AU1254100A - Chlamydia antigens and corresponding DNA fragments and uses thereof - Google Patents

Description

WO 00/24765 PCT/CA99/00992 TITLE OF INVENTION CHLAMYDIA ANTIGENS AND CORRESPONDING DNA FRAGMENTS AND USES THEREOF 5 REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 60/106034, filed October 28, 1998, U.S. Provisional Application No.60/106039, filed October 28, 1998,U.S. Provisional Application No. 60/106042, filed October 10 28, 1998, U.S. Provisional Application No. 60/106044, filed October 28, 1998, U.S. Provisional Application No. 60/106072, filed October 29, 1998, U.S. Provisional Application No. 60/106073, filed October 29, 1998, U.S. Provisional Application No. 60/106074, filed October 29, 1998,U.S. Provisional 15 Application No. 60/106087, filed October 29, 1998, U.S. Provisional Application No. 60/106587, filed November 2, 1998, U.S. Provisional Application No. 60/106588, filed November 2, 1998, U.S. Provisional Application No. 60/107089, filed November 2, 1998, U.S. Provisional Application No. 60/107034, filed 20 November 2, 1998 and U.S. Provisional Application No. 60/107035, filed November 2, 1998. FIELD OF INVENTION The present invention relates to Chlamydia antigens 25 and corresponding DNA molecules, which can be used to prevent and treat Chlamydia infection in mammals, such as humans. BACKGROUND OF THE INVENTION Chlamydiae are prokaryotes. They exhibit morphologic 30 and structural similarities to gram-negative bacteria including a trilaminar outer membrane, which contains lipopolysaccharide and several membrane proteins that are structurally and functionally analogous to proteins found in E coli. They are obligate intra-cellular parasites with a unique biphasic life RuRSTITUTE SHEET (RULE 261 WO 00/24765 PCT/CA99/00992 2 cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non-infectious intracellular stage. The replicative stage of the life-cycle takes place within a membrane-bound inclusion which sequesters 5 the bacteria away from the cytoplasm of the infected host cell. C. pneumoniae is a common human pathogen, originally described as the TWAR strain of Chlamydia psittaci but subsequently recognised to be a new species. C. pneumoniae is antigenically, genetically and morphologically distinct from 10 other chlamydia species (C. trachomatis, C. pecorum and C. psittaci). It shows 10% or less DNA sequence homology with either of C.trachomatis or C.psittaci. C. pneumoniae is a common cause of community acquired pneumonia, only less frequent than Streptococcus pneumoniae and 15 Mycoplasma pneumoniae (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Campos et al. (1995) Investigation of Ophthalmology and Visual Science 36:1477). It can also cause upper respiratory tract symptoms and disease, including bronchitis and sinusitis (Grayston et al. (1995) Journal of 20 Infectious Diseases 168:1231; Grayston et al (1990) Journal of Infectious Diseases 161:618; Marrie (1993) Clinical Infectious Diseases. 18:501; Wang et al (1986) Chlamydial infections). Cambridge University Press, Cambridge. p. 329The great majority of the adult population (over 60%) has antibodies to C. 25 pneumoniae (Wang et al (1986) Chlamydial infections. Cambridge University Press, Cambridge. p. 329), indicating past infection which was unrecognized or asymptomatic. C. pneumoniae infection usually presents as an acute respiratory disease (i.e., cough, sore throat, hoarseness, and 30 fever; abnormal chest sounds on auscultation). For most patients, the cough persists for 2 to 6 weeks, and recovery is slow. In approximately 10% of these cases, upper respiratory tract infection is followed by bronchitis or pneumonia. Furthermore, during a C. pneumoniae epidemic, subsequent SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 3 co-infection with pneumococcus has been noted in about half of these pneumonia patients, particularly in the infirm and the elderly. As noted above, there is more and more evidence that C. pneumoniae infection is also linked to diseases other than 5 respiratory infections. The reservoir for the organism is presumably people. In contrast to C. psittaci infections, there is no known bird or animal reservoir. Transmission has not been clearly defined. It may result from direct contact with secretions, from fomites, or 10 from airborne spread. There is a long incubation period, which may last for many months. Based on analysis of epidemics, C. pneumoniae appears to spread slowly through a population (case to-case interval averaging 30 days) because infected persons are inefficient transmitters of the organism. Susceptibility to C. 15 pneumoniae is universal. Reinfections occur during adulthood, following the primary infection as a child. C. pneumoniae appears to be an endemic disease throughout the world, noteworthy for superimposed intervals of increased incidence (epidemics) that persist for 2 to 3 years. C.trachomatis 20 infection does not confer cross-immunity to C. pneumoniae. Infections are easily treated with oral antibiotics, tetracycline or erythromycin (2 g/d, for at least 10 to 14 d). A recently developed drug, azithromycin, is highly effective as a single-dose therapy against chlamydial infections. 25 In most instances, C. pneumoniae infection is often mild and without complications, and up to 90% of infections are subacute or unrecognized. Among children in industrialized countries, infections have been thought to be rare up to the age of 5 y, although a recent study (E Normann et al, Chlamydia 30 pneumoniae in children with acute respiratory tract infections, Acta Paediatrica, 1998, Vol 87, Iss 1, pp 23-27) has reported that many children in this age group show PCR evidence of infection despite being seronegative, and estimates a prevalence of 17-19% in 2-4 y olds. In developing countries, the SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 4 seroprevalence of C. pneumoniae antibodies among young children is elevated, and there are suspicions that C. pneumoniae may be an important cause of acute lower respiratory tract disease and mortality for infants and children in tropical regions of the 5 world. From seroprevalence studies and studies of local epidemics, the initial C. pneumoniae infection usually happens between the ages of 5 and 20 y. In the USA, for example, there are estimated to be 30,000 cases of childhood pneumonia each 10 year caused by C. pneumoniae. Infections may cluster among groups of children or young adults (e.g., school pupils or military conscripts). C. pneumoniae causes 10 to 25% of community-acquired lower respiratory tract infections (as reported from Sweden, 15 Italy, Finland, and the USA). During an epidemic, C. pneumonia infection may account for 50 to 60% of the cases of pneumonia. During these periods, also, more episodes of mixed infections with S. pneumoniae have been reported. Reinfection during adulthood is common; the clinical 20 presentation tends to be milder. Based on population seroprevalence studies, there tends to be increased exposure with age, which is particularly evident among men. Some investigators have speculated that a persistent, asymptomatic C. pneumoniae infection state is common. 25 In adults of middle age or older, C. pneumoniae infection may progress to chronic bronchitis and sinusitis. A study in the USA revealed that the incidence of pneumonia caused by C. pneumoniae in persons younger than 60 years is 1 case per 1,000 persons per year; but in the elderly, the disease 30 incidence rose three-fold. C. pneumoniae infection rarely leads to hospitalization, except in patients with an underlying illness. Of considerable importance is the association of atherosclerosis and C. pneumoniae infection. There are several SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 5 epidemiological studies showing a correlation of previous infections with C. pneumoniae and heart attacks, coronary artery and carotid artery disease (Saikku et al.(1988) Lancet;ii:983; Thom et al. (1992) JAMA 268:68; Linnanmaki et al. (1993), 5 Circulation 87:1030; Saikku et al. (1992)Annals Internal Medicine 116:273; Melnick et al(1993) American Journal of Medicine 95:499). Moreover, the organisms has been detected in atheromas and fatty streaks of the coronary, carotid, peripheral arteries and aorta (Shor et al. (1992) South African. Medical 10 Journal 82:158; Kuo et al. (1993) Journal of Infectious Diseases 167:841; Kuo et al. (1993) Arteriosclerosis and Thrombosis 13:1500; Campbell et al (1995) Journal of Infectious Diseases 172:585; Chiu et al. Circulation, 1997 (In Press)). Viable C. pneumoniae has been recovered from the coronary and carotid 15 artery (Ramirez et al (1996) Annals of Internal Medicine 125:979; Jackson et al. Abst. K121, p272, 3 6 th ICAAC, 15-18 Sept. 1996, New Orleans). Furthermore, it has been shown that C. pneumoniae can induce changes of atherosclerosis in a rabbit model (Fong et al (1997) Journal of Clinical Microbiolology 20 35:48). Taken together, these results indicate that it is highly probable that C. pneumoniae can cause atherosclerosis in humans, though the epidemiological importance of chlamydial atherosclerosis remains to be demonstrated. A number of recent studies have also indicated an 25 association between C. pneumoniae infection and asthma. Infection has been linked to wheezing, asthmatic bronchitis, adult-onset asthma and acute exacerbations of asthma in adults, and small-scale studies have shown that prolonged antibiotic treatment was effective at greatly reducing the severity of the 30 disease in some individuals (Hahn DL, et al. Evidence for Chlamydia pneumoniae infection in steroid-dependent asthma. Ann Allergy Asthma Immunol. 1998 Jan; 80(1): 45-49.; Hahn DL, et al. Association of Chlamydia pneumoniae IgA antibodies with recently symptomatic asthma. Epidemiol Infect. 1996 Dec; SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 6 117(3): 513-517; Bjornsson E, et al. Serology of chlamydia in relation to asthma and bronchial hyperresponsiveness. Scand J Infect Dis. 1996; 28(1): 63-69.; Hahn DL. Treatment of Chlamydia pneumoniae infection in adult asthma: a before-after trial. J 5 Fam Pract. 1995 Oct; 41(4): 345-351.; Allegra L, et al. Acute exacerbations of asthma in adults: role of Chlamydia pneumoniae infection. Eur Respir J. 1994 Dec; 7(12): 2165-2168.; Hahn DL, et al. Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult-onset 10 asthma. JAMA. 1991 Jul 10; 266(2): 225-230). In light of these results a protective vaccine against C. pneumoniae infection would be of considerable importance. There is not yet an effective vaccine for any human chlamydial infection. It is conceivable that an effective vaccine can be 15 developed using physically or chemically inactivated Chlamydiae. However, such a vaccine does not have a high margin of safety. In general, safer vaccines are made by genetically manipulating the organism by attenuation or by recombinant means. Accordingly, a major obstacle in creating an effective and safe 20 vaccine against human chlamydial infection has been the paucity of genetic information regarding Chlamydia, specifically C. pneumoniae. Studies with C. trachomatis and C. psittaci indicate that safe and effective vaccine against Chlamydia is an 25 attainable goal. For example, mice which have recovered from a lung infection with C. trachomatis are protected from infertility induced by a subsequent vaginal challenge (Pal et al.