organisms by determination of total chlamydial lung loads.
Genetic immunization was again performed by ballistic delivery of recombinant mammalian expression vectors carrying individual bacterial genes under control of a eukaryotic promoter. This genetic immunization vector, pCMVi-UB is described in
The PCR generated fragments were dU cloned into the specially prepared pCMVi-UB vector. The vector was cleaved at BglII and HindIII sites and synthetic single stranded adapters were ligated to the imbedded 3′ ends of the cleavage sites. This resulted in generation of protruded 3′ ends. Adapter sequences were designed to compliment the ends of the PCR products added during the second phase of the protocol. To generate 3′ protruded ends on the PCR products they were treated with UGPase. This removed the primer incorporated dU bases from the 5′ ends of the PCR products and exposed complementary to the adaptors 3′ ends. The prepared vector and UDGase treated PCR product were mixed together and without any additional steps used for bacterial transformation. Correct integration and sequence of the assembled expression cassettes was confirmed by sequencing.
Plasmid-coated gold particles for gene gun immunization were prepared in a standard protocol (BioRad, Inc.) using endotoxin free plasmid DNA preparations. Each vaccine dose contained total of 1 μg of a plasmid DNA mix. The mix contained 0.9 μg of an antigen encoding plasmid and 0.1 μg of a genetic adjuvant. This adjuvant was a 1:4 mixture of two plasmids encoding the B and A subunits of E. coli heat-labile toxin. The coding sequence for subunit A was modified to change the R at position 192 to G to detoxify the gene. DNA was delivered by gene gun (BioRad, Inc.) into each ear lobe of each mouse (10 mice/group). An accelerated vaccination schedule was used to immunize mice on days 0, 3, 6, 20, and 34. Mice were challenged with 5×108 Chlamydia pneumoniae elementary bodies 4 weeks after the last immunization.
RESULTS. Referring now to
A fifteen day time course of infection was analyzed in both mouse strains, each strain having naïve and immune to Chlamydia pneumoniae mice. A/J mice had a lower incidence of disease than C57BL/6 mice, expressed as percent increase over the average lung weight of unchallenged mice. As shown in
Turning now to
Accordingly, conditions were identified that maximized the amplitude between chlamydial lung burden of naïve mice and of immune mice protected by a low-dose live Chlamydia pneumoniae inoculation. These preliminary results demonstrate that the optimum protection readout time point is ten days after challenge infection, and that A/J, but not C57BL/6 mice, are the host inbred mouse strain in the respiratory challenge model suitable for identification of protective Chlamydia pneumoniae protein antigens. The corollary of this finding is that an appropriate host genetic background will be essential for protective efficacy of any Chlamydia pneumoniae vaccine, presumably not only in inbred mouse strains, but also in an outbred human vaccine population. Vaccine candidates identified using this animal model of disease will be chlamydial antigens that are presented to and recognized by the immune system in a manner that stimulates a productive host response. However, successful use of vaccine antigens in individuals that are genetically refractory to immune protection against Chlamydia pneumoniae, as are C57BL/6 mice, will require an understanding of the factors that normally prevent immune protection. This will enable immunity to be manipulated more productively.
Referring now to
pneumoniae
C. pneumoniae
11
3
9
1.287
1
1.102
2
6
13
7
4
10
0.524
8
5
aBold numbers indicate significant difference (p < 0.05) from naïve controls in a post-hoc Dunnett's test for determination of the significant differences between a single control group mean and the remaining treatment group means in ANOVA.
bBold numbers indicate genes selected for further testing in round 3.
The 46 individual Chlamydia pneumoniae partial or full-length ORFs selected in round 1 were subsequently screened as individual LEEs in Round 2 as described above. Total lung Chlamydia pneumoniae protection scores, and the ranking of the genes based on these scores is shown in the last 2 columns of Table 3 above. The results of Round 2 selected the following Chlamydia pneumoniae genes, in this ranking, as candidates for final testing and confirmation in Round 3: cutE (SEQ ID NOS:1-4), Cpn0420 (SEQ ID NOS:5-6), ide (SEQ ID NOS:7-12), oppA—2 (SEQ ID NOS:17-20), ssb (SEQ ID NOS: 21-22), glgX (SEQ ID NOS:27-30), Cpn0020 (SEQ ID NOS:31-34), Cpn0509 (SEQ ID NOS:23-24), fabD (SEQ ID NOS:25-26), rl1 (SEQ ID NOS:37-38), atoC (SEQ ID NOS:35-36), and Cpn0095 (SEQ ID NOS:13-16).
In Round 3, the identified final 12 candidates were cloned as full-length genes into genetic immunization plasmid CMVi-UB (
The survival data is detailed in Table 4 below, and indicates that along with the calibration live vaccine, genes cutE, Cpn0420, and Cpn0020 prevented death of any inoculated animal while 43% of naïve mice died (P<0.05, Fisher Exact test). Table 4 demonstrates the survival of high-Chlamydia pneumoniae dose-challenged mice in Round 3 vaccinated with plasmid-cloned Chlamydia pneumoniae genes selected in Round 2 for further testing. Bold numbers indicate significant difference (p<0.05) from naïve controls in Fisher Exact test. In all groups vaccinated with the remaining constructs, one or more animals died, and the survival in these groups was not significantly different from naïve mice. Thus, genes cutE, Cpn0420, and Cpn0020 mediated significant protection from Chlamydia pneumoniae-induced death.
100
100
100
100
aBold numbers in red indicate significant difference (p < 0.05) from Naïve controls in Fisher Exact test.
Turning now to
0.002
0.029
0.009
0.002
0.005
aNaïve n = 8, live vaccine n = 15; genetic vaccine groups n = 3-10.
bDead mice were treated as missing data.
cBold numbers indicate significant difference (p < 0.05) from Naïve controls in Dunnett's post-hoc test.
Finally, referring to
C. pneumoniae
<0.001
<0.001
0.006
0.024
0.011
0.028
aNaïve, live vaccine groups n = 60; genetic vaccine groups n = 13-20.
bDead mice were treated as missing data.
cBold numbers indicate significant difference (p < 0.05) from Naïve controls in Dunnett's post-hoc test.
In summary, cutE and Cpn0420 are identified as genes individually protective by all criteria (survival, disease reduction, Chlamydia pneumoniae elimination). Gene oppA—2 was protective by dual criteria (disease reduction, Chlamydia pneumoniae elimination), and single criterion-protective genes were ssb (disease reduction), ide and Cpn0095 (Chlamydia pneumoniae elimination), and Cpn0020 (survival).
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this application have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
This application claims priority to co-pending U.S. Provisional Patent Application Ser. Nos. 60/875,920, filed on Dec. 19, 2006 and 60/872,694, filed on Dec. 4, 2006. The entire text of the above referenced disclosures are specifically incorporated herein by reference.
The Government may own rights in the present invention pursuant to National Institutes of Health (NIH) Grant No. AI47202.
| Number | Date | Country | |
|---|---|---|---|
| 60875920 | Dec 2006 | US | |
| 60872694 | Dec 2006 | US |
