HK1067381B - Compounds and methods for treatment and diagnosis of chlamydial infection - Google Patents

Description

Compounds and methods for the treatment and diagnosis of chlamydial infections

Technical Field

The present invention relates generally to the detection and treatment of chlamydial infections. In particular, the invention relates to polypeptides comprising a chlamydia antigen and the use of such polypeptides in the serological diagnosis and treatment of chlamydia infection.

Background

Chlamydiae (Chlamydiae) are intracellular bacterial pathogens responsible for many important human and animal infections. Chlamydia trachomatis (Chlamydia trachomatis) is one of the most common causes of sexually transmitted diseases, and can cause Pelvic Inflammation (PID), leading to tubal obstruction and infertility. Chlamydia trachomatis may also cause male infertility. In 1990, the cost of the U.S. treatment PID was estimated to be $ 40 billion. Trachoma, which is caused by chlamydia trachomatis infecting the eye, is a major cause of preventable blindness worldwide. Chlamydia pneumoniae (Chlamydia pneumoniae) is a major cause of acute respiratory infections in humans and is believed to play a role in the pathogenesis of atherosclerosis, particularly coronary heart disease. Individuals with high titers of chlamydia pneumoniae antibodies have been shown to be at least 2-fold more likely to develop coronary heart disease than seronegative individuals. Therefore, chlamydial infections are a significant health problem in the united states and throughout the world.

Chlamydial infections are often asymptomatic. For example, by the time a woman seeks medical treatment for PID, irreversible damage may have occurred, resulting in infertility. There is therefore a need in the art for improved vaccines and pharmaceutical compositions for the prevention and treatment of chlamydial infections. The present invention fulfills this need, and further provides other related advantages.

Summary of The Invention

The present invention provides compositions and methods for the diagnosis and treatment of chlamydial infections. In one aspect, the invention provides polypeptides comprising an immunogenic portion of a chlamydia antigen (or a variant of such an antigen). Certain portions and other variants are immunogenic such that the ability of the variant to react with antigen-specific antisera is not substantially diminished. In certain embodiments, the polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 358-361, 366-385, 406-430, 455-489, 516-517, 523-559, and 582-596; (b) the complement of said sequence; and (c) a sequence that hybridizes to the sequence of (a) or (b) under conditions of moderate to high stringency. In a specific embodiment, the polypeptide of the invention comprises a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 362-365, 386-405, 431-454, 490-515, 518-522, 560-581 and 597-599, or a variant thereof.

The invention further provides polynucleotides encoding the above polypeptides or portions thereof (e.g., portions encoding at least 15 amino acid residues of a chlamydia protein), expression vectors comprising such polynucleotides, and host cells transformed or transfected with such expression vectors.

In related aspects, polynucleotide sequences encoding the above polypeptides, recombinant expression vectors containing one or more of these polynucleotide sequences, and host cells transformed or transfected with such expression vectors are also provided.

In another aspect, the invention provides fusion proteins comprising a polypeptide of the invention, or alternatively, a polypeptide of the invention and a known chlamydia antigen, and polynucleotides encoding such fusion proteins, for use as pharmaceutical compositions and vaccines thereof, in combination with a physiologically acceptable carrier or an immunostimulant.

The present invention further provides a pharmaceutical composition comprising: (a) a polyclonal or monoclonal antibody, or antigen-binding fragment thereof, that specifically binds a chlamydia protein; and (b) a physiologically acceptable carrier. In other aspects, the invention provides pharmaceutical compositions comprising one or more chlamydia polypeptides disclosed herein, such as SEQ ID NO: 362-365, 386-405, 431-454, 490-515, 518-522, 560-581 and 597-599, or a polynucleotide molecule encoding such a polypeptide, such as the polypeptide shown in SEQ ID NO: 358-361, 366-385, 406-430, 455-489, 516-517, 523-559 and 582-596, and physiologically acceptable carriers. The invention also provides vaccines for prophylaxis or therapy comprising one or more polypeptides disclosed herein and an immunostimulant as defined herein, and vaccines comprising one or more polynucleotide sequences encoding such polypeptides and an immunostimulant.

In another aspect, the present invention provides a method of inducing protective immunity in a patient comprising administering to the patient an effective amount of one or more of the pharmaceutical compositions or vaccines described above.

In another aspect, the present invention provides a method of treating a chlamydia infection in a patient, the method comprising: obtaining Peripheral Blood Mononuclear Cells (PBMCs) from a patient, incubating the PBMCs with a polypeptide of the invention (or a polynucleotide encoding such a polypeptide) to provide incubated T cells, and administering the incubated T cells to the patient. The present invention also provides a method of treating a chlamydial infection comprising: incubating antigen presenting cells with a polypeptide of the invention (or a polynucleotide encoding such a polypeptide) to provide incubated antigen presenting cells, and administering the incubated antigen presenting cells to the patient. The proliferating cells may, but need not, be cloned prior to administration to a patient. In certain embodiments, the antigen presenting cell is selected from the group consisting of a dendritic cell, a macrophage, a monocyte, a B cell, and a fibroblast. Also provided are compositions for treating chlamydial infections comprising T cells or antigen presenting cells that have been incubated with a polypeptide or polynucleotide of the invention. In a related aspect, there is provided a vaccine comprising: (a) antigen presenting cells expressing the above polypeptides and (b) an immunostimulant.

In other aspects, the invention further provides methods of removing chlamydia infected cells from a biological sample comprising contacting the biological sample with T cells that specifically react with a chlamydia protein, wherein the contacting step is carried out under conditions and for a time sufficient to allow removal of cells expressing the protein from the sample.

In a related aspect, the invention provides a method of inhibiting the development of a chlamydia infection in a patient comprising administering to the patient a biological sample treated as described above. In another aspect, the invention provides methods and diagnostic kits for detecting chlamydial infection in a patient. In one embodiment, the method comprises: (a) contacting a biological sample with at least one polypeptide or fusion protein disclosed herein; and (b) detecting the presence of the binding agent that binds to the polypeptide or fusion protein in the sample, thereby detecting a chlamydial infection in the biological sample. Suitable biological samples include whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine. In one embodiment, a diagnostic kit comprises one or more polypeptides or fusion proteins disclosed herein and a detection reagent. In another embodiment, the diagnostic kit comprises a monoclonal or polyclonal antibody that binds to a polypeptide of the invention.

The present invention also provides a method of detecting chlamydial infection comprising: (a) obtaining a biological sample from a patient; (b) contacting the sample with at least two oligonucleotide primers in a polymerase chain reaction, at least one of the oligonucleotide primers being specific for a polynucleotide sequence disclosed herein; and (c) detecting in the sample the polynucleotide sequence amplified in the presence of the oligonucleotide primer. In one embodiment, the oligonucleotide primer contains at least about 10 contiguous nucleotides of a polynucleotide sequence disclosed herein or a sequence that hybridizes thereto.

In other aspects, the invention provides methods for detecting chlamydial infection in a patient, comprising: (a) obtaining a biological sample from the patient; (b) contacting the sample with an oligonucleotide probe specific for a polynucleotide sequence disclosed herein; and (c) detecting in the sample the polynucleotide sequence that hybridizes to the oligonucleotide probe. In one embodiment, an oligonucleotide probe comprises at least about 15 contiguous nucleotides of a polynucleotide sequence disclosed herein, or a sequence that hybridizes thereto.

These and other aspects of the invention will be apparent upon reference to the following detailed description. All references disclosed herein are incorporated by reference in their entirety as if each were individually incorporated.

Sequence identification

SEQ ID NO: 1 is the determined DNA sequence of Chlamydia trachomatis clone 1-B1-66.

SEQ ID NO: 2 is the determined DNA sequence of C.trachomatis clone 4-D7-28.

SEQ ID NO: 3 is the determined DNA sequence of C.trachomatis clone 3-G3-10.

SEQ ID NO: 4 is the determined DNA sequence of Chlamydia trachomatis clone 10-C10-31.

SEQ ID NO: and 5 is the predicted amino acid sequence of 1-B1-66.

SEQ ID NO: 6 is the predicted amino acid sequence of 4-D7-28.

SEQ ID NO: 7 is the first predicted amino acid sequence of 3-G3-10.

SEQ ID NO: and 8 is the second predicted amino acid sequence of 3-G3-10.

SEQ ID NO: 9 is the third predicted amino acid sequence of 3-G3-10.

SEQ ID NO: 10 is the fourth predicted amino acid sequence of 3-G3-10.

SEQ ID NO: 11 is the fifth predicted amino acid sequence of 3-G3-10.

SEQ ID NO: 12 is the predicted amino acid sequence of 10-C10-31.

SEQ ID NO: 13 is the amino acid sequence of synthetic peptide 1-B1-66/48-67.

SEQ ID NO: 14 is the amino acid sequence of synthetic peptide 1-B1-66/58-77.

SEQ ID NO: 15 is the determined DNA sequence of Chlamydia trachomatis serum variant LGV II clone 2C 7-8.

SEQ ID NO: 16 is the DNA sequence of the putative open reading frame of the region of the C7-8 mapping genome of C.trachomatis serovar D.

SEQ ID NO: 17 is SEQ ID NO: 16DNA sequence encoded predicted amino acid sequence.

SEQ ID NO: 18 is the amino acid sequence of the synthetic peptide CtC7.8-12.

SEQ ID NO: 19 is the amino acid sequence of the synthetic peptide CtC7.8-13.

SEQ ID NO: 20 is the predicted amino acid sequence encoded by the second putative open reading frame of chlamydia trachomatis serotype D.

SEQ ID NO: 21 is the determined DNA sequence of C9-18 of C.trachomatis LGV II clone.

SEQ ID NO: 22 is the determined DNA sequence homologous to Chlamydia trachomatis LGV II lipoamide dehydrogenase.

SEQ ID NO: 23 is the determined DNA sequence homologous to the C.trachomatis LGV II pseudoprotein.

SEQ ID NO: 24 is an assayed DNA sequence homologous to the Chlamydia trachomatis LGV II ubiquinone methyltransferase.

SEQ ID NO: 25 is the DNA sequence determined for C.trachomatis LGV II clone 4C9-18#2 BL21 pLysS.

SEQ ID NO: 26 is the predicted amino acid sequence of 4C9-18#2 of C.trachomatis LGV II.

SEQ ID NO: 27 is the determined DNA sequence of Cp-SWIB from the TWAR strain of Chlamydia pneumoniae.

SEQ ID NO: 28 is the predicted amino acid sequence of Cp-SWIB from the TWAR strain of Chlamydia pneumoniae.

SEQ ID NO: 29 is the determined DNA sequence of Cp-S13(CT509) from the TWAR strain of Chlamydia pneumoniae.

SEQ ID NO: 30 is the predicted amino acid sequence of Cp-S13 of the Chlamydia pneumoniae TWAR strain.

SEQ ID NO: 31 is the amino acid sequence of the 10mer (mer) consensus peptide of CtC7.8-12 and CtC7.8-13.

SEQ ID NO: 32 is the predicted amino acid sequence of clone 2C7-8 of C.trachomatis LGV II.

SEQ ID NO: 33 is a DNA sequence corresponding to nucleotide 597304-.

SEQ ID NO: 34 is SEQ ID NO: 33, or a predicted amino acid sequence encoded by the sequence of seq id no.

SEQ ID NO: 35 is the DNA sequence of C.p. SWIB Nde (5' primer) from Chlamydia pneumoniae.

SEQ ID NO: 36 is the DNA sequence of C.p. SWIB EcoRI (3' primer) from C.p. C.pneumoniae.

SEQ ID NO: 37 is the DNA sequence of C.p.S13 Nde (5' primer) of Chlamydia pneumoniae.

SEQ ID NO: 38 is the DNA sequence of C.p.S13 EcoRI (3' primer) from Chlamydia pneumoniae.

SEQ ID NO: 39 is the amino acid sequence of the CtSwib 52-67 peptide of C.trachomatis LGV II.

SEQ ID NO: 40 is the amino acid sequence of the CpSwib 53-68 peptide from Chlamydia pneumoniae.

SEQ ID NO: 41 is the amino acid sequence of the HuSwib 288-302 peptide of the human SWI domain.

SEQ ID NO: 42 is the amino acid sequence of the CtSWI-T822-837 peptide of a C.trachomatis topoisomerase-SWIB fusion.

SEQ ID NO: 43 is the amino acid sequence of the CpSWI-T828-842 peptide of a Chlamydia pneumoniae topoisomerase-SWIB fusion.

SEQ ID NO: 44 is the first determined DNA sequence of C.trachomatis LGV II clone 19783.3, jen. seq (1>509) CTL2#11-3 ', representing the 3' end.

SEQ ID NO: 45 is the second determined DNA sequence of C.trachomatis LGV II clone 19783.4, jen. seq (1>481) CTL2#11-5 ', representing the 5' end.

SEQ ID NO: 46 is the determined DNA sequence of C.trachomatis LGV II clone 19784CTL2-12consensus. seq (1>427) CTL2# 12.

SEQ ID NO: 47 is the determined DNA sequence of C.trachomatis LGVII clone 19785.4, jen.seq (1>600) CTL2#16-5 ', representing the 5' end.

SEQ ID NO: 48 is the first determined DNA sequence of C.trachomatis LGV II clone 19786.3, jen.seq (1>600) CTL2#18-3 ', representing the 3' end.

SEQ ID NO: 49 is the second determined DNA sequence of C.trachomatis LGV II clone 19786.4, jen.seq (1>600) CTL2#18-5 ', representing the 5' end.

SEQ ID NO: 50 is the determined DNA sequence of C.trachomatis LGV II clone 19788CTL2-21consensus. seq (1>406) CTL2# 21.

SEQ ID NO: 51 is the determined DNA sequence of C.trachomatis LGV II clone 19790CTL2-23consensus. seq (1>602) CTL2# 23.

SEQ ID NO: 52 is the determined DNA sequence of C.trachomatis LGV II clone 19791CTL2-24consensus. seq (1>145) CTL2# 24.

SEQ ID NO: 53 is the determined DNA sequence of C.trachomatis LGV II clone CTL2# 4.

SEQ ID NO: 54 is the determined DNA sequence of C.trachomatis LGV II clone CTL2#8 b.

SEQ ID NO: 55 is the determined DNA sequence of C.trachomatis LGV II clone 15-G1-89, having homology to the lipoamide dehydrogenase gene CT 557.

SEQ ID NO: 56 is a determined DNA sequence of C.trachomatis LGV II clone 14-H1-4, having homology to the thiol-specific antioxidant gene CT 603.

SEQ ID NO: 57 is a determined DNA sequence of C.trachomatis LGV II clone 12-G3-83, having homology to the pseudomimetic protein CT 622.

SEQ ID NO: 58 is the determined DNA sequence of C.trachomatis LGV II clone 12-B3-95, having homology to lipoamide dehydrogenase gene CT 557.

SEQ ID NO: 59 is the determined DNA sequence of C.trachomatis LGV II clone 11-H4-28, having homology to dnaK gene CT 396.

SEQ ID NO: 60 is the determined DNA sequence of C.trachomatis LGV II clone 11-H3-68, which has partial homology with PGP6-D virulence protein and LI ribosomal gene CT 318.

SEQ ID NO: 61 is the determined DNA sequence of C.trachomatis LGV II clone 11-G1-34, which has partial homology with malate dehydrogenase gene CT376 and glycohydrolase gene CT 042.

SEQ ID NO: 62 is a determined DNA sequence of C.trachomatis LGV II clone 11-G10-46, having homology to pseudomimetic protein CT 610.

SEQ ID NO: 63 is a determined DNA sequence of C12-91 of LGV II clone 11-C12-91 of Chlamydia trachomatis, having homology to the OMP2 gene CT 443.

SEQ ID NO: 64 is the determined DNA sequence of C.trachomatis LGV II clone 11-A3-93, having homology to the HAD superfamily gene CT 103.

SEQ ID NO: 65 is the determined amino acid sequence of C.trachomatis LGV II clone 14-H1-4, having homology to the thiol-specific antioxidant gene CT 603.

SEQ ID NO: 66 is the determined DNA sequence of C.trachomatis LGV II clone CtL2# 9.

SEQ ID NO: 67 is the determined DNA sequence of C.trachomatis LGV II clone CtL2# 7.

SEQ ID NO: 68 is the determined DNA sequence of C.trachomatis LGV II clone CtL2# 6.

SEQ ID NO: 69 is the determined DNA sequence of C.trachomatis LGV II clone CtL2# 5.

SEQ ID NO: 70 is the determined DNA sequence of C.trachomatis LGV II clone CtL2# 2.

SEQ ID NO: 71 is the determined DNA sequence of C.trachomatis LGV II clone CtL2# 1.

SEQ ID NO: 72 is the first determined DNA sequence of C.trachomatis LGV II clone 23509.2CtL2#3-5 ', representing the 5' end.

SEQ ID NO: 73 is the second determined DNA sequence of C.trachomatis LGV II clone 23509.1CtL2#3-3 ', representing the 3' end.

SEQ ID NO: 74 is the first determined DNA sequence of C.trachomatis LGV II clone 22121.2CtL2#10-5 ', representing the 5' end.

SEQ ID NO: 75 is the second determined DNA sequence of C.trachomatis LGV II clone 22121.1CtL2#10-3 ', representing the 3' end.