(1996) Infection and Immunity.64:5341). Similarly, sheep immunized with inactivated C. psittaci were protected from 30 subsequent chlamydial-induced abortions and stillbirths (Jones et al. (1995) Vaccine 13:715). Protection from chlamydial infections has been associated with Th1 immune responses, particularly the induction of INFg - producing CD4+T-cells (Igietsemes et al. (1993) Immunology 5:317). The adoptive SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 7 transfer of CD4+ cell lines or clones to nude or SCID mice conferred protection from challenge or cleared chronic disease (Igietseme et al (1993) Regional Immunology 5:317; Magee et al (1993) Regional Immunology 5: 305), and in vivo depletion of 5 CD4+ T cells exacerbated disease post-challenge (Landers et al (1991) Infection & Immunity 59:3774; Magee et al (1995) Infection & Immunity 63:516). However, the presence of sufficiently high titres of neutralising antibody at mucosal surfaces can also exert a protective effect (Cotter et al. 10 (1995) Infection and Immunity 63:4704). Antigenic variation within the species C. pneumoniae is not well documented due to insufficient genetic information, though variation is expected to exist based on C. trachomatis. Serovars of C. trachomatis are defined on the basis of antigenic 15 variation in MOMP, but published C. pneumoniae MOMP gene sequences show no variation between several diverse isolates of the organism (Campbell et al (1990) Infection and Immunity 58:93; McCafferty et al (1995) Infection and Immunity 63:2387-9; Knudsen et al (1996) Third Meeting of the European Society for 20 Chlamydia Research, Vienna). Regions of the protein known to be conserved in other chlamydial MOMPs are conserved in C. pneumoniae (Campbell et al (1990) Infection and Immunity 58:93; McCafferty et al (1995) Infection and Immunity 63:2387-9). One study has described a strain of C. pneumoniae with a MOMP of 25 greater that usual molecular weight, but the gene for this has not been sequenced (Grayston et al. (1995) Journal of Infectious Diseases 168:1231). Partial sequences of outer membrane protein 2 from nine diverse isolates were also found to be invariant (Ramirez et al (1996) Annals of Internal Medicine 125:979). The 30 genes for HSP60 and HSP70 show little variation from other chlamydial species, as would be expected. The gene encoding a 76kDa antigen has been cloned from a single strain of C. pneumoniae. It has no significant similarity with other known SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 8 chlamydial genes (Marrie (1993) Clinical Infectious Diseases. 18:501). Many antigens recognised by immune sera to C. pneumoniae are conserved across all chlamydiae, but 98kDa, 76 5 kDa and 54 kDa proteins appear to be C. pneumoniae-specific (Ref Campos et al. (1995) Investigation of Ophthalmology and Visual Science 36:1477; Marrie (1993) Clinical Infectious Diseases. 18:501; Wiedmann-Al-Ahmad M, et al. Reactions of polyclonal and neutralizing anti-p54 monoclonal antibodies with an isolated, 10 species-specific 54-kilodalton protein of Chlamydia pneumoniae. Clin Diagn Lab Immunol. 1997 Nov; 4(6): 700-704). Immunoblotting of isolates with sera from patients does show variation of blotting patterns between isolates, indicating that serotypes C. pneumoniae may exist (Ref 1,16). However, the 15 results are potentially confounded by the infection status of the patients, since immunoblot profiles of a patient's sera change with time post-infection. An assessment of the number and relative frequency of any serotypes, and the defining antigens, is not yet possible. 20 Accordingly, a need exists for identifying and isolating polynucleotide sequences of C. pneumoniae for use in preventing and treating Chlamydia infection. SUMMARY OF THE INVENTION 25 The present invention provides purified and isolated polynucleotide molecules that encode Chlamydia polypeptides which can be used in methods to prevent, treat, and diagnose Chlamydia infection. In one form of the invention, the polynucleotide molecules are selected from DNA that encode 30 polypeptides CPN100397 (SEQ ID Nos: 1 and 2), CPN100421 (SEQ ID Nos: 3 and 4), CPN100422 (SEQ ID Nos: 5 and 6), CPN100424 (SEQ ID Nos: 7 and 8), CPN100426 (SEQ ID Nos: 9 and 10), CPN100508 (SEQ ID Nos: 11 and 12), CPN100515 (SEQ ID Nos: 13 and 14), CPN100538 (SEQ ID Nos: 15 and 16), CPN100557 (SEQ ID Nos: 17 and .URqTIT1TF I4EHHT (RULE 26) WO 00/24765 PCT/CA99/00992 9 18), CPN100622 (SEQ ID Nos: 19 and 20), CPN100626 (SEQ ID Nos: 21 and, 22), CPN100628 (SEQ ID Nos: 23 and 24) and CPN100630 (SEQ ID Nos: 25 and 26). Another form of the invention provides polypeptides 5 corresponding to the isolated DNA molecules. The amino acid sequences of the corresponding encoded polypeptides are shown for CPN100397 as SEQ ID Nos: 27 and 28, CPN100421 as SEQ ID No: 29, CPN100422 as SEQ ID No: 30, CPN100424 as SEQ ID No: 31, CPN100426 as SEQ ID No: 32, CPN100508 as SEQ ID Nos: 33 and 34, 10 CPN100515 as SEQ ID Nos: 35 and 36, CPN100538 as SEQ ID No: 37, CPN100557 as SEQ ID Nos: 38 and 39, CPN100622 as SEQ ID Nos: 40 and 41, CPN100626 as SEQ ID No: 42, CPN100628 as SEQ ID No: 43 and CPN100630 as SEQ ID Nos: 44 and 45. Those skilled in the art will readily understand that the 15 invention, having provided the polynucleotide sequences encoding Chlamydia polypeptides, also provides polynucleotides encoding fragments derived from such peptides. Moreover, the invention is understood to provide mutants and derivatives of such polypeptides and fragments derived therefrom, which result from 20 the addition, deletion, or substitution of non-essential amino acids as described herein. Those skilled in the art would also readily understand that the invention, having provided the polynucleotide sequences encoding Chlamydia polypeptides, further provides monospecific antibodies that specifically bind 25 to such polypeptides The present invention has wide application and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention further provides (i) a method 30 for producing a polypeptide of the invention in a recombinant host system and related expression cassettes, vectors, and transformed or transfected cells; (ii) a vaccine, or a live vaccine vector such as a pox virus, Salmonella typhimurium, or Vibrio cholerae vector, containing a polynucleotide of the SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 10 invention, such vaccines and vaccine vectors being useful for, e.g., preventing and treating Chlamydia infection, in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or 5 prophylactic methods; (iii) a therapeutic and/or prophylactic use of an RNA or DNA molecule of the invention, either in a naked form or formulated with a delivery vehicle, a polypeptide or combination of polypeptides, or a monospecific antibody of the invention, and related pharmaceutical compositions; (iv) a 10 method for diagnosing the presence of Chlamydia in a biological sample, which can involve the use of a DNA or RNA molecule, a monospecific antibody, or a polypeptide of the invention; and (v) a method for purifying a polypeptide of the invention by antibody-based affinity chromatography. 15 BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be further understood from the following description with reference to the drawings, in which: Figure 1 shows the nucleotide sequence of the CPN100397 20 (SEQ ID No: 1 - entire sequence and SEQ ID No: 2 - coding sequence) and the deduced amino acid sequence of the CPN100397 protein from Chlamydia pneumoniae (SEQ ID No: 27 and 28). Figure 2 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100397 gene. 25 Figure 3 shows the nucleotide sequence of the CPN100421 (SEQ ID No: 3 - entire sequence and SEQ ID No: 4 - coding sequence) and the deduced amino acid sequence of the CPN100421 protein from Chlamydia pneumoniae (SEQ ID No: 29). Figure 4 shows the restriction enzyme analysis of the 30 gene encoding the C. pneumoniae CPN100421 gene. Figure 5 shows the nucleotide sequence of the CPN100422 (SEQ ID No: 5 - entire sequence and SEQ ID No: 6 - coding sequence) and the deduced amino acid sequence of the CPN100422 protein from Chlamydia pneumoniae (SEQ ID No: 30). SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 11 Figure 6 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100422 gene. Figure 7 shows the nucleotide sequence of the CPN100424 (SEQ ID No: 7 - entire sequence and SEQ ID No: 8 - coding 5 sequence) and the deduced amino acid sequence of the CPN100424 protein from Chlamydia pneumoniae (SEQ ID No: 31). Figure 8 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100424 gene. Figure 9 shows the nucleotide sequence of the CPN100426 10 (SEQ ID No: 9 - entire sequence and SEQ ID No: 10 - coding sequence) and the deduced amino acid sequence of the CPN100426 protein from Chlamydia pneumoniae (SEQ ID No: 32). Figure 10 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100426 gene. 15 Figure 11 shows the nucleotide sequence of the CPN100508 (SEQ ID No: 11 - entire sequence and SEQ ID No: 12 - coding sequence) and the deduced amino acid sequence of the CPN100508protein from Chlamydia pneumoniae (SEQ ID No: 33 - full length sequence and SEQ ID No: 34 - processed sequence). 20 Figure 12 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100508 gene. Figure 13 shows the nucleotide sequence of the CPN100515 (SEQ ID No: 13 - entire sequence and SEQ ID No: 14 - coding sequence) and the deduced amino acid sequence of the CPN100515 25 protein from Chlamydia pneumoniae (SEQ ID No: 35 - full length sequence and SEQ ID No: 36 - processed sequence). Figure 14 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100515 gene. Figure 15 shows the nucleotide sequence of the CPN100538 30 (SEQ ID No: 15 - entire sequence and SEQ ID No: 16 - coding sequence) and the deduced amino acid sequence of the CPN100538 protein from Chlamydia pneumoniae (SEQ ID No: 37). Figure 16 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100538 gene. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 12 Figure 17 shows the nucleotide sequence of the CPN100557 (SEQ ID No: 17 - entire sequence and SEQ ID No: 18 - coding sequence) and the deduced amino acid sequence of the CPN100557 protein from Chlamydia pneumoniae (SEQ ID No: 38 - full length 5 sequence and SEQ ID No: 39 - processed sequence). Figure 18 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100557 gene. Figure 19 shows the nucleotide sequence of the CPN100622 (SEQ ID No: 19 - entire sequence and SEQ ID No: 20 - coding 10 sequence) and the deduced amino acid sequence of the CPN100622 protein from Chlamydia pneumoniae (SEQ ID No: 40 - full length sequence and SEQ ID No: 41 - processed sequence). Figure 20 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100622 gene. 15 Figure 21 shows the nucleotide sequence of the CPN100626 (SEQ ID No: 21 - entire sequence and SEQ ID No: 22 - coding sequence) and the deduced amino acid sequence of the CPN100626 protein from Chlamydia pneumoniae (SEQ ID No: 42). Figure 22 shows the restriction enzyme analysis of the 20 gene encoding the C. pneumoniae CPN100626 gene. Figure 23 shows the nucleotide sequence of the CPN100628 (SEQ ID No: 23 - entire sequence and SEQ ID No: 24 - coding sequence) and the deduced amino acid sequence of the CPN100628 protein from Chlamydia pneumoniae (SEQ ID No: 43). 25 Figure 24 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100628 gene. Figure 25 shows the nucleotide sequence of the CPN100630 (SEQ ID No: 25 - entire sequence and SEQ ID No: 26 - coding sequence) and the deduced amino acid sequence of the CPN100630 30 protein from Chlamydia pneumoniae (SEQ ID No: 44 - full length sequence and SEQ ID No: 45 - processed sequence). Figure 26 shows the restriction enzyme analysis of the gene encoding the C. pneumoniae CPN100630 gene. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 13 Figures 27 through 39 show an identification of T and B cell epitopes from the amino acid sequences shown in the foregoing figures. 