SEQ ID NO: 76 is the determined DNA sequence of C.trachomatis LGV II clone 19787.6CtL2#19-5 ', representing the 5' end.

SEQ ID NO: 77 is the determined DNA sequence of C.pneumoniae LGV II clone CpS 13-His.

SEQ ID NO: 78 is the determined DNA sequence of C.pneumoniae LGV II clone Cp _ SWIB-His.

SEQ ID NO: 79 is a determined DNA sequence of C.trachomatis LGV II clone 23-G7-68, having partial homology to L11, L10 and L1 ribosomal proteins.

SEQ ID NO: 80 is the determined DNA sequence of C.trachomatis LGV II clone 22-F8-91, having homology to the pmpC gene.

SEQ ID NO: 81 is the determined DNA sequence of C.trachomatis LGV II clone 21-E8-95, having homology to the CT610-CT613 gene.

SEQ ID NO: 82 is the determined DNA sequence of clone 19-F12-57 of C.trachomatis LGV II, which has homology to CT858 and recA genes.

SEQ ID NO: 83 is the determined DNA sequence of C.trachomatis LGV II clone 19-F12-53 having homology to the CT445 gene encoding glutamyl tRNA synthetase.

SEQ ID NO: 84 is a determined DNA sequence of C.trachomatis LGV II clone 19-A5-54, having homology to the cryptic plasmid gene.

SEQ ID NO: 85 is the determined DNA sequence of C.trachomatis LGV II clone 17-E11-72, having partial homology to the OppC-2 and pmpD genes.

SEQ ID NO: 86 is a determined DNA sequence of clone LGV II from Chlamydia trachomatis 17-C1-77, having partial homology to the open reading frames of CT857 and CT 858.

SEQ ID NO: 87 is the determined DNA sequence of C.trachomatis LGV II clone 15-H2-76, with partial homology to the pmpD and SycE genes and the CT089 ORF (open reading frame).

SEQ ID NO: 88 is a determined DNA sequence of C.trachomatis LGV II clone 15-A3-26 having homology to CT858 ORF.

SEQ ID NO: 89 is the determined amino acid sequence of Chlamydia pneumoniae clone Cp _ SWIB-His.

SEQ ID NO: 90 is the determined amino acid sequence of C.trachomatis LGV II clone CtL2_ LPDA _ FL.

SEQ ID NO: 91 is the determined amino acid sequence of Chlamydia pneumoniae clone CpS 13-His.

SEQ ID NO: 92 is the determined amino acid sequence of Chlamydia trachomatis LGV II clone CtL2_ TSA _ FL.

SEQ ID NO: 93 is the amino acid sequence of the Ct-Swib 43-61 peptide of C.trachomatis LGV II.

SEQ ID NO: 94 is the amino acid sequence of the Ct-Swib 48-67 peptide of C.trachomatis LGV II.

SEQ ID NO: 95 is the amino acid sequence of the Ct-Swib 52-71 peptide of C.trachomatis LGV II.

SEQ ID NO: 96 is the amino acid sequence of the Ct-Swib 58-77 peptide of C.trachomatis LGV II.

SEQ ID NO: 97 is the amino acid sequence of the Ct-Swib 63-82 peptide of C.trachomatis LGV II.

SEQ ID NO: 98 is the amino acid sequence of the Ct-Swib 51-66 peptide of C.trachomatis LGV II.

SEQ ID NO: 99 is the amino acid sequence of the Cp-Swib 52-67 peptide from Chlamydia pneumoniae.

SEQ ID NO: 100 is the amino acid sequence of the Cp-Swib 37-51 peptide from Chlamydia pneumoniae.

SEQ ID NO: 101 is the amino acid sequence of the Cp-Swib 32-51 peptide of chlamydia pneumoniae.

SEQ ID NO: 102 is the amino acid sequence of the Cp-Swib 37-56 peptide from Chlamydia pneumoniae.

SEQ ID NO: 103 is the amino acid sequence of the Ct-Swib 36-50 peptide of Chlamydia trachomatis.

SEQ ID NO: 104 is the amino acid sequence of the Ct-S1346-65 peptide of Chlamydia trachomatis.

SEQ ID NO: 105 is the amino acid sequence of the Ct-S1360-80 peptide of Chlamydia trachomatis.

SEQ ID NO: 106 is the amino acid sequence of the Ct-S131-20 peptide of Chlamydia trachomatis.

SEQ ID NO: 107 is the amino acid sequence of the Ct-S1346-65 peptide of Chlamydia trachomatis.

SEQ ID NO: 108 is the amino acid sequence of the Ct-S1356-75 peptide of Chlamydia trachomatis.

SEQ ID NO: 109 is the amino acid sequence of the Cp-S1356-75 peptide of Chlamydia pneumoniae.

SEQ ID NO: 110 is the determined DNA sequence of C.trachomatis LGV II clone 21-G12-60, containing part of the open reading frame for the pseudoproteins CT875, CT229 and CT 228.

SEQ ID NO: 111 is the determined DNA sequence of C.trachomatis LGV II clone 22-B3-53, having homology to the CT110 ORF of GroEL.

SEQ ID NO: 112 is a determined DNA sequence of C.trachomatis LGV II clone 22-A1-49, having partial homology with CT660 and CT659 ORFs.

SEQ ID NO: 113 is a determined DNA sequence of clone 17-E2-9 of C.trachomatis LGV II, which has partial homology with CT611 and CT 610 ORFs.

SEQ ID NO: 114 is a determined DNA sequence of C10-31 clone 17-LGV II of C.trachomatis, which has partial homology to the CT858 ORF.

SEQ ID NO: 115 is a determined DNA sequence of C7-8 clone 21-LGV II of C.trachomatis, having homology to a dnaK-like gene (dnaK-like gene).

SEQ ID NO: 116 is a determined DNA sequence of C.trachomatis LGV II clone 20-G3-45, containing a portion of the pmpB gene CT 413.

SEQ ID NO: 117 is a determined DNA sequence of C5-2 from LGV II clone 18-C, C1, homologous to the ribosomal protein ORF of C1.

SEQ ID NO: 118 is a determined DNA sequence of clone 17-C5-19 of C.trachomatis LGV II, containing a portion of CT431 and CT430 ORFs.

SEQ ID NO: 119 is a determined DNA sequence of C.trachomatis LGV II clone 16-D4-22 containing partial sequences of ORF3 and ORF4 of the plasmid used for growth in mammalian cells.

SEQ ID NO: 120 is the determined full-length DNA sequence of Chlamydia trachomatis serovar LGV II Cap1 gene CT 529.

SEQ ID NO: 121 is the predicted full-length amino acid sequence of chlamydia trachomatis serovar LGV II Cap1 gene CT 529.

SEQ ID NO: 122 is the determined full-length DNA sequence of chlamydia trachomatis serum variant E Cap1 gene CT 529.

SEQ ID NO: 123 is the predicted full-length amino acid sequence of chlamydia trachomatis serovar E Cap1 gene CT 529.

SEQ ID NO: 124 is the determined full-length DNA sequence of Chlamydia trachomatis serum variant 1A Cap1 gene CT 529.

SEQ ID NO: 125 is the predicted full-length amino acid sequence of chlamydia trachomatis serovar 1A Cap1 gene CT 529.

SEQ ID NO: 126 is the determined full-length DNA sequence of chlamydia trachomatis serum variant G Cap1 gene CT 529.

SEQ ID NO: 127 is the predicted full-length amino acid sequence of CT529 from chlamydia trachomatis serum variant G Cap1 gene.

SEQ ID NO: 128 is the determined full-length DNA sequence of chlamydia trachomatis serum variant F1 NII Cap1 gene CT 529.

SEQ ID NO: 129 is the predicted full-length amino acid sequence of chlamydia trachomatis serum variant F1 NII Cap1 gene CT 529.

SEQ ID NO: 130 is the determined full-length DNA sequence of Chlamydia trachomatis serum variant L1 Cap1 gene CT 529.

SEQ ID NO: 131 is the predicted full-length amino acid sequence of chlamydia trachomatis serum variant L1 Cap1 gene CT 529.

SEQ ID NO: 132 is the determined full-length DNA sequence of Chlamydia trachomatis serum variant L3 Cap1 gene CT 529.

SEQ ID NO: 133 is the predicted full-length amino acid sequence of chlamydia trachomatis serum variant L3 Cap1 gene CT 529.

SEQ ID NO: 134 is the determined full-length DNA sequence of Chlamydia trachomatis serum variant Ba Cap1 gene CT 529.

SEQ ID NO: 135 is the predicted full-length amino acid sequence of CT529 from chlamydia trachomatis serovar Ba Cap1 gene.

SEQ ID NO: 136 is the determined full-length DNA sequence of chlamydia trachomatis serum variant MOPN Cap1 gene CT 529.

SEQ ID NO: 137 is the predicted full-length amino acid sequence of CT529 from chlamydia trachomatis serum variant MOPN Cap1 gene.

SEQ ID NO: 138 is the determined amino acid sequence of Cap1 CT529 ORF peptide #124-139 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 139 is the determined amino acid sequence of Cap1 CT529 ORF peptide #132-147 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 140 is the determined amino acid sequence of Cap1 CT529 ORF peptide #138-155 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 141 is the determined amino acid sequence of Cap1 CT529 ORF peptide #146-163 of the Chlamydia trachomatis serum variant L2.

SEQ ID NO: 142 is the determined amino acid sequence of Cap1 CT529 ORF peptide #154-171 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 143 is the determined amino acid sequence of Cap1 CT529 ORF peptide #162-178 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 144 is the determined amino acid sequence of Cap1 CT529 ORF peptide #138-147 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 145 is the determined amino acid sequence of Cap1 CT529 ORF peptide #139-147 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 146 is the determined amino acid sequence of Cap1 CT529 ORF peptide #140-147 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 147 is the determined amino acid sequence of Cap1 CT529 ORF peptide #138-146 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 148 is the determined amino acid sequence of Cap1 CT529 ORF peptide #138-145 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 149 is the determined amino acid sequence of Cap1 CT529 ORF peptide F140- > I of Chlamydia trachomatis serotype L2.

SEQ ID NO: 150 is a determined amino acid sequence of Cap1 CT529 ORF peptide # # S139> Ga of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 151 is the determined amino acid sequence of Cap1 CT529 ORF peptide # # S139> Gb of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 152 is the determined amino acid sequence of peptide #2C7.8-6 of the 216aa (amino acid) ORF of the Chlamydia trachomatis serovariant L2.

SEQ ID NO: 153 is the determined amino acid sequence of peptide #2C7.8-7 of the 216aa ORF of Chlamydia trachomatis serovariant L2.

SEQ ID NO: 154 is the determined amino acid sequence of peptide #2C7.8-8 of the 216aa ORF of Chlamydia trachomatis serovariant L2.

SEQ ID NO: 155 is the determined amino acid sequence of peptide #2C7.8-9 of the 216aa ORF of Chlamydia trachomatis serovariant L2.

SEQ ID NO: 156 is the determined amino acid sequence of peptide #2C7.8-10 of the 216aa ORF of Chlamydia trachomatis serovariant L2.

SEQ ID NO: 157 is the determined amino acid sequence of a 53 amino acid residue peptide of 216aaORF within clone 2C7.8 of Chlamydia trachomatis serum variant L2.

SEQ ID NO: 158 is a determined amino acid sequence of a 52 amino acid residue peptide of CT529ORF within clone 2C7.8 of chlamydia trachomatis serovar L2.

SEQ ID NO: 159 is the determined DNA sequence of the 5' (forward) primer used to clone the full length CT529 serovariant L2.

SEQ ID NO: 160 is the determined DNA sequence of the 5' (reverse) primer used to clone the full length CT529 serovariant L2.

SEQ ID NO: 161 is the determined DNA sequence of the 5' (forward) primer used to clone the full length CT529 serovariant, except L2 and MOPN.

SEQ ID NO: 162 is the determined DNA sequence of the 5' (reverse) primer used to clone the full length CT529 serovariant, except L2 and MOPN.

SEQ ID NO: 163 is the determined DNA sequence of the 5' (forward) primer used for cloning the full-length CT529 serovariant MOPN.

SEQ ID NO: 164 is the determined DNA sequence of the 5' (reverse) primer used for cloning the full-length CT529 serovariant MOPN.

SEQ ID NO: 165 is the determined DNA sequence of the 5' (forward) primer of pBIB-KS.

SEQ ID NO: 166 is the determined DNA sequence of the 5' (reverse) primer of pBIB-KS.

SEQ ID NO: 167 is the determined amino acid sequence of the 9-mer epitope peptide Cap1#139-147 of serovariant L2.

SEQ ID NO: 168 is the determined amino acid sequence of the 9-mer epitope peptide Cap1#139-147 of serovariant D.

SEQ ID NO: 169 is the determined full-length DNA sequence of the Chlamydia trachomatis pmpI (CT874) gene.

SEQ ID NO: 170 is the determined full-length DNA sequence of the Chlamydia trachomatis pmpG gene.

SEQ ID NO: 171 is the determined full-length DNA sequence of the Chlamydia trachomatis pmpE gene.

SEQ ID NO: 172 is the determined full-length DNA sequence of the Chlamydia trachomatis pmpD gene.

SEQ ID NO: 173 is the determined full-length DNA sequence of the Chlamydia trachomatis pmpC gene.

SEQ ID NO: 174 is the determined full-length DNA sequence of the chlamydia trachomatis pmpB gene.

SEQ ID NO: 175 is the predicted full-length amino acid sequence of the Chlamydia trachomatis pmpI gene.

SEQ ID NO: 176 is the predicted full-length amino acid sequence of the Chlamydia trachomatis pmpG gene.

SEQ ID NO: 177 is the predicted full-length amino acid sequence of the chlamydia trachomatis pmpE gene.

SEQ ID NO: 178 is the predicted full-length amino acid sequence of the Chlamydia trachomatis pmpD gene.

SEQ ID NO: 179 is the predicted full-length amino acid sequence of the chlamydia trachomatis pmpC gene.

SEQ ID NO: 180 is the predicted full-length amino acid sequence of the Chlamydia trachomatis pmpB gene.

SEQ ID NO: 181 is the determined DNA sequence of the Chlamydia trachomatis pmpI gene minus the signal sequence.

SEQ ID NO: 182 is the full-length DNA sequence of the Chlamydia trachomatis pmpG gene, which was later determined.

SEQ ID NO: 183 is the determined DNA sequence of the Chlamydia trachomatis pmpE gene minus the signal sequence.

SEQ ID NO: 184 is the first determined DNA sequence of the chlamydia trachomatis pmpD gene, representing the carboxy terminus.

SEQ ID NO: 185 is the second DNA sequence determined for the Chlamydia trachomatis pmpD gene, representing the amino terminal minus signal sequence.

SEQ ID NO: 186 is the first DNA sequence determined for the Chlamydia trachomatis pmpC gene, representing the carboxy terminus.

SEQ ID NO: 187 is the second determined DNA sequence of the Chlamydia trachomatis pmpC gene, representing the amino-terminal minus signal sequence.

SEQ ID NO: 188 is the determined DNA sequence representing the Chlamydomonas pneumoniae serum variant MOMPS pmp gene in a fusion molecule with Ra 12.

SEQ ID NO: 189 is the predicted amino acid sequence minus the signal sequence of the chlamydia trachomatis pmpI gene.

SEQ ID NO: 190 is the later predicted amino acid sequence of the Chlamydia trachomatis pmpG gene.

SEQ ID NO: 191 is the predicted amino acid sequence of the Chlamydia trachomatis pmpE gene minus the signal sequence.

SEQ ID NO: 192 is the first predicted amino acid sequence of the Chlamydia trachomatis pmpD gene, representing the carboxy terminus.

SEQ ID NO: 193 is the second predicted amino acid sequence of the Chlamydia trachomatis pmpD gene, representing the amino terminal minus signal sequence.

SEQ ID NO: 194 is the first predicted amino acid sequence of the Chlamydia trachomatis pmpC gene, representing the carboxy terminus.

SEQ ID NO: 195 is the second predicted amino acid sequence of the Chlamydia trachomatis pmpC gene, representing the amino terminus.

SEQ ID NO: 196 is the predicted amino acid sequence representing the chlamydia pneumoniae serum variant MOMPS pmp gene in a fusion molecule with Ra 12.

SEQ ID NO: 197 is the determined DNA sequence of the 5' oligo primer used to clone the Chlamydia trachomatis pmpC gene in SKB vaccine vectors.

SEQ ID NO: 198 is the determined DNA sequence of the 3' oligo primer used to clone the Chlamydia trachomatis pmpC gene in SKB vaccine vectors.

SEQ ID NO: 199 is the determined DNA sequence used to clone the insertion sequence of the Chlamydia trachomatis pmpC gene in the SKB vaccine vector.

SEQ ID NO: 200 is the determined DNA sequence of the 5' oligo primer used to clone the Chlamydia trachomatis pmpD gene in SKB vaccine vectors.

SEQ ID NO: 201 is the determined DNA sequence of the 3' oligo primer used to clone the Chlamydia trachomatis pmpD gene in SKB vaccine vectors.

SEQ ID NO: 202 is a determined DNA sequence for cloning the insertion sequence of the Chlamydia trachomatis pmpD gene in an SKB vaccine vector.