5 DETAILED DESCRIPTION OF INVENTION Open reading frames (ORFs) encoding chlamydial polypeptides have been identified from the C. pneumoniae genome. These polypeptides include polypeptides found permanently in the bacterial membrane structure, polypeptides present in the 10 external vicinity of the bacterial membrane, polypeptides found permanently in the inclusion membrane structure, polypeptides present in the external vicinity of the inclusion membrane, and polypeptides released into the cytoplasm of the infected cell. These polypeptides can be used to prevent and treat Chlamydia 15 infection. According to a first aspect of the invention, isolated polynucleotides are provided which encode the precursor and mature forms of Chlamydia polypeptides, whose amino acid sequences are selected from the group consisting of: SEQ ID 20 Nos: 27 to 45. The term "isolated polynucleotide" is defined as a polynucleotide removed from the environment in which it naturally occurs. For example, a naturally-occurring DNA molecule present in the genome of a living bacteria or as part 25 of a gene bank is not isolated, but the same molecule separated from the remaining part of the bacterial genome, as a result of, e.g., a cloning event (amplification), is isolated. Typically, an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous at the 5' or 3' 30 end, in the naturally occurring genome. Such isolated polynucleotides may be part of a vector or a composition and still be defined as isolated in that such a vector or composition is not part of the natural environment of such polynucleotide. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 14 The polynucleotide of the invention is either RNA or DNA (cDNA, genomic DNA, or synthetic DNA), or modifications, variants, homologs or fragments thereof. The DNA is either double-stranded or single-stranded, and, if single-stranded, is 5 either the coding strand or the non-coding (anti-sense) strand. Any one of the sequences that encode the polypeptides of the invention as shown in SEQ ID Nos: 1 to 26 is (a) a coding sequence, (b) a ribonucleotide sequence derived from transcription of (a), or (c) a coding sequence which uses the 10 redundancy or degeneracy of the genetic code to encode the same polypeptides. By "polypeptide" or "protein" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application. 15 Consistent with the first aspect of the invention, amino acid sequences are provided which are homologous to any one of SEQ ID Nos: 27 to 45. As used herein, "homologous amino acid sequence" is any polypeptide which is encoded, in whole or in part, by a nucleic acid sequence which hybridizes at 25-35"C 20 below critical melting temperature (Tm), to any portion of the nucleic acid sequences of SEQ ID Nos: 1 to 26. A homologous amino acid sequence is one that differs from an amino acid sequence shown in any one of SEQ ID Nos: 27 to 45 by one or more amino acid substitutions. Such a sequence also encompass 25 serotypic variants (defined below) as well as sequences containing deletions or insertions which retain inherent characteristics of the polypeptide such as immunogenicity. Preferably, such a sequence is at least 75%, more preferably 80%, and most preferably 90% identical to any one of SEQ ID 30 Nos: 27 to 45. Homologous amino acid sequences include sequences that are identical or substantially identical to SEQ ID Nos: 27 to 45. By "amino acid sequence substantially identical" is meant a sequence that is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 15 an amino acid sequence of reference and that preferably differs from the sequence of reference by a majority of conservative amino acid substitutions. Conservative amino acid substitutions are substitutions 5 among amino acids of the same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, such as 10 aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine. Homology is measured using sequence analysis software 15 such as Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705. Amino acid sequences are aligned to maximize identity. Gaps may be artificially introduced into the sequence to attain proper alignment. Once 20 the optimal alignment has been set up, the degree of homology is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions. Homologous polynucleotide sequences are defined in a 25 similar way. Preferably, a homologous sequence is one that is at least 45%, more preferably 60%, and most preferably 85% identical to any one of coding sequences SEQ ID Nos: 1 to 26. Consistent with the first aspect of the invention, polypeptides having a sequence homologous to any one of SEQ ID 30 Nos: 27 to 45 include naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants that retain the inherent characteristics of the polypeptide of SEQ ID Nos: 27 to 45. mRqTITUTF SmEET (RULE 261 WO 00/24765 PCT/CA99/00992 16 As is known in the art, an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide. 5 By "biological function" is meant the function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells. For example, the biological function of a porin is to allow the entry into cells of compounds present in the 10 extracellular medium. Biological function is distinct from antigenic property. A polypeptide can have more than one biological function. Allelic variants are very common in nature. For example, a bacterial species such as C. pneumoniae, is usually 15 represented by a variety of strains that differ from each other by minor allelic variations. Indeed, a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence (and polynucleotide sequence) that are not identical in each of the strains. Despite this 20 variation, an immune response directed generally against many allelic variants has been demonstrated. In studies of the Chlamydial MOMP antigen, cross-strain antibody binding plus neutralization of infectivity occurs despite amino acid sequence variation of MOMP from strain to strain, indicating that the 25 MOMP, when used as an immunogen, is tolerant of amino acid variations. Polynucleotides encoding homologous polypeptides or allelic variants are retrieved by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by 30 conventional methods. This involves the use of synthetic oligonucleotide primers matching upstream and downstream of the 5' and 3' ends of the encoding domain. Suitable primers are designed according to the nucleotide sequence information provided in SEQ ID Nos:1 to 26. The procedure is as follows: a SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 17 primer is selected which consists of 10 to 40, preferably 15 to 25 nucleotides. It is advantageous to select primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; i.e., an amount of C and G nucleotides 5 of at least 40%, preferably 50% of the total nucleotide content. An alternative method for retrieving polynucleotides encoding homologous polypeptides or allelic variants is by hybridization screening of a DNA or RNA library. Hybridization procedures are well-known in the art and are described in 10 Ausubel et al., (Ref 41), Silhavy et al. (Ref 43), and Davis et al. (ref 44). Important parameters for optimizing hybridization conditions are reflected in a formula used to obtain the critical melting temperature above which two complementary DNA strands separate from each other (Ref 45). For polynucleotides 15 of about 600 nucleotides or larger, this formula is as follows: Tm = 81.5 + 0.5 x (% G+C) + 1.6 log (positive ion concentration) - 0.6 x (% formamide). Under appropriate stringency conditions, hybridization temperature (Th) is approximately 20 to 400C, 20 to 250C, or, preferably 30 to 400C below the calculated Tm. 20 Those skilled in the art will understand that optimal temperature and salt conditions can be readily determined. For the polynucleotides of the invention, stringent conditions are achieved for both pre-hybridizing and hybridizing incubations (i) within 4-16 hours at 420C, in 6 x SSC containing 25 50% formamide, or (ii) within 4-16 hours at 650C in an aqueous 6 x SSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)). Useful homologs and fragments thereof that do not occur naturally are designed using known methods for identifying regions of an antigen that are likely to tolerate amino acid 30 sequence changes and/or deletions. As an example, homologous polypeptides from different species are compared; conserved sequences are identified. The more divergent sequences are the most likely to tolerate sequence changes. Alternatively, sequences are modified such that they become more reactive to T SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 18 and/or B-cells. (See Table below for identification of T- and B- epitopes.) Yet another alternative is to mutate a particular amino acid residue or sequence within the polypeptide in vitro, then screen the mutant polypeptides for their ability to prevent 5 or treat Chlamydia infection according to the method outlined below. A person skilled in the art will readily understand that by following the screening process of this invention, it will be determined without undue experimentation whether a particular 10 homolog of any of SEQ ID Nos: 27 to 45 may be useful in the prevention or treatment of Chlamydia infection. The screening procedure comprises the steps: (i) immunizing an animal, preferably mouse, with the test homolog or fragment; 15 (ii) inoculating the immunized animal with Chlamydia; and (iii) selecting those homologs or fragments which confer protection against Chlamydia. By "conferring protection" is meant that there is a 20 reduction is severity of any of the effects of Chlamydia infection, in comparison with a control animal which was not immunized with the test homolog or fragment. It has been previously demonstrated (Yang et. al., 1993) that mice are susceptible to intranasal infection with different 25 isolates of C. pneumoniae. Strain AR-39 (Grayston, 1989) was used in Balb/c mice as a challenge infection model to examine the capacity of chlamydia gene products delivered as naked DNA to elicit a protective response against a sublethal C. pneumoniae lung infection. Protective immunity is defined as an 30 accelerated clearance of pulmonary infection. Groups of 7 to 9 week old male Balb/c mice (6 to 10 per group) were immunized intramuscularly (i.m.) plus intranasally (i.n.) with plasmid DNA containing the coding sequence of a C.pneumoniae polypeptide. Saline or the plasmid vector lacking SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 19 an inserted chlamydial gene was given to groups of control animals. For i.m. immunization alternate left and right quadriceps were injected with 100pig of DNA in 50tl of PBS on 5 three occasions at 0, 3 and 6 weeks. For i.n. immunization, anaesthetized mice aspirated 50pLl of PBS containing 50 ptg DNA on three occasions at 0, 3 and 6 weeks. At week 8, immunized mice were inoculated i.n. with 5 x 105 IFU of C. pneumoniae, strain AR39 in 100ptl of SPG buffer to test their ability to limit the 10 growth of a sublethal C. pneumoniae challenge. Lungs were taken from mice at day 9 post-challenge and immediately homogenised in SPG buffer (7.5% sucrose, 5mM glutamate, 12.5mM phosphate pH7.5). The homogenate was stored frozen at -70*C until assay. Dilutions of the homogenate were 15 assayed for the presence of infectious chlamydia by inoculation onto monolayers of susceptible cells. The inoculum was centrifuged onto the cells at 3000rpm for 1 hour, then the cells were incubated for three days at 35 0 C in the presence of lpg/ml cycloheximide. After incubation the monolayers were fixed with 20 formalin and methanol then immunoperoxidase stained for the presence of chlamydial inclusions using convalescent sera from rabbits infected with C.pneumoniae and metal-enhanced DAB as a peroxidase substrate. Consistent with the first aspect of the invention, 25 polypeptide derivatives are provided that are partial sequences of SEQ ID Nos: 27 to 45, partial sequences of polypeptide sequences homologous to SEQ ID Nos: 27 to 45, polypeptides derived from full-length polypeptides by internal deletion, and fusion proteins. 