SEQ ID NO: 203 is the determined DNA sequence of the 5' oligo primer used to clone the Chlamydia trachomatis pmpE gene in SKB vaccine vectors.

SEQ ID NO: 204 is the determined DNA sequence of the 3' oligo primer used to clone the pmpe gene of Chlamydia trachomatis in SKB vaccine vectors.

SEQ ID NO: 205 is the determined DNA sequence of the 5' oligo primer used to clone the pmpG gene of Chlamydia trachomatis in SKB vaccine vectors.

SEQ ID NO: 206 is the determined DNA sequence of the 3' oligo primer used to clone the Chlamydia trachomatis pmpG gene in SKB vaccine vectors.

SEQ ID NO: 207 is the determined DNA sequence of the 5' oligo primer used to clone the amino-terminal portion of the pmpC gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 208 is the determined DNA sequence of the 3' oligo primer used to clone the amino-terminal portion of the pmpC gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 209 is the determined DNA sequence of the 5' oligo primer used to clone the carboxy-terminal portion of the pmpC gene of C.trachomatis in the pET17b vector.

SEQ ID NO: 210 is the determined DNA sequence of the 3' oligo primer used to clone the carboxy-terminal portion of the pmpC gene of C.trachomatis in the pET17b vector.

SEQ ID NO: 211 is the determined DNA sequence of the 5' oligo primer used to clone the amino terminal portion of the pmpD gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 212 is the determined DNA sequence of the 3' oligo primer used for cloning the amino-terminal part of the pmpD gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 213 is the determined DNA sequence of the 5' oligo primer used to clone the carboxy-terminal portion of the pmpD gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 214 is the determined DNA sequence of the 3' oligo primer used to clone the carboxy-terminal portion of the pmpD gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 215 is the determined DNA sequence of the 5' oligo primer used to clone the pmpE gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 216 is the determined DNA sequence of the 3' oligo primer used to clone the pmpE gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 217 is a DNA sequence for determination of the insertion sequence of the pmpE gene of Chlamydia trachomatis cloned in the pET17b vector.

SEQ ID NO: 218 is the amino acid sequence used to clone the inserted sequence of the chlamydia trachomatis pmpE gene in the pET17b vector.

SEQ ID NO: 219 is the determined DNA sequence of the 5' oligo primer used to clone the Chlamydia trachomatis pmpG gene in the pET17b vector.

SEQ ID NO: 220 is the determined DNA sequence of the 3' oligo primer used to clone the Chlamydia trachomatis pmpG gene in the pET17b vector.

SEQ ID NO: 221 is the amino acid sequence used to clone the inserted sequence of the chlamydia trachomatis pmpG gene in the pET17b vector.

SEQ ID NO: 222 is the determined DNA sequence of the 5' oligo primer used for cloning the pmpI gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 223 is the determined DNA sequence of the 3' oligo primer used for cloning the pmpI gene of Chlamydia trachomatis in the pET17b vector.

SEQ ID NO: 224 is the determined amino acid sequence of C.pneumoniae Swib peptides 1-20.

SEQ ID NO: 225 is the determined amino acid sequence of C.pneumoniae Swib peptide 6-25.

SEQ ID NO: 226 is the determined amino acid sequence of C.pneumoniae Swib peptide 12-31.

SEQ ID NO: 227 is the determined amino acid sequence of C.pneumoniae Swib peptide 17-36.

SEQ ID NO: 228 is the determined amino acid sequence of C.pneumoniae Swib peptide 22-41.

SEQ ID NO: 229 is the determined amino acid sequence of the C.pneumoniae Swib peptides 27-46.

SEQ ID NO: 230 is the determined amino acid sequence of C.pneumoniae Swib peptide 42-61.

SEQ ID NO: 231 is the determined amino acid sequence of C.pneumoniae Swib peptide 46-65.

SEQ ID NO: 232 is the determined amino acid sequence of C.pneumoniae Swib peptide 51-70.

SEQ ID NO: 233 is a determined amino acid sequence of C.pneumoniae Swib peptide 56-75.

SEQ ID NO: 234 is the determined amino acid sequence of C.pneumoniae Swib peptide 61-80.

SEQ ID NO: 235 is the determined amino acid sequence of C.pneumoniae Swib peptide 66-87.

SEQ ID NO: 236 is the determined amino acid sequence of C.trachomatis OMCB peptide 103-122.

SEQ ID NO: 237 is the determined amino acid sequence of Chlamydia trachomatis OMCB peptide 108-127.

SEQ ID NO: 238 is the determined amino acid sequence of C.trachomatis OMCB peptide 113-132.

SEQ ID NO: 239 is the determined amino acid sequence of C.trachomatis OMCB peptide 118-137.

SEQ ID NO: 240 is the determined amino acid sequence of the C.trachomatis OMCB peptide 123-143.

SEQ ID NO: 241 is the determined amino acid sequence of the C.trachomatis OMCB peptide 128-147.

SEQ ID NO: 242 is the determined amino acid sequence of the C.trachomatis OMCB peptide 133-152.

SEQ ID NO: 243 is the determined amino acid sequence of the C.trachomatis OMCB peptide 137-156.

SEQ ID NO: 244 is the determined amino acid sequence of C.trachomatis OMCB peptide 142-161.

SEQ ID NO: 245 is the determined amino acid sequence of the C.trachomatis OMCB peptide 147-166.

SEQ ID NO: 246 is the determined amino acid sequence of the C.trachomatis OMCB peptide 152-171.

SEQ ID NO: 247 is the determined amino acid sequence of the C.trachomatis OMCB peptide 157-176.

SEQ ID NO: 248 is the determined amino acid sequence of OMCB peptide 162-181 of Chlamydia trachomatis.

SEQ ID NO: 249 is the determined amino acid sequence of the C.trachomatis OMCB peptide 167-186.

SEQ ID NO: 250 is the determined amino acid sequence of the C.trachomatis OMCB peptide 171-190.

SEQ ID NO: 251 is the determined amino acid sequence of the C.trachomatis OMCB peptide 171-186.

SEQ ID NO: 252 is the determined amino acid sequence of C.trachomatis OMCB peptide 175-186.

SEQ ID NO: 252 is the determined amino acid sequence of C.trachomatis OMCB peptide 175-186.

SEQ ID NO: 253 is the determined amino acid sequence of C.pneumoniae OMCB peptide 185-198.

SEQ ID NO: 254 is the determined amino acid sequence of the Chlamydia trachomatis TSA peptide 96-115.

SEQ ID NO: 255 is the determined amino acid sequence of the C.trachomatis TSA peptide 101-120.

SEQ ID NO: 256 is the determined amino acid sequence of the C.trachomatis TSA peptide 106-125.

SEQ ID NO: 257 is the determined amino acid sequence of the C.trachomatis TSA peptide 111-130.

SEQ ID NO: 258 is the determined amino acid sequence of the C.trachomatis TSA peptide 116-135.

SEQ ID NO: 259 is the determined amino acid sequence of the C.trachomatis TSA peptide 121-140.

SEQ ID NO: 260 is the determined amino acid sequence of the C.trachomatis TSA peptide 126-145.

SEQ ID NO: 261 is the determined amino acid sequence of the C.trachomatis TSA peptide 131-150.

SEQ ID NO: 262 is the determined amino acid sequence of the C.trachomatis TSA peptide 136-155.

SEQ ID NO: 263 is the full-length DNA sequence of the assay for Chlamydia trachomatis CT529/Cap 1 gene serovariant I.

SEQ ID NO: 264 is the predicted full-length amino acid sequence of Chlamydia trachomatis CT529/Cap 1 gene serovar I.

SEQ ID NO: 265 is the determined full-length DNA sequence of the serovariant K of the CT529/Cap 1 gene of Chlamydia trachomatis.

SEQ ID NO: 266 is the predicted full-length amino acid sequence of Chlamydia trachomatis CT529/Cap 1 gene serovariant K.

SEQ ID NO: 267 is a determined DNA sequence of C.trachomatis clone 17-G4-36, having homology to a portion of the DNA-directed RNA polymerase beta subunit ORF (CT 315 in serD).

SEQ ID NO: 268 is the determined DNA sequence of the partial sequence of the chlamydia trachomatis CTO16 gene of clone 2E 10.

SEQ ID NO: 269 is a determined DNA sequence of a partial sequence of the Chlamydia trachomatis tRNA synthetase gene from clone 2E 10.

SEQ ID NO: 270 is the determined DNA sequence of the partial sequence of the c.trachomatis clpX gene in clone 2E 10.

SEQ ID NO: 271 is the first DNA sequence determined from Chlamydia trachomatis clone CtL2gam-30, representing the 5' end.

SEQ ID NO: 272 is the second DNA sequence determined from Chlamydia trachomatis clone CtL2gam-30, representing the 3' end.

SEQ ID NO: 273 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-28.

SEQ ID NO: 274 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-27.

SEQ ID NO: 275 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-26.

SEQ ID NO: 276 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-24.

SEQ ID NO: 277 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-23.

SEQ ID NO: 278 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-21.

SEQ ID NO: 279 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-18.

SEQ ID NO: 280 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-17.

SEQ ID NO: 281 is the first DNA sequence determined for Chlamydia trachomatis clone CtL2gam-15, representing the 5' end.

SEQ ID NO: 282 is the second determined DNA sequence of Chlamydia trachomatis clone CtL2gam-15, representing the 3' end.

SEQ ID NO: 283 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-13.

SEQ ID NO: 284 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-10.

SEQ ID NO: 285 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-8.

SEQ ID NO: 286 is the first DNA sequence determined for Chlamydia trachomatis clone CtL2gam-6, representing the 5' end.

SEQ ID NO: 287 is the second determined DNA sequence of Chlamydia trachomatis clone CtL2gam-6, representing the 3' end.

SEQ ID NO: 288 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-5.

SEQ ID NO: 289 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-2.

SEQ ID NO: 290 is the determined DNA sequence of Chlamydia trachomatis clone CtL2 gam-1.

SEQ ID NO: 291 is the determined full-length DNA sequence of the C.pneumoniae homolog (homologue) of the CT529 gene.

SEQ ID NO: 292 is the predicted full-length amino acid sequence of the chlamydia pneumoniae homolog of the CT529 gene.

SEQ ID NO: 293 is a DNA sequence for determination of the insertion sequence for cloning of the Chlamydia trachomatis pmpG gene in SKB vaccine vectors.

SEQ ID NO: 294 is the amino acid sequence of the open reading frame of clone CT 603.

SEQ ID NO: 295 is the amino acid sequence of the first open reading frame of clone CT 875.

SEQ ID NO: 296 is the amino acid sequence of the second open reading frame of clone CT 875.

SEQ ID NO: 297 is the amino acid sequence of the first open reading frame of clone CT 858.

SEQ ID NO: 298 is the amino acid sequence of the second open reading frame of clone CT 858.

SEQ ID NO: 299 is the amino acid sequence of the open reading frame of clone CT 622.

SEQ ID NO: 300 is the amino acid sequence of the open reading frame of clone CT 610.

SEQ ID NO: 301 is the amino acid sequence of the open reading frame of clone CT 396.

SEQ ID NO: 302 is the amino acid sequence of the open reading frame of clone CT 318.

SEQ ID NO: 304 is the amino acid sequence of Chlamydia trachomatis serum variant L2 rCt529c1-125, with a modified N-terminal sequence (6-His tag).

SEQ ID NO: 305 is the amino acid sequence of chlamydia trachomatis serovar L2 rCt529c 1-125.

SEQ ID NO: 306 is the sense primer used in the synthesis of the PmpA (N-term) fusion protein.

SEQ ID NO: 307 is an antisense primer used in the synthesis of the PmpA (N-term) fusion protein.

SEQ ID NO: 308 is a DNA sequence encoding the PmpA (N-term) fusion protein.

SEQ ID NO: 309 is the amino acid sequence of the PmpA (N-term) fusion protein.

SEQ ID NO: 310 is a sense primer used in the synthesis of the PmpA (C-term) fusion protein.

SEQ ID NO: 311 is an antisense primer used in the synthesis of the PmpA (C-term) fusion protein.

SEQ ID NO: 312 is a DNA sequence encoding the PmpA (C-term) fusion protein.

SEQ ID NO: 313 is the amino acid sequence of the PmpA (C-term) fusion protein.

SEQ ID NO: 314 is the sense primer used in the synthesis of the PmpF (N-term) fusion protein.

SEQ ID NO: 315 is the antisense primer used in the synthesis of the PmpF (N-term) fusion protein.

SEQ ID NO: 316 is a DNA sequence encoding a PmpF (N-term) fusion protein.

SEQ ID NO: 317 is the amino acid sequence of the PmpF (N-term) fusion protein.

SEQ ID NO: 318 is the sense primer used in the synthesis of the PmpF (C-term) fusion protein.

SEQ ID NO: 319 is an antisense primer used in the synthesis of the PmpF (C-term) fusion protein.

SEQ ID NO: 320 is a DNA sequence encoding a PmpF (C-term) fusion protein.

S EQID NO: 321 is the amino acid sequence of the PmpF (C-term) fusion protein.

SEQ ID NO: 322 is the sense primer used in the synthesis of the PmpH (CT412) (N-term) fusion protein.

SEQ ID NO: 323 is the antisense primer used in the synthesis of the PmpH (N-term) fusion protein.

SEQ ID NO: 324 is a DNA sequence encoding a PmpH (N-term) fusion protein.

SEQ ID NO: 325 is the amino acid sequence of the PmpH (N-term) fusion protein.

SEQ ID NO: 326 is the sense primer used in the synthesis of the PmpH (C-term) fusion protein.

SEQ ID NO: 327 is the antisense primer used in the synthesis of the PmpH (C-term) fusion protein.

SEQ ID NO: 328 is a DNA sequence encoding the PmpH (C-term) fusion protein.

SEQ ID NO: 329 is the amino acid sequence of the PmpH (C-term) fusion protein.

SEQ ID NO: 330 is a sense primer used in the synthesis of the PmpB (1) fusion protein.

SEQ ID NO: 331 is an antisense primer used for the synthesis of the PmpB (1) fusion protein.

SEQ ID NO: 332 is a DNA sequence encoding the PmpB (1) fusion protein.

SEQ ID NO: 333 is the amino acid sequence of the PmpB (1) fusion protein.

SEQ ID NO: 334 is the sense primer used in the synthesis of the PmpB (2) fusion protein.

SEQ ID NO: 335 is an antisense primer used for the synthesis of the PmpB (2) fusion protein.

SEQ ID NO: 336 is a DNA sequence encoding the PmpB (2) fusion protein.

SEQ ID NO: 337 is the amino acid sequence of the PmpB (2) fusion protein.

SEQ ID NO: 338 is a sense primer used in the synthesis of the PmpB (3) fusion protein.

SEQ ID NO: 339 is an antisense primer used in the synthesis of the PmpB (3) fusion protein.

SEQ ID NO: 340 is a DNA sequence encoding a PmpB (3) fusion protein.

SEQ ID NO: 341 is the amino acid sequence of the PmpB (3) fusion protein.

SEQ ID NO: 342 is a sense primer used in the synthesis of the PmpB (4) fusion protein.

SEQ ID NO: 343 is an antisense primer used for the synthesis of the PmpB (4) fusion protein.

SEQ ID NO: 344 is a DNA sequence encoding the PmpB (4) fusion protein.

SEQ ID NO: 345 is the amino acid sequence of the PmpB (4) fusion protein.

SEQ ID NO: 346 is the sense primer used in the synthesis of the PmpC (1) fusion protein.

SEQ ID NO: 347 is an antisense primer used for the synthesis of the PmpC (1) fusion protein.

SEQ ID NO: 348 is a DNA sequence encoding the PmpC (1) fusion protein.

SEQ ID NO: 349 is the amino acid sequence of the PmpC (1) fusion protein.

SEQ ID NO: 350 is the sense primer used in the synthesis of the PmpC (2) fusion protein.

SEQ ID NO: 351 is an antisense primer used for the synthesis of the PmpC (2) fusion protein.

SEQ ID NO: 352 is the DNA sequence encoding the PmpC (2) fusion protein.

SEQ ID NO: 353 is the amino acid sequence of the PmpC (2) fusion protein.

SEQ ID NO: 354 is the sense primer used in the synthesis of the PmpC (3) fusion protein.

SEQ ID NO: 355 is an antisense primer used for the synthesis of the PmpC (3) fusion protein.

SEQ ID NO: 356 is the DNA sequence encoding the PmpC (3) fusion protein.

SEQ ID NO: 357 is the amino acid sequence of the PmpC (3) fusion protein.

SEQ ID NO: 358 is the DNA sequence of oppA1 protein, lacking the first transmembrane domain.

SEQ ID NO: 359 is the full-length DNA sequence of CT 139.

SEQ ID NO: 360 is the full-length DNA sequence of ORF-3.

SEQ ID NO: 361 is the full-length DNA sequence of CT 611.

SEQ ID NO: 362 is the amino acid sequence of oppA1, starting with amino acid position 22.

SEQ ID NO: 363 is the amino acid sequence of CT 139.

SEQ ID NO: 364 is the amino acid sequence of ORF-3.

SEQ ID NO: 365 is the amino acid sequence of CT 611.