30 It is an accepted practice in the field of immunology to use fragments and variants of protein immunogens as vaccines, as all that is required to induce an immune response to a protein is a small (e.g., 8 to 10 amino acid) immunogenic region of the SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 20 protein. Various short synthetic peptides corresponding to surface-exposed antigens of pathogens other than Chlamydia have been shown to be effective vaccine antigens against their respective pathogens, e.g. an 11 residue peptide of murine 5 mammary tumor virus (Ref 38), a 16-residue peptide of Semliki Forest virus (Ref 39), and two overlapping peptides of 15 residues each from canine parvovirus (Ref 40). Accordingly, it will be readily apparent to one skilled in the art, having read the present description, that partial 10 sequences of SEQ ID Nos: 27 to 45 or their homologous amino acid sequences are inherent to the full-length sequences and are taught by the present invention. Such polypeptide fragments preferably are at least 12 amino acids in length. Advantageously, they are at least 20 amino acids, preferably at 15 least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length. Polynucleotides of 30 to 600 nucleotides encoding partial sequences of sequences homologous to SEQ ID Nos: 27 to 45 are retrieved by PCR amplification using the parameters outlined 20 above and using primers matching the sequences upstream and downstream of the 5' and 3' ends of the fragment to be amplified. The template polynucleotide for such amplification is either the full length polynucleotide homologous to one of SEQ ID Nos: 1 to 26, or a polynucleotide contained in a mixture 25 of polynucleotides such as a DNA or RNA library. As an alternative method for retrieving the partial sequences, screening hybridization is carried out under conditions described above and using the formula for calculating Tm. Where fragments of 30 to 600 nucleotides are to be retrieved, the 30 calculated Tm is corrected by subtracting (600/polynucleotide size in base pairs) and the stringency conditions are defined by a hybridization temperature that is 5 to 100C below Tm. Where oligonucleotides shorter than 20-30 bases are to be obtained, the formula for calculating the Tm is as follows: Tm = 4 x (G+C) SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 21 + 2 (A+T). For example, an 18 nucleotide fragment of 50% G+C would have an approximate Tm of 540C. Short peptides that are fragments of SEQ. ID Nos. 27 to 45 or their homologous sequences, are obtained directly by chemical synthesis (E. Gross 5 and H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques of Peptide Synthesis, John Wiley & Sons (1981), and M. Bodanzki, Principles of Peptide Synthesis, Springer -Verlag (1984)). Useful polypeptide derivatives, e.g., polypeptide 10 fragments, are designed using computer-assisted analysis of amino acid sequences. This identifies probable surface exposed, antigenic regions (Ref 37). An analysis of the 13 amino acid sequences contained in SEQ ID Nos: 27 to 45, based on the product of flexibility and hydrophobicity propensities using 15 the program SEQSEE (Wishart DS, et al. "SEQSEE: a comprehensive program suite for protein sequence analysis." Comput Appl Biosci. 1994 Apr;10(2):121-32), reveal a number of potential B and T-cell epitopes which may be used as a basis for selecting useful immunogenic fragments and variants. The results are 20 shown in Figures 27 to 39. This analysis uses a reasonable combination of external surface features that is likely to be recognized by antibodies. Probable T-cell epitopes for HLA A0201 MHC subclass were revealed by an algorithm written at Connaught Laboratories that emulates an approach developed at 25 the NIH (Parker KC, et al. "Peptide binding to MHC class I molecules: implications for antigenic peptide prediction." Immunol Res 1995;14(1):34-57 ). Epitopes which induce a protective T cell-dependent immune response are present throughout the length of the 30 polypeptide. However, some epitopes may be masked by secondary and tertiary structures of the polypeptide. To reveal such masked epitopes large internal deletions are created which remove much of the original protein structure and exposes the masked epitopes. Such internal deletions sometimes effects the SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 22 additional advantage of removing immunodominant regions of high variability among strains. Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions are constructed using standard methods (Ref 41). Such methods 5 include standard PCR, inverse PCR, restriction enzyme treatment of cloned DNA molecules, or the method of Kunkel et al. (Ref 42). Components for these methods and instructions for their use are readily available from various commercial sources such as Stratagene. Once the deletion mutants have been constructed, 10 they are tested for their ability to prevent or treat Chlamydia infection as described above. As used herein, a fusion polypeptide is one that contains a polypeptide or a polypeptide derivative of the invention fused at the N- or C-terminal end to any other polypeptide 15 (hereinafter referred to as a peptide tail). A simple way to obtain such a fusion polypeptide is by translation of an in frame fusion of the polynucleotide sequences, i.e., a hybrid gene. The hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or 20 transfect a host cell. Alternatively, the polynucleotide sequence encoding the polypeptide or polypeptide derivative is inserted into an expression vector in which the polynucleotide encoding the peptide tail is already present. Such vectors and instructions for their use are commercially available, e.g. 25 the pMal-c2 or pMal-p2 system from New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of 30 polypeptides and derivatives of the invention. An advantageous example of a fusion polypeptide is one where the polypeptide or homolog or fragment of the invention is fused to a polypeptide having adjuvant activity, such as subunit B of either cholera toxin or E. coli heat-labile toxin. Another SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 23 advantageous fusion is one where the polypeptide, homolog or fragment is fused to a strong T-cell epitope or B-cell epitope. Such an epitope may be one known in the art (e.g. the Hepatitis B virus core antigen, D.R. Millich et al., "Antibody production 5 to the nucleocapsid and envelope of the Hepatitis B virus primed by a single synthetic T cell site", Nature. 1987. 329:547-549), or one which has been identified in another polypeptide of the invention (Table ). Consistent with this aspect of the invention is a fusion polypeptide comprising T- or B-cell 10 epitopes from one of SEQ ID Nos: 27 to 45 or its homolog or fragment, wherein the epitopes are derived from multiple variants of said polypeptide or homolog or fragment, each variant differing from another in the location and sequence of its epitope within the polypeptide. Such a fusion is effective 15 in the prevention and treatment of Chlamydia infection since it optimizes the T- and B-cell response to the overall polypeptide, homolog or fragment. To effect fusion, the polypeptide of the invention is fused to the N-, or preferably, to the C-terminal end of the 20 polypeptide having adjuvant activity or T- or B-cell epitope. Alternatively, a polypeptide fragment of the invention is inserted internally within the amino acid sequence of the polypeptide having adjuvant activity. The T- or B-cell epitope may also be inserted internally within the amino acid sequence 25 of the polypeptide of the invention. Consistent with the first aspect, the polynucleotides of the invention also encode hybrid precursor polypeptides containing heterologous signal peptides, which mature into polypeptides of the invention. By "heterologous signal peptide" 30 is meant a signal peptide that is not found in naturally occurring precursors of polypeptides of the invention. A polynucleotide molecule according to the invention, including RNA, DNA, or modifications or combinations thereof, have various applications. A DNA molecule is used, for example, SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 24 (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Chlamydia 5 infection, (iii) as a vaccine agent (as well as an RNA molecule), in a naked form or formulated with a delivery vehicle and, (iv) in the construction of attenuated Chlamydia strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form. 10 Accordingly, a second aspect of the invention encompasses (i) an expression cassette containing a DNA molecule of the invention placed under the control of the elements required for expression, in particular under the control of an appropriate promoter; (ii) an expression vector containing an expression 15 cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a 20 procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the DNA molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture. 25 A recombinant expression system is selected from procaryotic and eucaryotic hosts. Eucaryotic hosts include yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant 30 cells. A preferred expression system is a procaryotic host such as E. coli. Bacterial and eucaryotic cells are available from a number of different sources including commercial sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Maryland). Commercial sources of SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 25 cells used for recombinant protein expression also provide instructions for usage of the cells. The choice of the expression system depends on the features desired for the expressed polypeptide. For example, it 5 may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form. One skilled in the art would redily understand that not all vectors and expression control sequences and hosts would be expected to express equally well the polynucleotides of this 10 invention. With the guidelines described below, however, a selection of vectors, expression control sequences and hosts may be made without undue experimentation and without departing from the scope of this invention. In selecting a vector, the host must be chosen that is 15 compatible with the vector which is to exist and possibly replicate in it. Considerations are made with respect to the vector copy number, the ability to control the copy number, expression of other proteins such as antibiotic resistance. In selecting an expression control sequence, a number of variables 20 are considered. Among the important variable are the relative strength of the sequence (e.g. the ability to drive expression under various conditions), the ability to control the sequence's function, compatibility between the polynucleotide to be expressed and the control sequence (e.g. secondary structures 25 are considered to avoid hairpin structures which prevent efficient transcription). In selecting the host, unicellular hosts are selected which are compatible with the selected vector, tolerant of any possible toxic effects of the expressed product, able to secrete the expressed product efficiently if 30 such is desired, to be able to express the product in the desired conformation, to be easily scaled up, and to which ease of purification of the final product. The choice of the expression cassette depends on the host system selected as well as the features desired for the SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 26 expressed polypeptide. Typically, an expression cassette includes a promoter that is functional in the selected host system and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; a region encoding a 5 signal peptide, e.g., a lipidation signal peptide; a DNA molecule of the invention; a stop codon; and optionally a 3' terminal region (translation and/or transcription terminator). The signal peptide encoding region is adjacent to the polynucleotide of the invention and placed in proper reading 10 frame. The signal peptide-encoding region is homologous or heterologous to the DNA molecule encoding the mature polypeptide and is compatible with the secretion apparatus of the host used for expression. The open reading frame constituted by the DNA molecule of the invention, solely or together with the signal 15 peptide, is placed under the control of the promoter so that transcription and translation occur in the host system. Promoters and signal peptide encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and 20 derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (as described in U.S. Patent No. 5,028,530 and in Cagnon et al., (Ref 46)); the promoter of the gene of bacteriophage T7 encoding RNA polymerase, that is functional in a number of E. coli 25 strains expressing T7 polymerase (described in U.S. Patent No. 4,952,496); OspA lipidation signal peptide ; and RlpB lipidation signal peptide (Ref 47). The expression cassette is typically part of an expression vector, which is selected for its ability to 30 replicate in the chosen expression system. Expression vectors (e.g., plasmids or viral vectors) can be chosen, for example, from those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual 1985, Supp. 1987). Suitable expression vectors can be purchased from various commercial sources. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 27 Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected as described in Ausubel et al., (Ref 41). Upon expression, a recombinant polypeptide of the 5 invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane. The polypeptide is recovered in a substantially purified form from the cell extract or from the 10 supernatant after centrifugation of the recombinant cell culture. Typically, the recombinant polypeptide is purified by antibody-based affinity purification or by other well-known methods that can be readily adapted by a person skilled in the art, such as fusion of the polynucleotide encoding the 15 polypeptide or its derivative to a small affinity binding domain. Antibodies useful for purifying by immunoaffinity the polypeptides of the invention are obtained as described below. A polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or 20 bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below. Accordingly, a third aspect of the invention provides (i) 25 a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition 30 containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Chlamydia in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Chlamydia infection of SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 28 animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit a protective or therapeutic immune response to Chlamydia ; and particularly, (v) a method 5 for preventing and/or treating a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumonia, C. pecorum) infection, which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an infected individual. Additionally, the third aspect of the invention 10 encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection. As used herein, a vaccine vector expresses one or several polypeptides or derivatives of the invention, as well as at 15 least one additional Chlamydia antigen (??), fragment, homolog, mutant, or derivative thereof. The vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response (adjuvant effect). It is understood that each of the components 20 to be expressed is placed under the control of elements required for expression in a mammalian cell. Consistent with the third aspect of the invention is a composition comprising several vaccine vectors, each of them capable of expressing a polypeptide or derivative of the 25 invention. A composition may also comprise a vaccine vector capable of expressing an additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; or a cytokine such as IL-2 or IL-12. Vaccination methods for treating or preventing infection 30 in a mammal comprises use of a vaccine vector of the invention to be administered by any conventional route , particularly to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 29 intravenous, or intraperitoneal) route. Preferred routes depend upon the choice of the vaccine vector. Treatment may be effected in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by 5 skilled artisans such as the vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (weight, age and the like). Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial 10 vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilie de Calmette-Gu6rin (BCG), and Streptococcus. An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a 15 DNA molecule of the invention, are described in U.S. Patent No. 4,920,209. Poxvirus vectors include vaccinia and canary pox virus, described in U.S. Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively. (Also see, e.g., Tartaglia et al., Virology (1992) 188:217) for a description of a vaccinia virus 20 vector and Taylor et al, Vaccine (1995) 13:539 for a reference of a canary pox.) Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al., Nature (1984) 312:163 so that the polynucleotide of the invention is inserted 25 in the viral genome under appropriate conditions for expression in mammalian cells. Generally, the dose of vaccine viral vector, for therapeutic or prophylactic use, can be of from about 1x10 4 to about 1x10 1 , advantageously from about 1x107 to about lx10 10 , preferably of from about 1x10 7 to about 1x10 9 30 plaque-forming units per kilogram. Preferably, viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart. It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 30 thereby minimizing the immune response to the viral vector itself. Non-toxicogenic Vibrio cholerae mutant strains that are useful as a live oral vaccine are known. Mekalanos et al., 5 Nature (1983) 306:551 and U.S. Patent No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced. WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation 10 can be combined in a single strain with ctxA mutations. WO 94/1533 describes a deletion mutant lacking functional ctxA and attRSl DNA sequences. These mutant strains are genetically engineered to express heterologous antigens, as described in WO 94/19482. An effective vaccine dose of a Vibrio cholerae 15 strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about 1x10 5 to about 1x10 9 , preferably about 1x10 6 to about 1x10 8 , viable bacteria in a volume appropriate for the selected route of administration. Preferred routes of administration include 20 all mucosal routes; most preferably, these vectors are administered intranasally or orally. Attenuated Salmonella typhimurium strains, genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral vaccines are described in 25 Nakayama et al. (Bio/Technology (1988) 6:693) and WO 92/11361. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally. Other bacterial strains used as vaccine vectors in the 30 context of the present invention are described in High et al., EMBO (1992) 11:1991 and Sizemore et al., Science (1995) 270:299 (Shigella flexneri); Medaglini et al., Proc. Natl. Acad. Sci. USA (1995) 92:6868 (Streptococcus gordonii) , Flynn J.L., Cell. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 31 Mol. Biol. (1994) 40 supply . I):31, WO 88/6626, WO 90/0594, WO 91/13157, WO 92/1796, and WO 92/21376 (Bacille Calmette Guerin). In bacterial vectors, the polynucleotide of the invention is inserted into the bacterial genome or remains in a free 5 state as part of a plasmid. The composition comprising a vaccine bacterial vector of the present invention may further contain an adjuvant . A number of adjuvants are known to those skilled in the art. Preferred adjuvants are selected from the list provided below. 10 Accordingly, a fourth aspect of the invention provides (i) a composition of matter comprising a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polynucleotide of the 15 invention; (iii) a method for inducing an immune response against Chlamydia in a mammal by administration of an immunogenically effective amount of a polynucleotide of the invention to elicit a protective immune response to Chlamydia; and particularly, (iv) a method for preventing and/or treating a 20 Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae, or C. pecorum) infection, by administering a prophylactic or therapeutic amount of a polynucleotide of the invention to an infected individual. Additionally, the fourth aspect of the invention encompasses the use of a polynucleotide of the 25 invention in the preparation of a medicament for preventing and/or treating Chlamydia infection. A preferred use includes the use of a DNA molecule placed under conditions for expression in a mammalian cell, especially in a plasmid that is unable to replicate in mammalian cells and to substantially integrate in a 30 mammalian genome. Use of the polynucleotides of the invention include their administration to a mammal as a vaccine, for therapeutic or prophylactic purposes. Such polynucleotides are used in the form of DNA as part of a plasmid that is unable to replicate in SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 32 a mammalian cell and unable to integrate into the mammalian genome. Typically, such a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter functions either ubiquitously or tissue 5 specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (described in Norton & Coffin, Molec. Cell Biol. (1985) 5:281). An example of a tissue-specific promoter is the desmin promoter 10 which drives expression in muscle cells (Li et al., Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403). Use of promoters is well-known to those skilled in the art. Useful vectors are described in numerous publications, specifically WO 94/21797 and 15 Hartikka et al., Human Gene Therapy (1996) 7:1205. Polynucleotides of the invention which are used as a vaccine encode either a precursor or a mature form of the corresponding polypeptide. In the precursor form, the signal peptide is either homologous or heterologous. In the latter 20 case, a eucaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred. As used herein, a composition of the invention contains one or several polynucleotides with optionally at least one additional polynucleotide encoding another Chlamydia antigen 25 such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof. The composition may also contain an additional polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) so that the immune response is enhanced. These additional polynucleotides 30 are placed under appropriate control for expression. Advantageously, DNA molecules of the invention and/or additional DNA molecules to be included in the same composition, are present in the same plasmid. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 33 Standard techniques of molecular biology for preparing and purifying polynucleotides are used in the preparation of polynucleotide therapeutics of the invention. For use as a vaccine, a polynucleotide of the invention is formulated 5 according to various methods outlined below. One method utililizes the polynucleotide in a naked form, free of any delivery vehicles. Such a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, with or without a 10 carrier. When present, the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose. An alternative method utilizes the polynucleotide in 15 association with agents that assist in cellular uptake. Examples of such agents are (i) chemicals that modify cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) liposomes for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten 20 microparticles which associate themselves with the polynucleotides. Anionic and neutral liposomes are well-known in the art (see, e.g., Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making 25 liposomes) and are useful for delivering a large range of products, including polynucleotides. Cationic lipids are also known in the art and are commonly used for gene delivery. Such lipids include Lipofectin