SEQ ID NO: 366 shows the DNA sequence of the Chlamydia pneumoniae homolog CPn0275 of the Chlamydia trachomatis gene CT 190.

SEQ ID NO: 367 shows the DNA sequence of Chlamydia pneumoniae homolog CPn0407 of Chlamydia trachomatis gene CT 103.

SEQ ID NO: 368 shows the DNA sequence of chlamydia pneumoniae homolog CPn0720 of chlamydia trachomatis gene CT 659.

SEQ ID NO: 369 shows the DNA sequence of Chlamydia pneumoniae homolog CPn0716 of the Chlamydia trachomatis gene CT 660.

SEQ ID NO: 370 shows the DNA sequence of Chlamydia pneumoniae homolog CPn0519 of the Chlamydia trachomatis gene CT 430.

SEQ ID NO: 371 shows the DNA sequence of chlamydia pneumoniae homolog CPn0520 of chlamydia trachomatis gene CT 431.

SEQ ID NO: 372 shows the DNA sequence of the C.pneumoniae homolog CPn0078 of the C.trachomatis gene CT 318.

SEQ ID NO: 373 shows the DNA sequence of the C.pneumoniae homolog CPn0628 of the C.trachomatis gene CT 509.

SEQ ID NO: 374 shows the DNA sequence of the C.pneumoniae homolog CPn0540 of the C.trachomatis gene CT 414.

SEQ ID NO: 375 shows the DNA sequence of the Chlamydia pneumoniae homolog pmp20 of the Chlamydia trachomatis gene CT 413.

SEQ ID NO: 376 shows the DNA sequence of the chlamydia pneumoniae homolog CPn0081 of the chlamydia trachomatis gene CT 315.

SEQ ID NO: 377 shows the DNA sequence of the Chlamydia pneumoniae homolog CPn0761 of the Chlamydia trachomatis gene CT 610.

SEQ ID NO: 378 shows the DNA sequence of the C.pneumoniae homolog CPn0557 of the C.trachomatis gene CT 443.

SEQ ID NO: 379 shows the DNA sequence of the C.pneumoniae homolog CPn0833 of C.pneumoniae gene CT 557.

SEQ ID NO: 380 shows the DNA sequence of the Chlamydia pneumoniae homolog CPn0134 of the Chlamydia trachomatis gene CT 604.

SEQ ID NO: 381 shows the DNA sequence of the Chlamydia pneumoniae homolog CPn0388 of the Chlamydia trachomatis gene CT 042.

SEQ ID NO: 382 shows the DNA sequence of the C.pneumoniae homolog CPn1028 of the C.trachomatis gene CT 376.

SEQ ID NO: 383 shows the DNA sequence of the Chlamydia pneumoniae homolog CPn0875 of the Chlamydia trachomatis gene CT 734.

SEQ ID NO: 384 shows the DNA sequence of the C.pneumoniae homolog CPn0908 of the C.trachomatis gene CT 764.

SEQ ID NO: 385 shows the DNA sequence of the chlamydia pneumoniae homolog CPn0728 of the chlamydia trachomatis gene CT 622.

SEQ ID NO: 386 shows the amino acid sequence of the C.pneumoniae homolog CPn0275 of the C.trachomatis gene CT 190.

SEQ ID NO: 387 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0407 of chlamydia trachomatis gene CT 103.

SEQ ID NO: 388 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0720 of chlamydia trachomatis gene CT 659.

SEQ ID NO: 389 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0716 of the chlamydia trachomatis gene CT 660.

SEQ ID NO: 390 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0519 of the chlamydia trachomatis gene CT 430.

SEQ ID NO: 391 shows the amino acid sequence of Chlamydia pneumoniae homolog CPn0520 of Chlamydia trachomatis gene CT 431.

SEQ ID NO: 392 shows the amino acid sequence of the C.pneumoniae homolog CPn0078 of the C.trachomatis gene CT 318.

SEQ ID NO: the amino acid sequence of the chlamydia pneumoniae homolog CPn0628 of the chlamydia trachomatis gene CT509 is shown at 393.

SEQ ID NO: 394 shows the amino acid sequence of the C.pneumoniae homolog CPn0540 of the C.trachomatis gene CT 414.

SEQ ID NO: 395, the amino acid sequence of the chlamydia pneumoniae homolog pmp20 of the chlamydia trachomatis gene CT413 is shown.

SEQ ID NO: 396 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0081 of the chlamydia trachomatis gene CT 315.

SEQ ID NO: 397 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0761 of the chlamydia trachomatis gene CT 610.

SEQ ID NO: 398 shows the amino acid sequence of the C.pneumoniae homolog CPn0557 of Chlamydia trachomatis gene CT 443.

SEQ ID NO: 399 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0833 of the chlamydia trachomatis gene CT 557.

SEQ ID NO: 400 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0134 of the chlamydia trachomatis gene CT 604.

SEQ ID NO: the amino acid sequence of the chlamydia pneumoniae homolog CPn0388 of the chlamydia trachomatis gene CT042 is shown at 401.

SEQ ID NO: 402 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn1028 of the chlamydia trachomatis gene CT 376.

SEQ ID NO: 403 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0875 of the chlamydia trachomatis gene CT 734.

SEQ ID NO: 404 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0908 of the chlamydia trachomatis gene CT 764.

SEQ ID NO: 405 shows the amino acid sequence of the chlamydia pneumoniae homolog CPn0728 of the chlamydia trachomatis gene CT 622.

SEQ ID NO: 406 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 287.

SEQ ID NO: 407 shows the full length serovariant DDNA sequence of the Chlamydia trachomatis gene CT 858.

SEQ ID NO: 408 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 764.

SEQ ID NO: the full-length serovariant DDNA sequence of the C.trachomatis gene CT734 is shown at 409.

SEQ ID NO: 410 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 660.

SEQ ID NO: the full-length serovariant DDNA sequence of the C.trachomatis gene CT659 is shown at 411.

SEQ ID NO: the full-length serovariant DDNA sequence of the C.trachomatis gene CT622 is shown at 412.

SEQ ID NO: 413 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 610.

SEQ ID NO: 414 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 604.

SEQ ID NO: 415 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 557.

SEQ ID NO: 416 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 509.

SEQ ID NO: 417 shows the full-length serovariant DDNA sequence of the C.trachomatis gene CT 443.

SEQ ID NO: 418 shows the full-length serovariant DDNA sequence of the Chlamydia trachomatis gene CT 431.

SEQ ID NO: 419 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 430.

SEQ ID NO: 420 shows the full length serovariant DDNA sequence of the chlamydia trachomatis gene CT 414.

SEQ ID NO: 421 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 413.

SEQ ID NO: 422 shows the full length serovariant DDNA sequence of the Chlamydia trachomatis gene CT 396.

SEQ ID NO: 423 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 376.

SEQ ID NO: 424 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 318.

SEQ ID NO: 425 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 315.

SEQ ID NO: 426 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 104.

SEQ ID NO: 427 shows the full-length serovariant DDNA sequence of the Chlamydia trachomatis gene CT 103.

SEQ ID NO: 428 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 102.

SEQ ID NO: 429 shows the full-length serovariant DDNA sequence of the Chlamydia trachomatis gene CT 098.

SEQ ID NO: 430 shows the full-length serovariant DDNA sequence of the chlamydia trachomatis gene CT 042.

SEQ ID NO: 431 shows the full length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 858.

SEQ ID NO: 432 shows the full length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 764.

SEQ ID NO: 433 shows the full-length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 734.

SEQ ID NO: 434 shows the full-length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 660.

SEQ ID NO: 435 shows the full length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 659.

SEQ ID NO: 436 shows the full-length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 622.

SEQ ID NO: 437 shows the full-length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 610.

SEQ ID NO: 438 shows the full-length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 604.

SEQ ID NO: 439 shows the full-length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 557.

SEQ ID NO: 440 shows the full-length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 509.

SEQ ID NO: 441 shows the full-length serovariant D amino acid sequence of the C.trachomatis gene CT 443.

SEQ ID NO: 442 shows the full-length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 431.

SEQ ID NO: 443 shows the full-length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 430.

SEQ ID NO: 444 shows the full length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 414.

SEQ ID NO: 445 shows the full length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 413.

SEQ ID NO: 446 shows the full length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 396.

SEQ ID NO: 447 shows the full length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 376.

SEQ ID NO: 448 shows the full-length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 318.

SEQ ID NO: 449 shows the full-length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 315.

SEQ ID NO: 450 shows the full-length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 104.

SEQ ID NO: 451 shows the full length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 103.

SEQ ID NO: 452 shows the full length serovariant D amino acid sequence of the chlamydia trachomatis gene CT 102.

SEQ ID NO: 453 shows the full-length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 098.

SEQ ID NO: 454 shows the full length serovariant D amino acid sequence of the Chlamydia trachomatis gene CT 042.

SEQ ID NO: 455 corresponds to the DNA sequence of CPn0894, which is a CP homolog of CT751 (ann) identified in clone CTL2-1, and CTL 2-5.

SEQ ID NO: 456 corresponds to the DNA sequence of CPn0074, which is the CP homologue of CT322(tuf) identified in clone CTL 2-2.

SEQ ID NO: 457 corresponds to the DNA sequence of CPn0122, which is a CP homologue of CT032(metG) identified in clones CTL2gam2, CTL2-3 (5') and CTL 2-4.

SEQ ID NO: 458 corresponds to the DNA sequence of CPn0121, which is a CP homolog of CT031 identified in clone CTL2-3(5 ') (3').

SEQ ID NO: 459 correspond to the DNA sequence of CPn0120, which is a CP homologue of CT030(gmK) identified in clones CTL2-3 (3') and CTL 2-21.

SEQ ID NO: 460 corresponds to the DNA sequence of CPn0359, which is the CP homologue of CT064(lepA) identified in clone CTL2gam 5.

SEQ ID NO: 461 corresponds to the DNA sequence of CPn0414, which is the CP homolog of CT265(accA) identified in clone CTL 2-6.

SEQ ID NO: 462 corresponds to the DNA sequence of CPn0413, which is the CP homolog of CT264(msbA) identified in clone CTL 2-6.

SEQ ID NO: 463 corresponds to the DNA sequence of CPn0394, which is the CP homologue of CT256 identified in clones CTL2gam6(5 ') and CTL2-11 (5').

SEQ ID NO: 464 corresponds to the DNA sequence of CPn0395, which is a CP homologue of CT257 identified in clones CTL2gam6(5 ') and CTL2-11 (5').

SEQ ID NO: 465 corresponding to the DNA sequence of CPn0487, which is a CP homologue of CT384 identified in clones CTL2gam6(3 ') and CTL2-11 (3').

SEQ ID NO: 466 corresponds to the DNA sequence of CPn0592, which is a CP homolog of CT473 identified in clone CTL2-8 b.

SEQ ID NO: 467 corresponds to the DNA sequence of CPn0593, which is the CP homologue of CT474 identified in clone CTL2-8 b.

SEQ ID NO: 468 corresponds to the DNA sequence of CPn0197, which is the CP homologue of CT139(oppA1) identified in clone CTL2-8 b.

SEQ ID NO: 469 corresponds to the DNA sequence of CPn0363, which is the CP homolog of CT060(flhA) identified in clone CTL2-8 b.

SEQ ID NO: 470 corresponds to the DNA sequence of CPn0301, which is a CP homologue of CT242 identified in clone CTL2gam 8.

SEQ ID NO: 471 corresponds to the DNA sequence of CPn0302, which is the CP homologue of CT243(lpxD) identified in clone CTL2gam 8.

SEQ ID NO: 472 corresponding to the DNA sequence of CPn0324, which is a CP homologue of CT089(lcrE) identified in clones CTL2-9, CTL2gam1, CTL2gam17 and CTL2-19 (5').

SEQ ID NO: 473 corresponds to the DNA sequence of CPn0761, which is the CP homologue of CT610 identified in clone CTL2-10(5 ') (3').

SEQ ID NO: 474 corresponds to the DNA sequence of CPn0760, which is the CP homologue of CT611 identified in clone CTL2-10 (5').

SEQ ID NO: 475 corresponds to the DNA sequence of CPn0329, which is a CP homologue of CT154 identified in clones CTL2gam10 and CTL2gam 21.

SEQ ID NO: 476 corresponds to the DNA sequence of CPn0990, which is the CP homologue of CT833(infC) identified in clone CTL 2-12.

SEQ ID NO: 477 corresponds to the DNA sequence of CPn0984, which is the CP homologue of CT827(nrdA) identified in clones CTL2-16(3 ') and CTL2gam15 (3').

SEQ ID NO: 478 corresponded to the DNA sequence of CPn0985, which is the CP homolog of CT828(nrdB) identified in clone CTL2-16(3 ') CTL2gam15 (3').

SEQ ID NO: 479 corresponds to the DNA sequence of CPn0349, which is the CP homologue of CT067(ytgA) identified in clone CTL2gam 18.

SEQ ID NO: 480 DNA sequence corresponding to CPn0325, which is the CP homolog of CT088(sycE) identified in clone CTL2-19 (5').

SEQ ID NO: 481 corresponds to the DNA sequence of CPn0326, which is the CP homologue of CT087(malQ) identified in clone CTL2-19 (5').

SEQ ID NO: 482 corresponds to the DNA sequence of CPn0793, which is a CP homologue of CT588(rbsu) identified in clone CTL2gam 23.

SEQ ID NO: 483 corresponds to the DNA sequence of CPn0199, which is the CP homologue of CT199(oppB1) identified in clone CTL2gam 24.

SEQ ID NO: 484 to CPn0666, which is a CP homologue of CT545(dnaE) identified in clone CTL 2-24.

SEQ ID NO: 485 corresponds to the DNA sequence of CPn0065, which is a CP homologue of CT288 identified in clone CTL2gam 27.

SEQ ID NO: 486 corresponds to the DNA sequence of CPn0444, which is the CP homologue of CT413(pmpB) identified in clone CTL2gam30(5 ') (3').

SEQ ID NO: 487 DNA sequence corresponding to CPn-ORF5, which is a CP homologue of CT-ORF3 identified in clones CTL2gam15(5 '), CTL2-16(5 '), CTL2-18(5 '), and CTL 2-23.

SEQ ID NO: 488 corresponds to the DNA sequence of CPn-ORF6, which is the CP homologue of CT-ORF4 identified in clone CTL2-18 (3').

SEQ ID NO: 489 DNA sequence corresponding to CP-ORF7, which is the CP homologue of CT-ORF5 identified in clone CTL2-18 (3').

SEQ ID NO: 490 corresponds to the amino acid sequence of CPn0894, which is a CP homolog of CT751 (ann) identified in clones CTL2-1 and CTL 2-5.

SEQ ID NO: 491 corresponds to the amino acid sequence of CPn0074, which is the CP homologue of CT332(tuf) identified in clone CTL 2-2.

SEQ ID NO: 492, corresponds to the amino acid sequence of CPn0122, which is the CP homolog of CT032(metG) identified in clones CTL2gam2, CTL2-3 (5'), and CTL 2-4.

SEQ ID NO: 493 corresponds to the amino acid sequence of CPn0121, which is a CP homolog of CT031 identified in clone CTL2-3(5 ') (3').

SEQ ID NO: 494 corresponds to the amino acid sequence of CPn0120, which is a CP homolog of CT030(gmK) identified in clones CTL2-3 (3') and CTL 2-21.

SEQ ID NO: 495 corresponds to the amino acid sequence of CPn0359, which is the CT064(lepA) CP homolog identified in clone CTL2gam 5.

SEQ ID NO: 496 corresponds to the amino acid sequence of CPn0414, which is the CP homolog of CT265(accA) identified in clone CTL 2-6.

SEQ ID NO: 497 corresponds to the amino acid sequence CPn0413, which is a CP homolog of CT264(msbA) identified in clone CTL 2-6.

SEQ ID NO: 498 corresponds to the amino acid sequence of CPn0394, which is a CP homologue of CT256 identified in clones CTL2gam6(5 ') and CTL2-11 (5').

SEQ ID NO: 499 corresponds to the amino acid sequence of CPn0395, which is a CP homologue of CT257 identified in clones CTL2gam6(5 ') and CTL2-11 (5').

SEQ ID NO: 500 corresponds to the amino acid sequence of CPn0487, which is the CP homologue of CT384 identified in clone CTL2gam6(3 ') and CTL2-11 (3').

SEQ ID NO: 501 corresponds to the amino acid sequence of CPn0592, which is the CP homologue of CT473 identified in clone CTL2-8 b.

SEQ ID NO: 502 corresponds to the amino acid sequence of CPn0593, which is the CP homologue of CT474 identified in clone CTL2-8 b.

SEQ ID NO: 503 corresponds to the amino acid sequence of CPn0197, which is the CP homologue of CT139(oppA1) identified in clone CTL2-8 b.

SEQ ID NO: 504 corresponds to the amino acid sequence of CPn0363, which is the CP homolog of CT060(flhA) identified in clone CTL2-8 b.

SEQ ID NO: 505 corresponds to the amino acid sequence of CPn0301, which is the CP homologue of CT242 identified in clone CTL2gam 8.

SEQ ID NO: 506 corresponds to the amino acid sequence of CPn0302, which is the CP homologue of CT243(lpxD) identified in clone CTL2gam 8.