T

4 also known as DOTMA (N-[1-(2,3 dioleyloxy)propyll-N,N,N-trimethylammonium chloride), DOTAP 30 (1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N-(N',N'-dimethyl aminomethane) carbamoyl) cholesterol). A description of these cationic lipids SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 34 can be found in EP 187,702, WO 90/11092, U.S. Patent No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Patent No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl 5 phosphatidylethanolamine), as described in WO 90/11092 as an example. Formulation containing cationic liposomes may optionally contain other transfection-facilitating compounds. A number of them are described in WO 93/18759, WO 93/19768, WO 94/25608, and 10 WO 95/2397. They include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768). 15 Gold or tungsten microparticles are used for gene delivery, as described in WO 91/359, WO 93/17706, and Tang et al. (Nature (1992) 356:152). The microparticle-coated polynucleotide is injected via intradermal or intraepidermal routes using a needleless injection device ("gene gun"), such as 20 those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263. The amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the 25 condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of administration, and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 pg to about 1 mg, preferably, from about 10 pg to about 800 pg 30 and, more preferably, from about 25 pg to about 250 pg, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals. The route of administration is any conventional route used in the vaccine field. As general guidance, a SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 35 polynucleotide of the invention is administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, 5 intraperitoneal, intradermal, intraepidermal, or intramuscular route. The choice of administration route depends on the formulation that is selected. A polynucleotide formulated in association with bupivacaine is advantageously administered into muscles. When a neutral or anionic liposome or a cationic 10 lipid, such as DOTMA or DC-Chol, is used, the formulation can be advantageously injected via intravenous, intranasal (aerosolization), intramuscular, intradermal, and subcutaneous routes. A polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or sub 15 cutaneous routes. Although not absolutely required, such a composition can also contain an adjuvant. If so, a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21, which is 20 described in U.S. Patent No. 5,057,546. The sequence information provided in the present application enables the design of specific nucleotide probes and primers that are used for diagnostic purposes. Accordingly, a fifth aspect of the invention provides a nucleotide probe or 25 primer having a sequence found in or derived by degeneracy of the genetic code from a sequence shown in any one of SEQ ID Nos:1 to 26. The term "probe" as used in the present application refers to DNA (preferably single stranded) or RNA molecules (or 30 modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to nucleic acid molecules having SEQ ID Nos: 1 to 26 or to sequences homologous to SEQ ID Nos:1 to 26, or to their complementary or anti-sense sequences. Generally, probes are significantly shorter than SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 36 full-length sequences . Such probes contain from about 5 to about 100, preferably from about 10 to about 80, nucleotides. In particular, probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a 5 portion of any of SEQ ID Nos:l to 26 or that are complementary to such sequences. Probes may contain modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5 deoxyuridine, or diamino-2, 6-purine. Sugar or phosphate residues may also be modified or substituted. For example, a 10 deoxyribose residue may be replaced by a polyamide (Nielsen et al., Science (1991) 254:1497) and phosphate residues may be replaced by ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters. In addition, the 2'-hydroxyl group on ribonucleotides may be modified by 15 including such groups as alkyl groups. Probes of the invention are used in diagnostic tests, as capture or detection probes. Such capture probes are conventionally immobilized on a solid support, directly or indirectly, by covalent means or by passive adsorption. A 20 detection probe is labelled by a detection marker selected from: radioactive isotopes, enzymes such as peroxidase, alkaline phosphatase, and enzymes able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate, compounds that are chromogenic, fluorogenic, or luminescent, nucleotide base 25 analogs, and biotin. Probes of the invention are used in any conventional hybridization technique, such as dot blot (Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), Southern blot 30 (Southern, J. Mol. Biol. (1975) 98:503), northern blot (identical to Southern blot with the exception that RNA is used as a target), or the sandwich technique (Dunn et al., Cell (1977) 12:23). The latter technique involves the use of a specific capture probe and/or a specific detection probe with SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 37 nucleotide sequences that at least partially differ from each other. A primer is a probe of usually about 10 to about 40 nucleotides that is used to initiate enzymatic polymerization 5 of DNA in an amplification process (e.g., PCR), in an elongation process, or in a reverse transcription method. Primers used in diagnostic methods involving PCR are labeled by methods known in the art. As described herein, the invention also encompasses (i) a 10 reagent comprising a probe of the invention for detecting and/or identifying the presence of Chlamydia in a biological material; (ii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or 15 RNA is extracted from the material and denatured, and (c) exposed to a probe of the invention, for example, a capture, detection probe or both, under stringent hybridization conditions, such that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of 20 Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is primed with at least one, and preferably two, primers of the invention and amplified by polymerase chain reaction, and (d) the amplified 25 DNA fragment is produced. It is apparent that disclosure of polynucleotide sequences of SEQ ID Nos: 1 to 26, their homolog, and partial sequences of either enable their corresponding amino acid sequences. Accordingly, a sixth aspect of the invention 30 features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention. A "substantially purified polypeptide" as used herein is defined as a polypeptide that is separated from the environment SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 38 in which it naturally occurs and/or that is free of the majority of the polypeptides that are present in the environment in which it was synthesized. For example, a substantially purified polypeptide is free from cytoplasmic polypeptides. Those 5 skilled in the art would readily understand that the polypeptides of the invention may be purified from a natural source, i.e., a Chlamydia strain, or produced by recombinant means. Consistent with the sixth aspect of the invention are 10 polypeptides, homologs or fragments which are modified or treated to enhance their immunogenicity in the target animal, in whom the polypeptide, homolog or fragments are intended to confer protection against Chlamydia. Such modifications or treatments include: amino acid substitutions with an amino acid 15 derivative such as 3-methyhistidine, 4-hydroxyproline, 5 hydroxylysine etc., modifications or deletions which are carried out after preparation of the polypeptide, homolog or fragment, such as the modification of free amino, carboxyl or hydroxyl side groups of the amino acids. 20 Identification of homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention which have specific antigenicity is achieved by screening for cross reactivity with an antiserum raised against the polypeptide of reference having an amino acid sequence of any one of SEQ ID 25 Nos: 27 to 45. The procedure is as follows: a monospecific hyperimmune antiserum is raised against a purified reference polypeptide, a fusion polypeptide (for example, an expression product of MBP, GST, or His-tag systems), or a synthetic peptide predicted to be antigenic. Where an antiserum is raised 30 against a fusion polypeptide, two different fusion systems are employed. Specific antigenicity can be determined according to a number of methods, including Western blot (Towbin et al., Proc. Natl. Acad. Sci. USA (1979) 76:4350), dot blot, and ELISA, as described below. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 39 In a Western blot assay, the product to be screened, either as a purified preparation or a total E. coli extract, is submitted to SDS-Page electrophoresis as described by Laemmli (Nature (1970) 227:680). After transfer to a nitrocellulose 5 membrane, the material is further incubated with the monospecific hyperimmune antiserum diluted in the range of dilutions from about 1:5 to about 1:5000, preferably from about 1:100 to about 1:500. Specific antigenicity is shown once a band corresponding to the product exhibits reactivity at any of 10 the dilutions in the above range. In an ELISA assay, the product to be screened is preferably used as the coating antigen. A purified preparation is preferred, although a whole cell extract can also be used. Briefly, about 100 pl of a preparation at about 10 pg protein/ml 15 are distributed into wells of a 96-well polycarbonate ELISA plate. The plate is incubated for 2 hours at 37'C then overnight at 4*C. The plate is washed with phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer). The wells are saturated with 250 pl PBS containing 1% bovine serum 20 albumin (BSA) to prevent non-specific antibody binding. After 1 hour incubation at 370C, the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA. 100 pl of dilutions are added per well. The plate is incubated for 90 minutes at 370C, washed and 25 evaluated according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when specific antibodies were raised in rabbits. Incubation is carried out for 90 minutes at 370C and the plate is washed. The reaction is developed with the appropriate substrate and the 30 reaction is measured by colorimetry (absorbance measured spectrophotometrically). Under the above experimental conditions, a positive reaction is shown by O.D. values greater than a non immune control serum. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 40 In a dot blot assay, a purified product is preferred, although a whole cell extract can also be used. Briefly, a solution of the product at about 100 pg/ml is serially two-fold diluted in 50 mM Tris-HCl (pH 7.5). 100 pl of each dilution are 5 applied to a nitrocellulose membrane 0.45 pm set in a 96-well dot blot apparatus (Biorad). The buffer is removed by applying vacuum to the system. Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried. The membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M 10 NaCl, 10 g/L skim milk) and incubated with an antiserum dilution from about 1:50 to about 1:5000, preferably about 1:500. The reaction is revealed according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when rabbit antibodies are used. Incubation is carried 15 out 90 minutes at 370C and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is measured visually by the appearance of a colored spot, e.g., by colorimetry. Under the above experimental conditions, a positive reaction is shown once a colored spot is 20 associated with a dilution of at least about 1:5, preferably of at least about 1:500. Therapeutic or prophylactic efficacy of a polypeptide or derivative of the invention can be evaluated as described below. A seventh aspect of the invention provides (i) a composition of 25 matter comprising a polypeptide of the invention together with a diluent or carrier; specifically (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (iii) a method for inducing an immune response against Chlamydia in a 30 mammal, by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit a protective immune response to Chlamydia; and particularly, (iv) a method for preventing and/or treating a Chlamydia (e.g., C. trachomatis. C. psittaci, C. pneumoniae. or C. pecorum) qIRmTITITT= R4FFT (RULE 261 WO 00/24765 PCT/CA99/00992 41 infection, by administering a prophylactic or therapeutic amount of a polypeptide of the invention to an infected individual. Additionally, the seventh aspect of the invention encompasses the use of a polypeptide of the invention in the preparation of 5 a medicament for preventing and/or treating Chlamydia infection. As used herein, the immunogenic compositions of the invention are administered by conventional routes known the vaccine field, in particular to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, 10 vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. The choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, 15 the intranasal or oral route is preferred. If a lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-cutaneous or intramuscular route being most preferred. The choice also depends upon the nature of the vaccine agent. For example, a polypeptide of the 20 invention fused to CTB or LTB is best administered to a mucosal surface. As used herein, the composition of the invention contains one or several polypeptides or derivatives of the invention. The composition optionally contains at least one additional 25 Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof. For use in a composition of the invention, a polypeptide or derivative thereof is formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, 30 or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response. These compounds are readily available to one skilled in the art; for example, see Liposomes: A Practical Approach (supra). SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 42 Adjuvants other than liposomes and the like are also used and. are known in the art. Adjuvants may protect the antigen from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete 5 factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can conventionally be made by those skilled in the art, for example, from those described below. Treatment is achieved in a single dose or repeated as 10 necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals. An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine 15 antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art. In general, a vaccine antigen of the invention is administered by a mucosal route in an amount from 20 about 10 pg to about 500 mg, preferably from about 1 mg to about 200 mg. For the parenteral route of administration, the dose usually does not exceed about 1 mg, preferably about 100 pg. When used as vaccine agents, polynucleotides and polypeptides of the invention may be used sequentially as part 25 of a multistep immunization process. For example, a mammal is initially primed with a vaccine vector of the invention such as a pox virus, e.g., via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e.g., via the mucosal route. In another example, liposomes associated 30 with a polypeptide or derivative of the invention is also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT). SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 43 A polypeptide derivative of the invention is also used in accordance with the seventh aspect as a diagnostic reagent for detecting the presence of anti-Chlamydia antibodies, e.g., in a blood sample. Such polypeptides are about 5 to about 80, 5 preferably about 10 to about 50 amino acids in length. They are either labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below. Upon expression of a DNA molecule of the invention, a 10 polypeptide or polypeptide derivative is produced and purified using known laboratory techniques. As described above, the polypeptide or polypeptide derivative may be produced as a fusion protein containing a fused tail that facilitates purification. The fusion product is used to immunize a small 15 mammal, e.g., a mouse or a rabbit, in order to raise antibodies against the polypeptide or polypeptide derivative (monospecific antibodies). Accordingly, an eighth aspect of the invention provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention. 