SEQ ID NO: 507 corresponds to the amino acid sequence of CPn0324, which is a CP homologue of CT089(lcrE) identified in clones CTL2-9, CTL2gam1, CTL2gam17 and CTL2-19 (5').

SEQ ID NO: 508 corresponds to the amino acid sequence of CPn0761, which is the CP homologue of CT610 identified in clone CTL2-10(5 ') (3').

SEQ ID NO: 509 corresponds to the amino acid sequence CPn0760, which is the CP homologue of CT611 identified in clone CTL2-10 (5').

SEQ ID NO: 510 corresponds to the amino acid sequence CPn0329, which is a CP homologue of CT154 identified in clones CTL2gam10 and CTL2gam 21.

SEQ ID NO: 511 corresponds to the amino acid sequence of CPn0990, which is the CP homologue of CT833(infC) identified in clone CTL 2-12.

SEQ ID NO: 512 corresponding to the amino acid sequence of CPn-ORF5, which is a CP homologue of CT ORF3 identified in clones CTL2gam15(5 '), CTL2-16(5 '), CTL2-18(5 '), and CTL 2-23.

SEQ ID NO: 513 corresponds to the amino acid sequence CPn0984, which is a CP homologue of CT827(nrdA) identified in clones CTL2-16(3 ') and CTL2gam15 (3').

SEQ ID NO: 514 corresponds to the amino acid sequence of CPn0985, which is the CP homologue of CT828(nrdB) identified in clone CTL2-16(3 ') CTL2gam15 (3').

SEQ ID NO: 515 corresponds to the amino acid sequence of CPn0349, which is the CP homologue of CT067(ytgA) identified in clone CTL2gam 18.

SEQ ID NO: 516 corresponds to the DNA sequence of CPn-ORF6, which is the CP homologue of CT-ORF4 identified in clone CTL2-18 (3').

SEQ ID NO: 517 DNA sequence corresponding to CP-ORF7, which is a CP homologue of CT-ORF5 identified in clone CTL2-18 (3').

SEQ ID NO: 518 corresponds to the amino acid sequence of CPn0326, which is the CP homolog of CT087(malQ) identified in clone CTL2-19 (5').

SEQ ID NO: 519 corresponds to the amino acid sequence of CPn0325, which is the CP homologue of CT088(sycE) identified in clone CTL2-19 (5').

SEQ ID NO: 520 corresponds to the amino acid sequence of CPn0793, which is a CP homologue of CT588(rbsu) identified in clone CTL2gam 23.

SEQ ID NO: 521 corresponds to the amino acid sequence of CPn0199, which is the CP homologue of CT199(oppB1) identified in clone CTL2gam 24.

SEQ ID NO: 522 corresponds to the amino acid sequence of CPn0666, which is the CP homologue of CT545(dnaE) identified in clone CTL 2-24.

SEQ ID NO: 523 corresponds to the DNA sequence of CPn0065, which is a CP homologue of CT288 identified in clone CTL2gam 27.

SEQ ID NO: 524 corresponds to the DNA sequence of CPn0444, which is the CP homologue of CT413(pmpB) identified in clone CTL2gam30(5 ') (3').

SEQ ID NO: 525 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the Chlamydia trachomatis LGV II sequence CT751(amn) identified from clones CTL2-1 and CTL 2-5.

SEQ ID NO: 526 shows the full length C.trachomatis serum variant D DNA sequence homologous to the C.trachomatis LGV II sequence CT322(tuff) identified from clone CTL 2-2.

SEQ ID NO: 527 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the Chlamydia trachomatis LGV II sequence CT032(metG) identified from clones CTL2gam2, CTL2-3 (5') and CTL 2-4.

SEQ ID NO: 528 shows a full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT031 identified from clone CTL2-3(5 ') (3').

SEQ ID NO: 529 shows a full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT030(gmK) identified from clones CTL2-3 (3') and CTL 2-21.

SEQ ID NO: 530 shows the full length chlamydia trachomatis serum variant DDNA sequence homologous to the chlamydia trachomatis LGV II sequence CT064(lepA) identified from clone CTL2gam 5.

SEQ ID NO: 531 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT265(accA) identified from clone CTL 2-6.

SEQ ID NO: 532 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT624(msbA) identified from clone CTL 2-6.

SEQ ID NO: 533 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the Chlamydia trachomatis LGV II sequence CT256 identified from clones CTL2gam6(5 ') and CTL2-11 (5').

SEQ ID NO: 534 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to CT257 from the sequence of Chlamydia trachomatis LGV II identified in clones CTL2gam6(5 ') and CTL2-11 (5').

SEQ ID NO: 535 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the C.trachomatis LGV II sequence CT384 identified from clones CTL2gam6(3 ') and CTL2-11 (3').

SEQ ID NO: 536 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT473 identified from clone CTL2-8 b.

SEQ ID NO: 537 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT474 identified from clone CTL2-8 b.

SEQ ID NO: 538 shows the full length C.trachomatis serum variant D DNA sequence homologous to the C.trachomatis LGV II sequence CT139(oppA1) identified from clone CTL2-8 b.

SEQ ID NO: 539 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the Chlamydia trachomatis LGV II sequence CT060(flhA) identified from clone CTL2-8 b.

SEQ ID NO: 540 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT242 identified from clone CTL2gam 8.

SEQ ID NO: 541 shows a full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT243(lpxD) identified from clone CTL2gam 8.

SEQ ID NO: 542 shows the full length chlamydia trachomatis serovar D DNA sequence homologous to the C.trachomatis LGV II sequence CT089 identified from clones CTL2-9, CTL2gam1, CTL2gam17, and CTL2-19 (5').

SEQ ID NO: 543 shows the full length chlamydia trachomatis serum variant DDNA sequence homologous to the chlamydia trachomatis LGV II sequence CT610 identified from clone CTL2-10(5 ') (3').

SEQ ID NO: 544 shows the full length C.trachomatis serum variant D DNA sequence homologous to the C.trachomatis LGV II sequence CT611 identified from clone CTL2-10 (5').

SEQ ID NO: 545 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT154 identified from clones CTL2gam10 and CTL2gam 21.

SEQ ID NO: 546 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the C.trachomatis LGV II sequence CT833(infC) identified from clone CTL 2-12.

SEQ ID NO: 547 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the Chlamydia trachomatis LGV II sequence CT827(nrdA) identified from clones CTL2-16(3 ') and CTL2gam15 (3').

SEQ ID NO: 548 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the sequence CT828(nrdB) of the LGV II sequence of Chlamydia trachomatis identified from clones CTL2-16(3 ') and CTL2gam15 (3').

SEQ ID NO: 549 shows the full-length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT067(ytgA) identified from clone CTL2gam 18.

SEQ ID NO: 550 shows the full length chlamydia trachomatis serum variant DDNA sequence homologous to the chlamydia trachomatis LGV II sequence CT088(sycE) identified from clone CTL2-19 (5').

SEQ ID NO: 551 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT087 identified from clone CTL2-19 (5').

SEQ ID NO: 552 shows the full length chlamydia trachomatis serum variant DDNA sequence homologous to the chlamydia trachomatis LGV II sequence CT588(rsbu) identified from clone CTL2gam 23.

SEQ ID NO: 553 shows a full length Chlamydia trachomatis serum variant DDNA sequence homologous to the Chlamydia trachomatis LGV II sequence CT199(oppB1) identified from clone CTL2gam 24.

SEQ ID NO: 554 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the C.trachomatis LGV II sequence CT545(dnaE) identified from clone CTL 2-4.

SEQ ID NO: 555 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT288 identified from clone CTL2gam 27.

SEQ ID NO: 556 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the Chlamydia trachomatis LGV II sequence CT413(pmpB) identified from clone CTL2gam30(5 ') (3').

SEQ ID NO: 557 shows the full length chlamydia trachomatis serovar D DNA sequence homologous to the C T-ORF3 sequence of Chlamydia trachomatis LGV II identified in clones CTL2gam15(5 '), CTL2-16(5 '), CTL2-18(5 ') and CTL 2-23.

SEQ ID NO: 558 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence pCT-ORF4 identified from clone CTL2-18 (3').

SEQ ID NO: 559 shows the full length C.trachomatis serovar variant D DNA sequence homologous to the C.trachomatis LGV II sequence CT-ORF5 identified from clone CTL2-18 (3').

SEQ ID NO: 560 shows the full length chlamydia trachomatis serum variant D amino acid sequence homologous to the chlamydia trachomatis LGV II sequence CT751 (arn) identified from clones CTL2-1 and CTL 2-5.

SEQ ID NO: 561 shows the full length chlamydia trachomatis serum variant D amino acid sequence homologous to the C.trachomatis LGV II sequence CT322(tuff) identified from clone CTL 2-2.

SEQ ID NO: 562 shows the full length chlamydia trachomatis serum variant D amino acid sequence homologous to the C.trachomatis LGV II sequence CT032(metG) identified from clones CTL2gam2, CTL2-3 (5') and CTL 2-4.

SEQ ID NO: 563 shows the full length chlamydia trachomatis serovar D amino acid sequence homologous to the C.trachomatis LGV II sequence CT031 identified from clone CTL2-3(5 ') (3').

SEQ ID NO: 564 shows the full length chlamydia trachomatis serum variant D amino acid sequence homologous to the chlamydia trachomatis LGV II sequence CT030(gmK) identified from clones CTL2-3 (3') and CTL 2-21.

SEQ ID NO: 565 shows the amino acid sequence of the full-length chlamydia trachomatis serum variant D homologous to the sequence CT064(lepA) of chlamydia trachomatis LGV II identified from clone CTL2gam 5.

SEQ ID NO: 566 shows the full length Chlamydia trachomatis serum variant D amino acid sequence homologous to the Chlamydia trachomatis LGV II sequence CT265(accA) identified from clone CTL 2-6.

SEQ ID NO: 567 shows the amino acid sequence of the full length chlamydia trachomatis serovariant D homologous to the C.trachomatis LGV II sequence CT624(msbA) identified from clone CTL 2-6.

SEQ ID NO: 568 shows the full length Chlamydia trachomatis serum variant D amino acid sequence homologous to the C.trachomatis LGV II sequence CT256 identified from clones CTL2gam6(5 ') and CTL2-11 (5').

SEQ ID NO: 569 shows the full length Chlamydia trachomatis serum variant D amino acid sequence homologous to CT257 from the sequence of Chlamydia trachomatis LGV II identified in clones CTL2gam6(5 ') and CTL2-11 (5').

SEQ ID NO: 570 shows the full length Chlamydia trachomatis serum variant D amino acid sequence homologous to the C.trachomatis LGV II sequence CT384 identified from clones CTL2gam6(3 ') and CTL2-11 (3').

SEQ ID NO: 571 shows the full length chlamydia trachomatis serum variant D amino acid sequence homologous to the C.trachomatis LGV II sequence CT473 identified from clone CTL2-8 b.

SEQ ID NO: 572 shows the full length chlamydia trachomatis serum variant D amino acid sequence homologous to the chlamydia trachomatis LGV II sequence CT474 identified from clone CTL2-8 b.

SEQ ID NO: 573 shows the amino acid sequence of the full-length chlamydia trachomatis serum variant D homologous to the LGV II sequence CT139(oppA1) of chlamydia trachomatis identified from clone CTL2-8 b.

SEQ ID NO: 574 shows the amino acid sequence of the full length chlamydia trachomatis serum variant D homologous to the C.trachomatis LGV II sequence CT060(flhA) identified from clone CTL2-8 b.

SEQ ID NO: 575 shows the full length chlamydia trachomatis serum variant D amino acid sequence homologous to the C.trachomatis LGV II sequence CT242 identified from clone CTL2gam 8.

SEQ ID NO: 576 shows the amino acid sequence of the full length chlamydia trachomatis serum variant D homologous to the chlamydia trachomatis LGV II sequence CT243(lpxD) identified from clone CTL2gam 8.

SEQ ID NO: 577 shows the full length chlamydia trachomatis serovar D amino acid sequence homologous to the C.trachomatis LGV II sequence CT089 identified from clones CTL2-9, CTL2gam1, CTL2gam17, and CTL2-19 (5').

SEQ ID NO: 578 shows the amino acid sequence of the full length chlamydia trachomatis serum variant D homologous to the LGV II sequence CT610 for chlamydia trachomatis identified from clone CTL2-10(5 ') (3').

SEQ ID NO: 579 shows the amino acid sequence of the full length chlamydia trachomatis serum variant D homologous to the C.trachomatis LGV II sequence CT611 identified from clone CTL2-10 (5').

SEQ ID NO: 580 shows the full length chlamydia trachomatis serum variant D amino acid sequence homologous to the Chlamydia trachomatis LGV II sequence CT154 identified from clones CTL2gam10 and CTL2gam 21.

SEQ ID NO: 581 shows the full length Chlamydia trachomatis serovariant D amino acid sequence homologous to the C.trachomatis LGV II sequence CT833(infC) identified from clone CTL 2-12.

SEQ ID NO: 582 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the C.trachomatis LGV II sequence CT-ORF3 identified in clones CTL2gam15(5 '), CTL2-16(5 '), CTL2-18(5 ') and CTL 2-23.

SEQ ID NO: 583 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the Chlamydia trachomatis LGV II sequence CT827(nrdA) identified from clones CTL2-16(3 ') and CTL2gam15 (3').

SEQ ID NO: 584 shows the full length C.trachomatis serovariant D DNA sequence homologous to the C.trachomatis LGV II sequence CT828(nrdB) identified from clones CTL2-16(3 ') and CTL2gam15 (3').

SEQ ID NO: 585 shows the full length chlamydia trachomatis serum variant DDNA sequence homologous to the chlamydia trachomatis LGV II sequence CT067(ytgA) identified from clone CTL2gam 18.

SEQ ID NO: 586 shows the full length C.trachomatis serum variant D DNA sequence homologous to the C.trachomatis LGV II sequence pCT-ORF4 identified from clone CTL2-18 (3').

SEQ ID NO: 587 shows the full length C.trachomatis serovariant D DNA sequence homologous to the C.trachomatis LGV II sequence CT-ORF5 identified from clone CTL2-18 (3').

SEQ ID NO: 588 shows the full length Chlamydia trachomatis serum variant D DNA sequence homologous to the sequence CT087 for the LGV II sequence of Chlamydia trachomatis identified from clone CTL2-19 (5').

SEQ ID NO: 589 shows the full length chlamydia trachomatis serum variant DDNA sequence homologous to the C.trachomatis LGV II sequence CT088(sycE) identified from clone CTL2-19 (5').

SEQ ID NO: 590 shows the full length chlamydia trachomatis serum variant DDNA sequence homologous to the chlamydia trachomatis LGV II sequence CT588(rsbu) identified from clone CTL2gam 23.

SEQ ID NO: 591 shows the full length chlamydia trachomatis serum variant DDNA sequence homologous to the chlamydia trachomatis LGV II sequence CT199(oppB1) identified from clone CTL2gam 24.

SEQ ID NO: 592 shows a full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT545(dnaE) identified from clone CTL 2-4.

SEQ ID NO: 593 shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the chlamydia trachomatis LGV II sequence CT288 identified from clone CTL2gam 27.

SEQ ID NO: 594 to et al shows the full length chlamydia trachomatis serum variant D DNA sequence homologous to the sequence CT413(pmpB) for the chlamydia trachomatis LGV II sequence identified from clone CTL2gam30(5 ') (3').

SEQ ID NO: 595 shows the DNA sequence of the Chlamydia pneumoniae homolog CPn0406 of the Chlamydia trachomatis gene CT 102.

SEQ ID NO: 596 shows the DNA sequence of Chlamydia pneumoniae homolog CPn0315 of Chlamydia trachomatis gene CT 098.

SEQ ID NO: 597 shows the amino acid sequence of Chlamydia pneumoniae homolog CPn0406 of Chlamydia trachomatis gene CT 102.

SEQ ID NO: 598 shows the amino acid sequence of Chlamydia pneumoniae homolog CPn0315 of Chlamydia trachomatis gene CT 098.

SEQ ID NO: 599 shows the amino acid sequence of chlamydia trachomatis serovar D CT287 protein.

Brief Description of Drawings

FIG. 1 is a graph showing the induction of INF-gamma by a Chlamydia-specific T cell line activated from target cells expressing clone 4C9-18# 2.

FIG. 2 is a schematic representation of the retroviral vector pBIB-KS1, 2, 3 modified to contain a Kosak translation start site and a stop codon.

FIG. 3 shows the specific lysis of P815 cells pulsed with the Chlamydia peptides CtC7.8-12(SEQ ID NO: 18) and CtC7.8-13(SEQ ID NO: 19) in the chromium release assay.

FIG. 4 shows antibody isotype titers in C57B1/6 mice immunized with the C.trachomatis SWIB protein.

FIG. 5 shows a Chlamydia-specific T cell proliferation response in splenocytes from C3H mice immunized with the SWIB protein of Chlamydia trachomatis.

FIG. 6 shows the 5 'and 3' primer sequences designed from C.pneumoniae to isolate the SWIB and S13 genes from C.pneumoniae.

FIGS. 7A and 7B show IFN- γ induction in a human anti-Chlamydia T cell line (TCL-8) that cross-reacts with Chlamydia trachomatis and Chlamydia pneumoniae following activation by monocyte-derived dendritic cells expressing Chlamydia protein.