20 By "monospecific antibody" is meant an antibody that is capable of reacting with a unique naturally-occurring Chlamydia polypeptide. An antibody of the invention is either polyclonal or monoclonal. Monospecific antibodies may be recombinant, e.g., chimeric (e.g., constituted by a variable region of murine 25 origin associated with a human constant region), humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain. Both polyclonal and monospecific antibodies may also be in the form of immunoglobulin fragments, e.g., F(ab)'2 30 or Fab fragments. The antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes. Antibodies against the polypeptides, homologs or fragments of the present invention are generated by immunization SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 44 of a mammal with a composition comprising said polypeptide, homolog or fragment. Scu antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see 5 "Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. E. Harlow and D. Lane (1988), and D.E. Yelton et al., 1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, see Kohl and Milstein?... The antibodies of the invention, which are raised to a 10 polypeptide or polypeptide derivative of the invention, are produced and identified using standard immunological assays, e.g., Western blot analysis, dot blot assay, or ELISA (see, e.g., Coligan et al., Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, NY). The antibodies are used 15 in diagnostic methods to detect the presence of a Chlamydia antigen in a sample, such as a biological sample. The antibodies are also used in affinity chromatography for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, such antibodies may 20 be used in prophylactic and therapeutic passive immunization methods. Accordingly, a ninth aspect of the invention provides (i) a reagent for detecting the presence of Chlamydia in a biological sample that contains an antibody, polypeptide, or 25 polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Chlamydia in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, such that an immune complex is formed, and by detecting such complex 30 to indicate the presence of Chlamydia in the sample or the organism from which the sample is derived. Those skilled in the art will readily understand that the immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, whichever SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 45 is used, and that any unbound material is removed prior to detecting the complex. It is understood that a polypeptide reagent is useful for detecting the presence of anti-Chlamydia antibodies in a sample, e.g., a blood sample, while an antibody 5 of the invention is used for screening a sample, such as a gastric extract or biopsy, for the presence of Chlamydia polypeptides. For diagnostic applications, the reagent (i.e., the antibody, polypeptide, or polypeptide derivative of the 10 invention) is either in a free state or immobilized on a solid support, such as a tube, a bead, or any other conventional support used in the field. Immobilization is achieved using direct or indirect means. Direct means include passive adsorption (non-covalent binding) or covalent binding between 15 the support and the reagent. By "indirect means" is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support. For example, if a polypeptide reagent is used, an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not 20 involved in the recognition of antibodies in biological samples. Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the polypeptide reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin 25 streptavidin system. Alternatively, a peptide tail is added chemically or by genetic engineering to the reagent and the grafted or fused product immobilized by passive adsorption or covalent linkage of the peptide tail. Such diagnostic agents may be included in a kit which 30 also comprises instructions for use. The reagent are labeled with a detection means which allows for the detection of the reagent when it is bound to its target. The detection means may be a fluorescent agent such as fluorescein isocyanate or fluorescein isothiocyanate, or an enzyme such as horse radish SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 46 peroxidase or luciferase or alkaline phosphatase, or a radioactive element such as 1251 or 51 Cr. Accordingly, a tenth aspect of the invention provides a process for purifying, from a biological sample, a polypeptide 5 or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention. For use in a purification process of the invention, the 10 antibody is either polyclonal or monospecific, and preferably is of the IgG type. Purified IgGs is prepared from an antiserum using standard methods (see, e.g., Coligan et al., supra). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described in, e.g., 15 Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988) and outlined below. Briefly, a biological sample, such as an C. pneumoniae extract preferably in a buffer solution, is applied to a chromatography material, preferably equilibrated with the buffer 20 used to dilute the biological sample so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material. The chromatography material, such as a gel or a resin coupled to an antibody of the invention, is in either a batch form or a column. The unbound 25 components are washed off and the antigen is then eluted with an appropriate elution buffer, such as a glycine buffer or a buffer containing a chaotropic agent, e.g., guanidine HCl, or high salt concentration (e.g., 3 M MgCl 2 ) . Eluted fractions are recovered and the presence of the antigen is detected, e.g., by measuring 30 the absorbance at 280 nm. An eleventh aspect of the invention provides (i) a composition of matter comprising a monospecific antibody of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 47 prophylactically effective amount of a monospecific antibody of the invention, and (iii) a method for treating or preventing a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae or C. pecorum) infection, by administering a therapeutic or 5 prophylactic amount of a monospecific antibody of the invention to an infected individual. Additionally, the eleventh aspect of the invention encompasses the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Chlamydia infection. 10 The monospecific antibody is either polyclonal or monoclonal, preferably of the IgA isotype (predominantly). In passive immunization, the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, advantageously, in the presence of a 15 bicarbonate buffer. Alternatively, systemic administration, not requiring a bicarbonate buffer, is carried out. A monospecific antibody of the invention is administered as a single active component or as a mixture with at least one monospecific antibody specific for a different Chlamydia polypeptide. The 20 amount of antibody and the particular regimen used are readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibodies over one week, or three doses per day of about 100 to 1,000 mg of antibodies over two or three days, are effective regimens for 25 most purposes. Therapeutic or prophylactic efficacy are evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the C. pneumoniae mouse 30 model. Those skilled in the art will readily recognize that the C. pneumoniae strain of the model may be replaced with another Chlamydia strain. For example, the efficacy of DNA molecules and polypeptides from C. pneumoniae is preferably evaluated in a mouse model using C. pneumoniae strain. Protection is SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 48 determined by comparing the degree of Chlamydia infection to that of a control group. Protection is shown when infection is reduced by comparison to the control group. Such an evaluation is made for polynucleotides, vaccine vectors, polypeptides and 5 derivatives thereof, as well as antibodies of the invention. Adjuvants useful in any of the vaccine compositions described above are as follows. Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and 10 aluminum hydroxy phosphate. The antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, MT), is used in parenteral administration. Adjuvants for mucosal administration include bacterial 15 toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB). Fragments, homologs, derivatives, and fusions 20 to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). 25 Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69 Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella 30 flexneri; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration. Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/2415), DC-chol (3 SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 49 b-(N-(N',N'-dimethyl aminomethane)-carbamoyl) cholesterol; U.S. Patent No. 5,283,185 and WO 96/14831) and QS-21 (WO 88/9336). Any pharmaceutical composition of the invention containing a polynucleotide, a polypeptide, a polypeptide 5 derivative, or an antibody of the invention, is manufactured in a conventional manner. In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and 10 route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in the 15 USP/NF. The invention also includes methods in which Chlamydia infection are treated by oral administration of a Chlamydia polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antacid, sucralfate, or a 20 combination thereof. Examples of such compounds that can be administered with the vaccine antigen and the adjuvant are antibiotics, including, e.g., macrolides, tetracyclines, and derivatives thereof (specific examples of antibiotics that can be used include azithromycin or doxicyclin or immunomodulators 25 such as cytokines or steroids). In addition, compounds containing more than one of the above-listed components coupled together, are used. The invention also includes compositions for carrying out these methods, i.e., compositions containing a Chlamydia antigen (or antigens) of the invention, an adjuvant, 30 and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent. Amounts of the above-listed compounds used in the methods and compositions of the invention are readily determined by one skilled in the art. Treatment/immunization schedules are also SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 50 known and readily designed by one skilled in the art. For example, the non-vaccine components can be administered on days 1-14, and the vaccine antigen + adjuvant can be administered on days 7, 14, 21, and 28. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 51 REFERENCES 1. Grayston et al. (1995) Journal of Infectious Diseases 168:1231 2. Campos et al. (1995) Investigation of Ophthalmology and 5 Visual Science 36:1477 3. Grayston et al (1990) Journal of Infectious Diseases 161:618 4. Marrie (1993) Clinical Infectious Diseases. 18:501 5. Wang et al (1986) Chlamydial infections. Cambridge University Press, Cambridge. p. 329 10 6. Saikku et al.(1988) Lancet;ii:983 7. Thom et al. (1992) JAMA 268:68 8. Linnanmaki et al. (1993), Circulation 87:1030 9. Saikku et al. (1992)Annals Internal Medicine 116:273 10. Melnick et al(1993) American Journal of Medicine 95:499 11 15 Shor et al. (1992) South African. Medical Journal 82:158 12. Kuo et al. (1993) Journal of Infectious Diseases 167:841 13. Kuo et al. (1993) Arteriosclerosis and Thrombosis 13:1500 14. Campbell et al (1995) Journal of Infectious Diseases 172:585 15. Chiu et al. Circulation, 1997 (In Press). 20 16. Ramirez et al (1996) Annals of Internal Medicine 125:979 17. Jackson et al. Abst. K121, p272, 36th ICAAC, 15-18 Sept. 1996, New Orleans. 18. Fong et al (1997) Journal of Clinical Microbiolology 35:48 19. Hahn DL, et al. Evidence for Chlamydia pneumoniae infection 25 in steroid-dependent asthma. Ann Allergy Asthma Immunol. 1998 Jan; 80(1): 45-49. 20. Hahn DL, et al. Association of Chlamydia pneumoniae IgA antibodies with recently symptomatic asthma. Epidemiol Infect. 1996 Dec; 117(3): 513-517. 30 21. Bjornsson E, et al. Serology of chlamydia in relation to asthma and bronchial hyperresponsiveness. Scand J Infect Dis. 1996; 28(1): 63-69. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 52 22. Hahn DL. Treatment of Chlamydia pneumoniae infection in adult asthma: a before-after trial. J Fam Pract. 1995 Oct; 41(4): 345-351. 23. Allegra L, et al. Acute exacerbations of asthma in adults: 5 role of Chlamydia pneumoniae infection. Eur Respir J. 1994 Dec; 7(12): 2165-2168. 24. Hahn DL, et al. Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult onset asthma. JAMA. 1991 Jul 10; 266(2): 225-230. 10 25. Pal et al.(1996) Infection and Immunity.64:5341 26. Jones et al. (1995) Vaccine 13:715 27. Igietsemes et al. (1993) Immunology 5:317 28. Igietseme et al (1993) Regional Immunology 5:317 29. Magee et al (1993) Regional Immunology 5: 305 15 30. Landers et al (1991) Infection & Immunity 59:3774 31. Magee et al (1995) Infection & Immunity 63:516 32. Cotter et al. (1995) Infection and Immunity63:4704 33. Campbell et al (1990) Infection and Immunity 58:93 34. McCafferty et al (1995) Infection and Immunity 63:2387-9. 20 35. Knudsen et al (1996)Third Meeting of the European Society for Chlamydia Research, Vienna 36. Wiedmann-Al-Ahmad M, et al. Reactions of polyclonal and neutralizing anti-p54 monoclonal antibodies with an isolated, species-specific 54-kilodalton protein of Chlamydia pneumoniae. 25 Clin Diagn Lab Immunol. 1997 Nov; 4(6): 700-704 37. Hughes et al., 1992. Infect. Immun. 60(9):3497, 38. Dion et al., 1990. Virology 179:474-477, 39. Snijders et al., 1991. J. Gen. Virol. 72:557-565, 40. Langeveld et al., Vaccine 12(15):1473-1480, 1994 30 41. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994 42. Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448 43. Silhavy et al. Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, 1984 SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 53 44. Davis et al. A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, 1980) 45. Casey & Davidson, Nucl. Acid Res. (1977) 4:1539 46. Cagnon et al., Protein Engineering (1991) 4(7):843 5 47. Takase et al., J. Bact. (1987) 169:5692 SUBSTITUTE SHEET (RULE 26)