FIG. 8 shows the identification of T cell epitopes in Chlamydia ribosome S13 protein using the T cell line TCL8 EB/DC.

FIGS. 9A and B illustrate the proliferative response of CP-21T cells generated against C.pneumoniae-infected dendritic cells to recombinant C.pneumoniae-SWIB protein, but not C.trachomatis-SWIB protein.

FIG. 10 shows the Chlamydia trachomatis-specific SWIB proliferative response of primary T cell lines (TCT-10 EB) from asymptomatic donors.

FIG. 11 illustrates the identification of T-cell epitopes in Chlamydia trachomatis SWIB using an antigen-specific T-cell line (TCL-10 EB).

Fig. 12 shows chlamydia trachomatis specific proliferative responses of primary T cell lines generated by two patients against CT specific antigens CT622, CT875 and CT EB.

Detailed Description

As noted above, the present invention relates generally to compositions and methods for diagnosing and treating chlamydial infections. In one aspect, the compositions of the invention include a polypeptide comprising at least one immunogenic portion of a chlamydia antigen or variant thereof.

In particular embodiments, the invention discloses polypeptides comprising an immunogenic portion of a chlamydia antigen, wherein said chlamydia antigen comprises an amino acid sequence encoded by a polynucleotide molecule disclosed herein, a complement of said nucleotide sequence, and variants of these sequences.

As used herein, the term "polypeptide" includes amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked together by covalent peptide bonds. Thus, a polypeptide comprising an immunogenic portion of an antigen of the invention may consist entirely of the immunogenic portion, or may contain other sequences. The additional sequence may be derived from a native chlamydia antigen or may be heterologous, and the sequence may (but need not) be immunogenic.

The term "polynucleotide" as used herein refers to single-or double-stranded polymers of deoxyribonucleic acid or ribonucleic acid bases, including DNA and corresponding RNA molecules (including HnRNA and mRNA molecules), including both sense and antisense strands, and including cDNA, genomic DNA, and recombinant DNA, as well as fully or partially synthetic polynucleotides. The HnRNA molecules contain introns and correspond to DNA molecules in a generally one-to-one correspondence. The mRNA molecules correspond to the HnRNA and DNA molecules from which introns have been excised. A polynucleotide may be composed of the entire gene or any portion thereof. An operable antisense polynucleotide can comprise a fragment of the corresponding polynucleotide, whereby the definition of "polynucleotide" includes all such operable antisense fragments.

An "immunogenic portion" of an antigen is a portion that is capable of reacting with serum obtained from a chlamydia-infected individual (i.e., that produces an absorbance reading with serum from an infected individual that is at least three standard deviations higher than the absorbance reading obtained with serum from an uninfected individual in the representative ELISA assay described herein). The immunogenic portion generally comprises at least about 5 amino acid residues, more preferably at least about 10, and most preferably at least about 20 amino acid residues. Methods for preparing and identifying immunogenic portions of antigens of known sequence are known in the art and include Paul, Fundamental Immunology, 3 rd edition, Raven Press, 1993, pp243-247 and those summarized in the references cited therein. These techniques include screening for polypeptides capable of reacting with antigen-specific antibodies, anti-serum and/or T cell lines or clones. Antisera and antibodies herein are "antigen-specific" if they can specifically bind to an antigen (i.e., react with an antigenic protein in an ELISA or other immunoassay, but do not detectably react with unrelated proteins). The antisera and antibodies can be as described herein and using well known techniques And (4) preparing. An immunogenic portion of a native chlamydia protein is a portion that reacts with the anti-serum and/or T cells at a level that is substantially no less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T cell reactivity assay). The immunogenic portion can react in these assays at a level similar to or greater than the reactivity of the full-length polypeptide. This screening can generally be performed using methods well known to those of ordinary skill in the art, such as Harlow and Lane, antibodies: those methods described in Laboratory manuals (Antibodies: A Laboratory Manual) (Cold spring harbor Laboratory, 1988). For example, the polypeptide may be immobilized on a solid support and contacted with patient serum to allow binding of antibodies in the serum to the immobilized polypeptide. Unbound serum is then removed, for example, by¹²⁵I labeled protein a detects bound antibody.

Examples of immunogenic portions of antigens contemplated by the invention include, for example, the T cell stimulatory epitopes provided in SEQ ID NO9, 10, 18, 19, 31, 39, 93-96, 98, 100-102, 106, 108, 138-140, 158, 167, 168, 246, 247, and 254-256. Polypeptides comprising at least an immunogenic portion of one or more chlamydia antigens as described herein can generally be used alone or in combination to detect chlamydia infection in a patient.

The compositions and methods of the invention also include variants of the above-described polypeptide and polynucleotide molecules. Such variants include, but are not limited to, naturally occurring allelic variants of the sequences of the invention. In particular, variants include other chlamydia serum variants having homology with the polypeptide and polynucleotide molecules of the invention described herein, such as serovariants D, E and F, as well as several LGV serum variants. Preferably, the serovariant homologue (homologue) exhibits 95-99% homology with the corresponding polypeptide sequence described herein.

A polypeptide "variant" as used herein refers to a polypeptide that differs from the polypeptide in question only by conservative substitutions and/or modifications, such that the antigenic properties of the polypeptide in question are retained. In preferred embodiments, the variant polypeptide differs from the indicated sequence by 5 or fewer amino acid substitutions, deletions or insertions. Such variants can generally be identified by modifying one of the above polypeptide sequences using, for example, the representative methods described herein, and then evaluating the antigenic properties of the modified polypeptide. In other words, the ability of the variant to react with antigen-specific antisera may be enhanced or unaltered relative to the native protein, or may be reduced by less than 50%, preferably less than 20%, relative to the native protein. Such variants can generally be identified by modifying one of the polypeptide sequences described above and then evaluating the reactivity of the modified polypeptide with an antigen-specific antibody or antisera described herein. Preferred variants include those in which one or more portions, such as the N-terminal leader sequence or transmembrane domain, have been removed. Other preferred variants include those in which a small portion of the N-and/or C-terminus of the mature protein has been removed (e.g., 1-30 amino acids, preferably 5-15 amino acids).

Herein, "conservative substitutions" refer to substitutions in which one amino acid is substituted for another with similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophilic properties of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with similar hydrophilicity values with uncharged polar head groups include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine, and tyrosine. Other groups of amino acids that may exhibit conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Variants may also, or alternatively, contain non-conservative changes. In a preferred embodiment, the variant polypeptide differs from the native sequence by 5 or fewer amino acid substitutions, deletions or additions. Variants may also (or alternatively) be modified by, for example, deletion or addition of amino acids that have minimal impact on the immunogenicity, secondary structure and hydrophilic properties of the polypeptide. Variants may also, or alternatively, contain other modifications, including deletions or additions of amino acids that have minimal effect on the antigenic, secondary structure, and hydrophilic properties of the polypeptide. For example, the polypeptide may be conjugated to a (conjugate) signal (or leader) sequence at the N-terminus of the protein that will co-or post-translationally direct transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence that facilitates synthesis, purification, or identification of the polypeptide (e.g., poly-His) or enhances binding of the polypeptide to a solid support. For example, the polypeptide may be conjugated to an immunoglobulin Fc region.

A polynucleotide "variant" is a sequence that differs from the recited nucleotide sequence by one or more nucleotide deletions, substitutions, or additions such that the immunogenicity of the encoded polypeptide is not diminished relative to the native protein. The effect on the immunogenicity of the encoded polypeptide can generally be assessed as described herein. These modifications can be readily introduced by using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis as taught by Adelman et al (DNA, 2:183, 1983). The nucleotide variant may be a natural allelic variant, discussed below, or a non-natural variant. The polypeptides provided by the invention include variants encoded by polynucleotide sequences substantially homologous to one or more of the polynucleotide sequences specifically described herein. "substantially homologous" refers herein to a polynucleotide sequence that is capable of hybridizing under moderately stringent conditions. Suitable moderately stringent conditions include a prewash in a solution of 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridization overnight in 5 XSSC at 50 ℃ to 65 ℃ or in the case of cross species homology, hybridization using 0.5 XSSC at 45 ℃; then, 2X, 0.5X and 0.2 XSSC containing 0.1% SDS were used to wash at 65 ℃ for 2 times 20 minutes each. Such hybrid polynucleotide sequences are also within the scope of the present invention, as are nucleotide sequences that, due to the degeneracy of the code, encode polypeptides identical to a polypeptide of the present invention.

Two nucleotide or polypeptide sequences are said to be "identical" if the sequences of nucleotide or amino acid residues in the two sequences are identical when aligned for maximum correspondence as described below. The two sequences are compared, typically by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. "comparison window" refers herein to a segment of at least about 20 contiguous positions, usually 30 to about 75 positions, 40 to about 50 positions, in which a sequence can be compared to a reference sequence having the same number of contiguous positions after optimal alignment (alignment) of the two sequences.

For comparison, sequences can be optimally aligned using the Lasergene software package of bioinformatics software (DNASTAR, inc., Madison, WI) and default parameters. This program embodies several alignment strategies as described in the following references; day Dayhoff, m.o. (1978), Protein evolution alteration model-matrices for detecting distant relations, Dayhoff, M.O. (eds.) "Atlas of Protein sequences and structures", National biological research foundation, Washington d.c. vol.5, suppl.3, pp.345-358; hein J. (1990) unified Methods of alignment and phylogeny pp.626-645 enzymology Methods (Methods in enzymology) vol.183, Academic Press, inc., San Diego, calif.; higgins, D.G. and Sharp, P.M, (1989) Rapid and sensitive multiple sequence alignment on a microcomputer, CABIOS 5: 151-; optimal alignment in Myers, E.W. and Muller W. (1988) Linear space, CABIOS 4: 11-17; robinson, E.D, (1971) comb. Theor 11: 105; santou, N.Nes, M. (1987) Adjacent ligation method, a new method for the reconstruction of phylogenetic trees, mol.biol.Evol.4: 406-425; sneath, p.h.a., and Sokal, R.R, (1973) Principles and practice of Numerical Taxonomy-Numerical Taxonomy (Numerical taxomy-the Principles and practice of Numerical taxomy), Freeman Press, San Francisco, Cal if.; wi lbur, W.J. and Lipman, D.J. (1983) Rapid similarity search of nucleic acid and protein databases, Proc. Natl.Acad., Sci. USA 80: 726-.

Alternatively, the optical properties can be measured by Smith and Waterman (1981) add. 482, local consistency algorithm; the identity alignment algorithm of Needleman and Wunsch (1970) J.Mol.biol.48: 433; similarity search methods of Pearson and Lipman (1988) Proc.Natl.Acad.Sci. (U.S.A.)85: 2444; computer-implemented programs for these algorithms (GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.); or visually aligning the sequences for comparison.

An illustrative example of an algorithm suitable for determining sequence identity and percent sequence similarity is the BLAST and BLAST2.0 algorithms, described in Altschul et al (1977) Nuc. acids Res.25; 3389 and 3402 and Altschul et al (1990) J.mol.biol.215:403 and 410. Percent sequence identity for polynucleotides and polypeptides of the invention can be determined using BLAST and BLAST2.0, for example, using the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov /). In one illustrative example, cumulative scores can be calculated for nucleotide sequences using the parameters M (reward score for paired matching residues; total >0) and N (penalty score for mismatching residues; total < 0). For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. When: when the cumulative alignment score decreases by an amount X from its maximum gain value; (ii) when the cumulative score approaches zero or less due to one or more negative-scoring residue alignments; or reaching the end of either sequence, the extension of the character pair (word hit) in each direction is terminated. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) used a word length of 11 (W), an expectation of 10 (E), and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) proc. natl. acad. sci. usa89: 10915) alignment with 50's expectation (B), 10's expectation (E), M5, N4, and double-stranded comparisons as defaults.

Preferably, the "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window having at least 20 positions, wherein the polynucleotide or amino acid sequence portion in the comparison window may comprise additions or insertions (i.e., gaps) of 20% or less, typically 5% to 15%, or 10% to 12%, as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. This percentage is determined by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to give the number of matched positions, then dividing this number of matched positions by the total number of positions in the reference sequence (i.e., the size of the window) and multiplying the result by 100 to give the percentage of sequence identity.

Thus, the invention provides polynucleotide and polypeptide sequences having substantial identity to the sequences disclosed herein, e.g., those comprising at least 50% or more sequence identity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a polynucleotide or polypeptide sequence of the invention using the methods disclosed herein (e.g., BLAST analysis using standard parameters, see below). It will be appreciated by those skilled in the art that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by the two polynucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame position, and the like.

In other embodiments, the invention provides isolated polynucleotides or polypeptides comprising contiguous sequence segments of various lengths that are identical or complementary to one or more of the sequences disclosed herein. For example, polynucleotides and polypeptides encompassed by the invention may comprise at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleotides of one or more of the disclosed sequences, and all intervening lengths of contiguous nucleotides therebetween. It is readily understood that "intermediate length" in this context means any length between the referenced values, e.g. 16, 17, 18, 19, etc.; 21. 22, 23, etc.; 30. 31, 32, etc.; 50. 51, 52, 53, etc.; 100. 101, 102, 103, etc.; 150. 151, 152, 153, etc.; from 200 to 500; all integers from 500 to 1,000, and the like.

The polynucleotides of the invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. Thus, nucleic acid fragments of almost any length are contemplated for use herein, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA procedure. For example, illustrative DNA segments having a total length of about 10,000, about 5000, about 3000, about 2000, about 1000, about 500, about 200, about 100, about 50 base pairs are contemplated for use in many embodiments of the present invention.

Allelic sequences of the genes encoding the nucleotide sequences described herein are also included within the scope of the invention. Herein, an "allele" or "allelic sequence" is an alternative form of a gene, which may be caused by at least one mutation in a nucleic acid sequence. Alleles may result in altered mRNA or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutations that give rise to alleles can generally be classified as natural deletions, additions or substitutions of nucleotides. All of these types of changes can occur one or more times in a given sequence, either alone or in combination with other changes.

In particular embodiments, the invention discloses polypeptides comprising at least an immunogenic portion of a chlamydia antigen (or a variant of the antigen) comprising one or more amino acid sequences encoded by the following polynucleotide sequences: (a) selected from the group consisting of SEQ ID NO: 358-361, 407-430, 525-559, 582-598; (b) the complement of these DNA sequences; or (c) a DNA sequence substantially homologous to the sequence in (a) or (b). As discussed in the examples below, several of the chlamydia antigens disclosed herein can recognize T cell lines that recognize monocyte-derived dendritic cells infected with chlamydia trachomatis and chlamydia pneumoniae, suggesting that these antigens may represent immunoreactive epitopes common to chlamydia trachomatis and chlamydia pneumoniae. Thus, the antigen may be used in vaccines for genital tract infection of chlamydia trachomatis and chlamydia pneumoniae infection. To determine the extent of cross-reactivity, further characterization of these chlamydia antigens from chlamydia trachomatis and chlamydia pneumoniae is described in example 6. Furthermore, example 4 describes cDNA fragments (SEQ ID NOS: 15, 16 and 33) isolated from Chlamydia trachomatis encoding proteins capable of stimulating the Chlamydia-specific mouse CD8+ T cell line (SEQ ID NOS: 17-19 and 32).

In general, chlamydia antigens and polynucleotide sequences encoding such antigens can be prepared using any of a variety of methods. For example, polynucleotide molecules encoding chlamydia antigens can be isolated from chlamydia genomic or cDNA expression libraries by screening with a chlamydia-specific T cell line as described below and sequenced using techniques well known to those skilled in the art. In addition, polynucleotides can be identified by screening cDNA microarrays for chlamydia-associated expression (i.e., at least two-fold higher expression in chlamydia-infected cells than in controls, as determined using the representative assays provided herein) as described in more detail below. The screening can be carried out using Synteni microarrays (Palo Alto, CA) according to the manufacturer's instructions (and essentially as described by Schena et al, Proc. Natl. Acad. Sci. USA 93: 10614-. Alternatively, the polypeptide can be amplified from a cDNA prepared from a cell expressing a protein described herein. The polynucleotide may be amplified by Polymerase Chain Reaction (PCR). For this method, sequence specific primers can be designed based on the sequences provided herein, and can also be purchased or synthesized.

An antigen can be recombinantly produced by inserting a polynucleotide sequence encoding the antigen into an expression vector and then expressing the antigen in an appropriate host, as described below. The desired properties of the antigen, such as the ability to react with serum obtained from an individual infected with chlamydia, can be assessed as described herein and the antigen can be sequenced using, for example, conventional Edman chemistry. See Edman and Berg, Eur.J.biochem.80: 116-.

The polynucleotide sequence encoding the antigen may also be obtained by screening an appropriate cDNA or genomic DNA library of Chlamydia for a polynucleotide sequence which hybridizes with degenerate oligonucleotides derived from part of the amino acid sequence of the isolated antigen. The Molecular Cloning can be performed, for example, as described in Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor laboratories, Cold Spring Harbor, N.Y. (and references cited therein) describe methods for designing and synthesizing degenerate nucleotide sequences for use in this screen, and performing this screen. The nucleic acid probes can also be isolated from cDNA or genomic libraries using the above oligonucleotides in methods well known in the art using the Polymerase Chain Reaction (PCR). The library is then screened using the isolated probe.