Claims (24)

1. A nucleic acid molecule comprising a nucleic acid sequence which encodes a polypeptide selected from any of: 5 (a) SEQ ID Nos: 27 to 45; (b) an immunogenic fragment comprising at least 12 consecutive amino acids from a polypeptide of (a); and (c) a polypeptide of (a) or (b) which has been modified to improve its immunogenicity, wherein said modified 10 polypeptide is at least 75% identical in amino acid sequence to the corresponding polypeptide of (a) or (b).

2. A nucleic acid molecule comprising a nucleic acid 15 sequence selected from any of: (a) SEQ ID Nos: 1 to 26; (b) a sequence which encodes a polypeptide encoded by any one of SEQ ID Nos: 1 to 26; (c) a sequence comprising at least 38 consecutive 20 nucleotides from any one of the nucleic acid sequences of (a) and (b); and (d) a sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to any one of the polypeptides encoded by SEQ ID Nos: 1 to 26. 25 SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 55

3. A nucleic acid molecule comprising a nucleic acid sequence which encodes a fusion protein, said fusion protein comprising a polypeptide encoded by a nucleic acid molecule according to claim 1 and an additional 5 polypeptide.

4. A nucleic acid molecule according to claim 1, operatively linked to one or more expression control sequences. 10

5. A vaccine comprising at least one first nucleic acid according to any one of claims 1 to 4 and a vaccine vector wherein each first nucleic acid is expressed as a polypeptide, the vaccine optionally comprising a second 15 nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by said first nucleic acid.

6. The vaccine of claim 5 wherein the second nucleic acid 20 encodes an additional Chlamydia polypeptide.

7. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier. 25 SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 56

8. A pharmaceutical composition comprising a vaccine according to claim 5 or 6 and a pharmaceutically acceptable carrier. 5

9. A unicellular host transformed with the nucleic acid molecule of claim 4.

10. A nucleic acid probe of 5 to 100 nucleotides which hybridizes under stringent conditions to any one of nucleic 10 acid molecules of SEQ ID Nos: 1 to 26, or to a homolog or complementary or anti-sense sequence of said nucleic acid molecule.

11. A primer of 10 to 40 nucleotides which hybridizes 15 under stringent conditions to any one of nucleic acid molecules of SEQ ID Nos: 1 to 26, or to a homolog or complementary or anti-sense sequence of said nucleic acid molecule. 20

12. A polypeptide encoded by a nucleic acid sequence according to any one of claims 1 to 4.

13. A polypeptide comprising an amino acid sequence selected from any of: 25 (a) SEQ ID Nos: 27 to 45; SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 57 (b) an immunogenic fragment comprising at least 12 consecutive amino acids from a polypeptide of (a); and (c) a polypeptide of (a) or (b) which has been modified to improve its immunogenicity, wherein said modified 5 polypeptide is at least 75% identical in amino acid sequence to the corresponding polypeptide of (a) or (b).

14. A fusion polypeptide comprising a polypeptide of claim 10 12 or 13 and an additional polypeptide.

15. A method for producing a polypeptide of claim 12 or 13, comprising the step of culturing a unicellular host according to claim 9. 15

16. An antibody against the polypeptide of any one of claims 12 to 14.

17. A vaccine comprising at least one first polypeptide 20 according to any one of claims 12 to 14 and a pharmaceutically acceptable carrier, optionally comprising a second polypeptide which enhances the immune response to the first polypeptide. 25

18. The vaccine of claim 17 wherein the second polypeptide comprises an additional Chlamydia polypeptide. SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 58

19. A pharmaceutical composition comprising a polypeptide according to any one of claims 12 to 14 and a pharmaceutically acceptable carrier. 5

20. A pharmaceutical composition comprising a vaccine according to claim 17 or 18 and a pharmaceutically acceptable carrier. 10

21. A pharmaceutical composition comprising an antibody according to claim 16 and a pharmaceutically acceptable carrier.

22. A method for preventing or treating Chlamydia 15 infection using: (a) the nucleic acid of any one of claims 1 to 4; (b) the vaccine of any one of claims 5, 6, 17 and 18; (c) the pharmaceutical composition of any one of claims 7, 8, 19 to 21; 20 (d) the polypeptide of any one of claims 12 to 14; or (e) the antibody of claim 16.

23. A method of detecting Chlamydia infection comprising the step of assaying a body fluid of a mammal to be tested, 25 with a component selected from any one of: (a) the nucleic acid of any one of claims 1 to 4; SUBSTITUTE SHEET (RULE 26) WO 00/24765 PCT/CA99/00992 59 (b) the polypeptide of any one of claims 12 to 14; and (c) the antibody of claim 16.

24. A diagnostic kit comprising instructions for use and a 5 component selected from any one of: (a) the nucleic acid of any one of claims 1 to 4; (b) the polypeptide of any one of claims 12 to 14; and the antibody of claim 16. SUBSTITUTE SHEET (RULE 26)