The amplified portion can be used to isolate the full-length gene from a suitable library (e.g., a chlamydia cDNA library) using well-known techniques. In these techniques, libraries (cDNA or genomic) may be screened using one or more polynucleotide probes or primers suitable for amplification. Libraries are preferably screened by size to include larger molecules. The 5' and upstream regions of the gene may also preferably be identified using random primordial libraries. Preferably, genomic libraries are used to obtain introns and extended 5' sequences.

For hybridization techniques, the partial sequence may be labeled using well-known techniques (e.g., by nick translation or with³²P end-labeled). The bacteria or phage library is then screened by hybridizing labeled probes to filters containing denatured bacterial colonies (or plaques containing) by hybridization (see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Colonies or plaques which hybridized were selected and the DNA isolated for further analysis. The cDNA clones can be analyzed to determine the amount of other sequences, for example by PCR using primers from the partial sequence and primers from the vector. Restriction maps and partial sequences can be prepared to identify one or more overlapping clones. Standard techniques (which may involve the generation of a series of deletion clones) may then be used to confirm And determining the complete sequence. The resulting overlapping sequences are then assembled into a contiguous sequence. Full-length cDNA molecules can be generated by ligating appropriate fragments using well-known techniques.

Alternatively, a number of amplification techniques can be used to obtain the full-length coding sequence from a partial cDNA sequence. In these techniques, amplification is generally performed by PCR. The amplification step can be carried out using any of a variety of commercial kits. Primers can be designed using techniques well known in the art (see, e.g., Mullis et al, Cold Spring Harbor Symp. Quant. biol.51:263, 1987; Erlich eds., PCR Technology, Stockton Press, NY, 1989), and software well known in the art can also be used. The primer is preferably 22-30 nucleotides long, has a GC content of at least 50%, and can anneal to the target sequence at a temperature of about 68 ℃ to 72 ℃. The amplified regions can be sequenced as described above, and the overlapping sequences can then be assembled into one contiguous sequence.

One such amplification technique is inverse PCR (see Triglia et al, Nucl. acids Res.16:8186, 1988), which uses restriction enzymes to generate a fragment in a known region of a gene. This fragment was then circularized by intramolecular ligation, used as a template for PCR, and amplified using primers that are backed from this known region. In another alternative method, sequences adjacent to the partial sequence may be obtained by amplification using primers directed to the adaptor sequence and primers specific for known regions. A second round of amplification of the amplified sequence is typically performed using the same adapter primer and a second primer specific for the known region. A variant of this method uses two primers which extend in opposite directions from a known sequence, see WO 96/38591. Other techniques include capture PCR (Lagerstrom et al, PCR Methods applied.1: 111-19, 1991) and walk PCR (Parker et al, Nucl. acids. Res.19:3055-60, 1991). Transcription mediated amplification or TMA is another method that can be used to amplify DNA, rRNA or mRNA and is described in patent PCT/US 91/03184. This non-PCR based autocatalytic isothermal approach utilizes two primers and two enzymes: RNA polymerase and reverse transcriptase. One primer contains the promoter sequence of RNA polymerase. In the first amplification, a promoter primer hybridizes to the target rRNA at a defined site. Reverse transcriptase generates a DNA copy of the target rRNA by extension from the 3' end of the promoter primer. The RNA in the resulting complex is degraded and a second primer binds to the DNA copy. Reverse transcriptase synthesizes a new DNA strand from the end of the primer, thereby generating double-stranded DNA. The RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription. Each newly synthesized RNA amplicon re-enters the TMA program and serves as a template for a new round of replication, resulting in exponential amplification of the RNA amplicon. Other methods using amplification can also be used to obtain the full-length cDNA sequence.

In some cases, the full-length cDNA sequence can be obtained by analyzing sequences provided in an Expressed Sequence Tag (EST) database (e.g., available from GenBank). Overlapping ESTs can generally be searched using well known programs (e.g., NCBI BLAST search) and can then be used to generate contiguous full-length sequences. The full-length cDNA sequence can also be obtained by analyzing genomic fragments.

Polynucleotide variants can generally be prepared by any method known in the art, including chemical synthesis, e.g., solid phase phosphoramidite chemical synthesis. Modifications can also be introduced into a polynucleotide sequence using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al, DNA 2:183, 1983). Alternatively, an RNA molecule may be prepared by in vitro or in vivo transcription of a DNA sequence encoding a Chlamydia protein or part thereof, provided that the DNA is inserted into a vector having a suitable RNA polymerase promoter (e.g., T7 or SP 6). Certain portions may be used to prepare the encoded polypeptides, as described herein. In addition, or as an alternative, a moiety may be administered to the patient to produce the encoded polypeptide in vivo (e.g., by transfecting antigen presenting cells, such as dendritic cells, with a cDNA construct encoding a Chlamydia polypeptide and then administering the transfected cells to the patient).

The portion of the sequence complementary to the coding sequence (i.e., the antisense polynucleotide) can also be used as a probe or to modulate gene expression. A cDNA construct capable of being transcribed into antisense RNA can also be introduced into tissue cells to facilitate the production of antisense RNA. As described herein, antisense polynucleotides can be used to inhibit chlamydia protein expression. Gene expression can be controlled using antisense technology by the ability of triple helix formation to disrupt the double helix opening sufficiently large to bind polymerases, transcription factors or regulatory molecules (see Gee et al, Molecular and immunological applications (Huber and Carr eds.), Futura Publishing Co. (mt. kisco, n.y.; 1994)). Alternatively, antisense molecules can be designed to hybridize to a control region of a gene (e.g., a promoter, enhancer, or transcription initiation site), thereby blocking gene transcription; or by inhibiting the binding of transcripts to ribosomes.

A portion of the coding sequence or complementary sequence may also be designed as a probe or primer to detect gene expression. The probes may be labeled with a variety of reporter groups, such as radionuclides and enzymes, and are preferably at least 10 nucleotides in length, more preferably at least 20 nucleotides, and even more preferably at least 30 nucleotides in length. As mentioned above, the primer is preferably 22 to 30 nucleotides in length.

Any polynucleotide may be further modified to increase in vivo stability. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and/or 3' ends; the use of phosphorothioate or 2' O-methyl groups in the backbone rather than phosphodiester linkages; and/or include unconventional bases such as inosine, queosine, and wybutosine, as well as acetyl, methyl, thio, and other modified forms of adenine, cytosine, guanine, thymine, and uracil.

The nucleotide sequences described herein may be joined to a variety of other nucleotide sequences by established recombinant DNA techniques. For example, the polynucleotide may be cloned in any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe preparation vectors and sequencing vectors. Typically, the vector contains an origin of replication functional in at least one organism, a convenient restriction endonuclease site, and one or more selectable markers. Other elements will depend on the desired application and will be apparent to those of ordinary skill in the art.

Synthetic polypeptides having less than about 100 amino acids, typically less than about 50 amino acids, can be produced using techniques well known in the art. For example, such polypeptides may be synthesized using any one of the commercial solid phase techniques, such as the Merrifield solid phase synthesis method (in which amino acids are added sequentially to a growing amino acid chain). See Merrifield, J.Am.chem.Soc.85: 2149-C2146, 1963. An apparatus for automated synthesis of polypeptides is commercially available from suppliers such as Perkinelmer/Applied BioSystems Division (Foster City, Calif.) and may be operated according to the manufacturer's instructions.

As noted above, immunogenic portions of chlamydial antigens can be prepared and characterized using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3 rd edition, Raven Press, 1993, pp.243-247 and the references cited therein. These techniques include screening for polypeptide portions of the native antigen that have immunogenic properties. Representative ELISAs described herein may generally be used in these screens. The immunogenic portion of the polypeptide is that portion which produces a substantially similar level of signal to the full-length antigen in these representative assays. In other words, in the model ELISA described herein, the signal produced by the immunogenic portion of the chlamydia antigen is at least about 20%, preferably about 100%, of the signal induced by the full-length antigen.

Portions and other variants of chlamydia antigens may be prepared synthetically or recombinantly. Variants of the native antigen can generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Some segments of the polynucleotide sequence may also be removed using standard techniques that allow for the preparation of truncated polypeptides.

Recombinant polypeptides containing portions and/or variants of a native antigen can be readily prepared from polynucleotides encoding the polypeptides using a variety of techniques well known to those of ordinary skill in the art. For example, the supernatant from an appropriate host/vector system that secretes the recombinant protein into the culture medium can be first concentrated using a commercial filter. After concentration, the concentrate can be loaded onto a suitable purification matrix such as an affinity matrix or ion exchange resin. Finally, the recombinant protein may be further purified using one or more reverse phase HPLC steps.

A variety of expression vectors known to those of ordinary skill in the art can be used to express the recombinant polypeptides described herein. Expression may be effected in a suitable host cell which has been transformed or transfected with an expression vector containing a polynucleotide molecule encoding a recombinant polypeptide. Suitable host cells include prokaryotic cells, yeast or mammalian cell lines, such as COS or CHO. The DNA sequence expressed in this manner may encode a native antigen, a portion of a native antigen, or other variants thereof.

In general, the polypeptides disclosed herein can be prepared in isolated, substantially pure form, regardless of the method of preparation used. Preferably, the polypeptide has at least about 80% purity, more preferably at least about 90% purity, and most preferably at least about 99% purity.

In certain embodiments, the polypeptide can be a fusion protein comprising a plurality of polypeptides as described herein, or a fusion protein comprising at least one polypeptide as described herein and an unrelated sequence (e.g., a known chlamydia protein). The fusion partner may, for example, help to provide T helper epitopes (immunological fusion partners), preferably T helper epitopes that can be recognized by humans, or may help to express the protein in higher yields (compared to the original recombinant protein) (expression enhancers). Certain preferred fusion partners are both immunological and expression-enhancing fusion partners. Other fusion partners may be selected to increase the solubility of the protein or to enable the protein to be targeted to a desired intracellular compartment. Still other fusion partners include affinity tags that facilitate protein purification. The DNA sequence encoding the fusion protein of the invention may be constructed using known recombinant DNA techniques to assemble the isolated DNA sequences encoding, for example, the first and second polypeptides in a suitable expression vector. The 3 'end of the DNA sequence encoding the first polypeptide and the 5' end of the DNA sequence encoding the second polypeptide are linked, with or without a peptide linker, so that the reading frames of these sequences may be in phase to allow translation of the two DNA sequences into a single fusion protein that retains the biological activity of the first and second polypeptides.

The peptide linker may be used to separate the first and second polypeptides by a distance large enough to ensure that each polypeptide folds into its secondary and tertiary structures. The peptide linker sequence may be incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be selected based on the following factors: (1) capable of adopting a flexible extended conformation; (2) it cannot adopt a secondary structure that would interact with functional epitopes on the first and second polypeptides; and (3) lack of hydrophobic or charged residues that can react with functional epitopes of these polypeptides. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other only neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that can be commonly used as linkers include those disclosed in Maratea et al, Gene 40:39-46, 1985; murphy et al, Proc.Natl.Acad.Sci.USA83:8258-8562, 1986; those of U.S. patents 4,935,233 and 4,751,180. The linker sequence may be 1 to about 50 amino acids in length. As an alternative to using peptide linker sequences (if desired), non-essential N-terminal amino acid regions on the first and second polypeptides (if present) may be used to separate the functional domains and prevent steric hindrance.

These ligated DNA sequences are operably linked to appropriate transcriptional or translational regulatory elements. The regulatory elements responsible for the expression of the DNA are located only 5' to the DNA sequence encoding the first polypeptide. Similarly, the stop codon and transcription termination signal required to terminate translation are only present 3' of the DNA sequence encoding the second polypeptide.

The invention also provides fusion proteins comprising a polypeptide of the invention and an unrelated immunogenic protein. Preferably, the immunogenic protein is capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis, and hepatitis proteins (see, e.g., Stoute et al New Engl. J. Med., 336:86-91, 1997).

In a preferred embodiment, the immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (wo 91/18926). Preferably, the D protein derivative comprises about the first third of the protein (e.g., the N-terminal 100-110 amino acids), and the D protein derivative may be lipidated. In certain preferred embodiments, the first 109 residues of the lipoprotein D fusion partner are included on the N-terminus to provide additional foreign T cell epitopes to the polypeptide and to enhance expression (and thus function as an expression enhancer) in e. The lipid tail ensures optimal presentation of antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenza virus, NS1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, but different fragments including T helper epitopes may also be used.

In another embodiment, the immunological fusion partner is a protein known as LYTA, or a portion (preferably the C-terminal portion) thereof. LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase called the amidase LYTA (encoded by the LytA Gene; Gene43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-domain of the LYTA protein is responsible for the affinity to choline or some choline analogs such as DEAE. This property has been exploited in the development of E.coli C-LYTA expression plasmids for expression of fusion proteins. Purification of hybrid proteins containing a fragment of C-LYTA at the amino terminus has been described (see Biotechnology 10:795-798, 1992). In preferred embodiments, the repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion exists in the C-segment region starting at residue 178. Particularly preferred repeat portions contain residues 188-305.

In another embodiment, a Ra12 polynucleotide derived from mycobacterium tuberculosis (mycobacterium tuberculosis) is linked to at least the immunogenic portion of the polynucleotide of the invention. Ra12 compositions and methods for their use to enhance expression of heterologous polynucleotide sequences are described in U.S. patent application 60/158,585, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ra12 refers to a polynucleotide region that is a subsequence of mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a serine protease with a molecular weight of 32kD, encoded by a gene in a virulent and avirulent strain of Mycobacterium tuberculosis. Such nucleotide and amino acid sequences for MTB32A have been described (U.S. patent application 60/158,585; see also, Skeiky et al, Infection and Immun. (1999)67:3998-4007, incorporated herein by reference). In one embodiment, the Ra12 polypeptide used to make the fusion polypeptide comprises a C-terminal fragment of the MTB32A coding sequence that is effective to enhance expression and/or immunogenicity of the heterologous chlamydia antigen polypeptide fused thereto. In another embodiment, the Ra12 polypeptide corresponds to the approximately 14kDC terminal fragment of MTB32A that comprises some or all of the amino acid residues 192-323 of MTB 32A.

Recombinant nucleic acids encoding fusion polypeptides comprising the Ra12 polypeptide and a heterologous chlamydia polypeptide of interest can be readily constructed using conventional genetic engineering techniques. The recombinant nucleic acid is constructed such that preferably the Ra12 polynucleotide sequence is located 5' to the selected heterologous chlamydia polynucleotide sequence. It may also be appropriate to place the Ra12 polynucleotide sequence 3' to the selected heterologous polynucleotide sequence, or to insert the heterologous polynucleotide sequence at a position within the Ra12 polynucleotide sequence.

In addition, any suitable polynucleotide encoding Ra12 or a portion or other variant thereof may be used to construct a recombinant fusion polynucleotide comprising Ra12 and one or more chlamydia polynucleotides described herein. Preferred Ra12 polynucleotides generally comprise at least about 15 contiguous polynucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides encoding a portion of a Ra12 polypeptide.

The Ra12 polynucleotide may comprise the native sequence (i.e., the endogenous sequence encoding Ra12 polypeptide or portion thereof) or may comprise a variant of that sequence. A Ra12 polynucleotide variant may contain one or more substitutions, additions, deletions, and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished relative to the biological activity of a fusion polypeptide comprising a native Ra12 polypeptide. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity to a polynucleotide sequence encoding a native Ra12 polypeptide or portion thereof.

In another aspect, the invention provides methods of inducing protective immunity in a patient against chlamydial infection using one or more of the above polypeptides or fusion proteins (or polynucleotides encoding such polypeptides or fusion proteins). Herein, "patient" refers to any warm-blooded animal, preferably a human. The patient may have a disease, or may be absent a detectable disease and/or infection. In other words, protective immunity can be induced to prevent or treat chlamydial infection.

In this regard, the polypeptide, fusion protein or polynucleotide molecule is typically present in a pharmaceutical composition or vaccine. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. A vaccine may comprise one or more of the above polypeptides and an immunostimulant, such as an adjuvant or liposome (in which the polypeptide is contained). These pharmaceutical compositions and vaccines may also contain other chlamydia antigens, which may be incorporated into the combined polypeptide or may be present in separate polypeptides.

Alternatively, the vaccine may contain a polynucleotide encoding one or more of the polypeptides or fusion proteins described above, such that the polypeptide may be produced in situ. In such vaccines, the polynucleotide may be present in any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Suitable nucleic acid expression systems contain polynucleotides necessary for expression in the patient (e.g., suitable promoters and termination signals). The bacterial delivery system involves the administration of bacteria (e.g., bcg) that express an immunogenic portion of the polypeptide on the cell surface. In a preferred embodiment, the polynucleotide may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective) virus. Techniques for incorporating polynucleotides into these expression systems are well known to those of ordinary skill in the art. These polynucleotides may also be administered in the form of "naked" plasmid vectors, as described, for example, in the reviews by Ulmer et al, Science259: 1745-. Techniques for incorporating DNA into these vectors are well known to those of ordinary skill in the art. In addition, the retrovirus may transfer or incorporate genes encoding a selectable marker (to aid in identifying or screening transduced cells) and/or a targeting moiety, such as a gene encoding a ligand for a receptor on a specific target cell to confer targeting specificity to the vector. Targeting can also be achieved using antibodies by methods known to those of ordinary skill in the art.

Other formulations for therapeutic purposes include colloidally dispersed systems such as polymer complexes, nanocapsules, microspheres, beads and lipid-based systems (including oil-in-water emulsions), micelles, mixed micelles and liposomes. The preferred colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (i.e., artificial membrane vesicles). Uptake of naked polynucleotides can be increased by adding the polynucleotide to and/or onto biodegradable beads that can be efficiently transported into cells. The preparation and use of such systems is well known in the art.

In a related aspect, the polynucleotide vaccine described above may be administered simultaneously or sequentially with the polypeptide of the invention or a known chlamydia antigen. For example, a polynucleotide encoding a polypeptide of the invention may be administered ("naked" or in the delivery systems described above) followed by administration of the antigen to enhance the protective immune effect of the vaccine.

The polypeptides and polynucleotides disclosed herein may also be used in adoptive immunotherapy to treat chlamydial infections. Adoptive immunotherapy can be broadly classified as active or passive immunotherapy. In active immunotherapy, treatment relies on stimulating the endogenous host immune system in vivo by administering immune response modifiers (e.g., vaccines, bacterial adjuvants, and/or cytokines).

In passive immunotherapy, treatment involves the delivery of biological agents (e.g., effector cells or antibodies) with defined immunoreactivity that can mediate anti-chlamydial effects directly or indirectly without relying on the intact host immune system. Examples of effector cells include T lymphocytes (e.g., CD8+ cytotoxic T lymphocytes, CD4+ T helper cells), killer cells (e.g., natural killer cells, lymphokine-activated killer cells), B cells, or antigen presenting cells expressing the disclosed antigens (e.g., dendritic cells and macrophages). The polypeptides disclosed herein can also be used to make antibodies or anti-idiotype antibodies (e.g., U.S. patent 4,918,164) for passive immunotherapy.

The main method to obtain sufficient numbers of T cells for adoptive immunotherapy is to culture immune T cells in vitro. Culture conditions for expanding the number of individual antigen-specific T cells to billions and maintaining antigen recognition in vivo are well known in the art. These in vitro culture conditions typically employ intermittent stimulation with antigens and are often performed in the presence of cytokines such as IL-2 and non-dividing feeder cells. As described above, antigen-specific T cell cultures can be rapidly expanded using the immunoreactive polypeptides described herein in order to generate sufficient cell numbers for immunotherapy. In particular, an antigen presenting cell, such as a dendritic cell, macrophage, monocyte, fibroblast, or B cell, can be pulsed with an immunoreactive polypeptide or the polynucleotide sequence can be introduced into an antigen presenting cell using a variety of standard techniques well known in the art. For example, an antigen presenting cell may be transfected or transduced with a polynucleotide sequence containing a promoter region suitable for enhanced expression and capable of being expressed as part of a recombinant virus or other expression system. Antigen presenting cells can be transduced with several viral vectors, including poxviruses, vaccinia viruses, and adenoviruses; antigen presenting cells may also be transfected with the polynucleotide sequences disclosed herein, wherein transfection is performed by a variety of means, including gene gun technology, lipid-mediated delivery, electroporation, osmotic shock, and particle delivery mechanisms, resulting in effective and acceptable expression levels as determined by one of ordinary skill in the art. In order for cultured T cells to be effective in therapy, the cultured T cells must be able to grow and widely distribute in vivo and survive for long periods. Studies have demonstrated that repeated stimulation with IL-2-supplemented antigens can induce growth and long-term survival of cultured T cells in vivo in significant numbers (see, e.g., Cheever, M., et al, "treatment with cultured T cells: re-visit principles," Immunological Reviews, 157:177, 1997).

The polypeptides disclosed herein may also be used to prepare and/or isolate chlamydia-reactive T cells, which may then be administered to a patient. In one technique, antigen-specific T cell lines can be prepared by in vivo immunization with short peptides corresponding to immunogenic portions of the disclosed polypeptides. The resulting antigen-specific CD8+ or CD4+ T cell clones can be isolated from the patient, expanded using standard tissue culture techniques, and then returned to the patient.

Alternatively, chlamydia-reactive T cell subsets can be prepared by selective stimulation and expansion of autologous T cells in vitro using peptides corresponding to immunogenic portions of these polypeptides, providing antigen-specific T cells that can subsequently be transferred into a patient, see, e.g., Chang et al (crit. Cells of the immune System, such as T cells, can be isolated from the peripheral blood of a patient using a commercial cell isolation System (e.g., isolex (tm) System, available from Nexell Therapeutics, inc. The isolated cells are stimulated with one or more immunoreactive polypeptides contained in a delivery vehicle, such as a microsphere, to provide antigen-specific T cells. The antigen-specific T cell population is then expanded using standard techniques and the cells are re-administered back into the patient.

In other embodiments, T cells and/or antibody receptors specific for the polypeptides herein can be cloned, expanded, and transferred to other vectors or effector cells for adoptive immunotherapy. In particular, T cells can be transfected with appropriate genes to express the variable regions from a chlamydia-specific monoclonal antibody as an extracellular recognition element and linked to the T cell receptor signal chain, resulting in T cell activation, specific lysis and cytokine release. This enables T cells to redirect their specificity in an MHC independent manner. See, e.g., Eshhar, z., Cancer Immunol immunoher, 45 (3-4): 131-6, 1997 and Hwu, P., et al, Cancer Res, 55(15):3369-73, 1995. Another embodiment may include transfection of Chlamydia antigen-specific alpha and beta T cell receptor chains into other T cells, see Cole, D J, et al, Cancer Res, 55(4):748-52, 1995.

In yet another embodiment, syngeneic or autologous dendritic cells can be pulsed with peptides corresponding to at least immunogenic portions of the polypeptides disclosed herein. The obtained antigen-specific dendritic cells can be transferred to a patient or used to stimulate T cells to provide antigen-specific T cells, which can then be administered to the patient. The use of peptide-pulsed dendritic cells for the preparation of antigen-specific T cells in a mouse model and the subsequent use of these antigen-specific T cells for disease eradication has been demonstrated by Cheever et al (Immunological Reviews, 157:177, 1997). In addition, vectors expressing the disclosed polynucleotides can be introduced into stem cells taken from a patient, expanded in vitro clones, and used for autologous transplantation back into the same patient.

In certain aspects, the polypeptides, polynucleotides, T cells, and/or binding agents disclosed herein can be incorporated into a pharmaceutical composition or an immunogenic composition (i.e., a vaccine). Alternatively, the pharmaceutical composition may comprise an antigen presenting cell (e.g., a dendritic cell) transfected with a chlamydia polynucleotide so as to express a chlamydia polypeptide. Pharmaceutical compositions may comprise one or more such compounds and a physiologically acceptable carrier. Vaccines may comprise one or more of such compounds and an immunostimulant. The immunostimulant may be any substance that enhances or potentiates an immune response to the foreign antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., lactic-co-glycolic acid), and liposomes (containing such compounds therein; see, e.g., Fullerton, U.S. patent No. 4,235,877). Vaccine formulations can be found generally, for example, in M.F. Powell and M.J. Newman, eds., "Vaccine Design (the subbunit and adjuvant Vaccine)," Plenum Press (NY, 1995). The pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other chlamydia antigens may be present in the composition or vaccine as integrated in the fusion polypeptide or as separate compounds.

The pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides described above, such that the polypeptide may be produced in situ. As noted above, the DNA may be present in any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. A number of gene delivery techniques are well known in the art, see, for example, Roll and, Crit. Rev. therapy. drug Carrier Systems 15:143-198, 1998 and references cited therein. Suitable nucleic acid expression systems contain DNA sequences necessary for expression in the patient (e.g., suitable promoter and termination signals). Bacterial delivery systems involve the application of bacteria (e.g., bcg) that express an immunogenic portion of the polypeptide on their cell surface or secrete the epitope.

In a preferred embodiment, the DNA may be introduced using viral expression systems (e.g., vaccinia or other poxviruses, retroviruses, adenoviruses, baculoviruses, togaviruses, bacteriophages, etc.), which often involve the use of non-pathogenic (defective) replication-competent viruses.

For example, many viral expression vectors are derived from viruses of the family retroviridae. This family includes murine leukemia virus, murine breast cancer virus, human foamy virus, rous sarcoma virus and immunodeficiency virus, including human, simian and feline immunodeficiency viruses. Considerations in designing reverse transcription expression vectors are discussed in Comstock et al (1997).

Kim et al (1998) developed excellent viral expression vectors based on Murine Leukemia Virus (MLV). In constructing MLV vectors, Kim et al found that the entire gag sequence plus the immediately upstream region could be deleted without significantly affecting viral packaging or gene expression. Furthermore, it was found that almost all of the U3 region could be replaced with the immediate early promoter of human cytomegalovirus without deleterious effects. Furthermore, MCR and an Internal Ribosome Entry Site (IRES) can also be added without adverse effects. Based on their observations, Kim et al designed a series of MLV-based expression vectors that contain one or more of the features described above.

With increased awareness of Human Foamy Virus (HFV), features in HFV that favor its use as an expression vector were discovered. These features include expression of pol by splicing and initiation of translation from a defined start codon. For further aspects of HIF virus expression vectors, see review by Bodem et al (1997).

Murakami et al (1997) describe Rous Sarcoma Virus (RSV) -based replication competent avian retroviral vectors, IR1 and IR2, capable of high level expression of heterologous genes. In these vectors, IRES derived from encephalomyocarditis virus (EMCV) was inserted between env gene and heterologous gene. IR1 retained the splice acceptor site, which is present downstream of the env gene, whereas the IR2 vector lacks this site. Murakami et al demonstrated that these vectors can express several different heterologous genes at high levels.

Recently, many lentivirus-based retroviral expression vectors have been developed. Kafri et al (1997) demonstrated that Human Immunodeficiency Virus (HIV) -based expression vectors can persistently express genes delivered directly into the liver and muscle. One advantage of this system is the inherent ability of HIV to transduce non-dividing cells. Since the viruses of Kafri et al are pseudotyped viruses with vesicular stomatitis virus G glycoprotein (VSVG), they can transduce a broad spectrum of tissues and cell types.

A vast number of adenovirus-based expression vectors have been developed, primarily due to the advantages these vectors offer in gene therapy applications. Adenoviral expression vectors and methods of using these vectors are the subject of a number of U.S. patents (including U.S. patent 5,698,202, U.S. patent 5,616,326, U.S. patent 5,585,362, and U.S. patent 5,518,913, all of which are incorporated herein by reference).

Other adenoviral structures are described in Khatri et al (1997) and Tomanin et al (1997). Khatri et al describe novel ovine adenovirus expression vectors and their ability to infect bovine turbinate cells and rabbit kidney cells as well as a range of human cell types including lung and foreskin fibroblasts and liver, prostate, breast, colon and retinal cell lines. Tomanin et al describe adenoviral expression vectors containing the T7RNA polymerase gene. When introduced into cells containing a heterologous gene operably linked to the T7 promoter, the vector is capable of driving gene expression from the T7 promoter. The authors propose that this system can be used to clone and express genes encoding cytotoxic proteins.

Poxviruses are widely used to express heterologous genes in mammalian cells. Over the years, the vector has been improved to allow high expression of heterologous genes and to simplify the process of integrating multiple heterologous genes into a single molecule. Vaccinia virus mutants and other poxviruses that are abortive infected in mammalian cells are of particular interest in an effort to eliminate cytopathic effects and increase safety (oetli et al, 1997). For the use of poxviruses as expression vectors, see Carroll and Moss (1997) for a review.

Togavirus expression vectors, including alphavirus expression vectors, have been used to study the structure and function of proteins and for protein production purposes. The attractive features possessed by togavirus expression vectors are rapid and efficient gene expression, broad host range and RNA genome (Huang, 1996). Moreover, recombinant vaccines based on alphavirus expression vectors have been shown to induce strong humoral and cellular immune responses with good immunological memory and protective effects (Tubulikas et al, 1997). A discussion of alphavirus expression vectors and their use is found, for example, in Lundstrom (1997).

In one study, Li and Garoff (1996) used Semliki Forest Virus (SFV) expression vectors to express retroviral genes and prepare retroviral particles in BHK-21 cells. The particles prepared by this method have protease and reverse transcriptase activity and are infectious. Moreover, helper virus could not be detected in the virus stock. Thus, the system has features that facilitate its use in gene therapy procedures.

Baculovirus expression vectors have traditionally been used to express heterologous proteins in insect cells. Examples of proteins include mammalian chemokine receptors (Wang et al, 1997), reporter proteins such as green fluorescent protein (Wu et al, 1997), and FLAG fusion protein (Wu et al, 1997; Koh et al, 1997). Recent advances in baculovirus expression vector technology, including its use in virion display vectors and expression in mammalian cells, can be reviewed in Possee (1997). Other reviews of baculovirus expression vectors include Jones and Morikawa (1996) and O' Reilly (1997).

Other suitable viral expression systems are disclosed, for example, in Fisher-Hoch et al, Proc. Natl. Acad. Sci. USA 86: 317-; flexner et al, Ann.N.Y.Acad.Sci.569:86-103, 1989; flexner et al, Vaccine 8:17-21, 1990; U.S. patents 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. patent 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; berkner, Biotechniques 6: 616-; rosenfeld et al, Science252: 431-; kolls et al, Proc.Natl.Acad.Sci.USA91: 215-; Kass-Eisler et al, Proc.Natl.Acad.Sci.USA90: 11498-; guzman et al, Circulation 88:2838-2848, 1993; and Guzman et al, cir. Res.73: 1202. 1207, 1993. Techniques for incorporating DNA into these expression vectors are well known to those of ordinary skill in the art. In other systems, the DNA may be introduced as "naked" DNA, as described, for example, in Ulmer et al, reviews by Science 259: 1745-. Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that can be efficiently transported into cells.

Obviously, the vaccine may include polynucleotide and/or polypeptide components, if desired. It will also be apparent that vaccines can contain pharmaceutically acceptable salts of the polynucleotides and/or polypeptides provided herein. These salts can be prepared from pharmaceutically acceptable non-toxic bases including organic bases such as salts of primary, secondary and tertiary amines and salts of basic amino acids and inorganic bases such as sodium, potassium, lithium, ammonium, calcium and magnesium salts. Although any suitable carrier known to those of ordinary skill in the art may be used in the pharmaceutical compositions of the present invention, the type of carrier will vary with the mode of administration. The compositions of the present invention may be formulated to accommodate any suitable mode of administration, including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration. For parenteral administration, e.g., subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above carriers or solid carriers may be employed, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose and magnesium carbonate. Biodegradable microspheres (e.g., polylactate polyglycolite) may also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. nos. 4,897,268 and 5,075,109.

These compositions may also contain buffers (e.g., a neutral buffered saline solution or a phosphate buffered saline solution), sugars (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic, or slightly hypertonic compared to the blood of the recipient, suspending agents, thickening agents, and/or preservatives. Alternatively, the compositions of the present invention may be formulated as a lyophilizate. The compounds may also be encapsulated in liposomes using well known techniques.

Any of a variety of immunostimulants can be used in the vaccines of the present invention. For example, adjuvants may be included. Most adjuvants contain substances designed to protect the antigen from rapid metabolism, such as aluminum hydroxide or mineral oil, and stimulators of the immune response, such as lipid a, Bortadella pertussis or mycobacterium tuberculosis-derived proteins. Suitable adjuvants are commercially available, for example Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); merck adjuvant 65(Merck and Company, inc., Rahway, n.j.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; calcium, iron or zinc salts; an insoluble suspension of acylated tyrosine; acylated sugars; a cationically or anionically derivatized polysaccharide; polyphosphazene; biodegradable microspheres; monophosphoryl lipid a and quil a. Cytokines, such as GM-CSF or interleukin-2, -7 or-12, may also be used as adjuvants.

In the vaccines provided herein, the adjuvant compositions may be designed to induce an immune response that is predominantly of the Th1 or Th2 type under selective conditions. High levels of Th1 type cytokines (e.g., IFN-. gamma., TNF. alpha., IL-2, and IL-12) tend to promote the induction of cell-mediated immune responses to administered antigens. In contrast, high levels of Th 2-type cytokines (e.g., IL-4, IL-5, IL-6, and IL-10) tend to promote the induction of humoral immune responses. After application of the vaccines provided herein, patients will support immune responses including Th1 and Th2 type responses. In a preferred embodiment, when the response is predominantly of the Th1 type, the level of Th1 type cytokines will increase to a greater extent than the level of Th2 type cytokines. The levels of these cytokines can be readily assessed using standard assays. For a review of the cytokine family, see Mosmann and Coffman, Ann. Rev. Immunol.7:145-173, 1989.

Preferred adjuvants for eliciting a Th 1-type predominant response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), and an aluminum salt. MPL adjuvant is available from Corixa corporation (Seattle, Wash.; see U.S. Pat. Nos.4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a Th 1-type predominant response. Such oligonucleotides are well known and described, for example, in WO 96/02555 and WO 99/33488. Immunostimulatory DNA sequences have also been described, for example, in Sato et al, Science273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21(aquila biopharmaceuticals inc., Framingham, MA), which may be used alone or in combination with other adjuvants. For example, enhancement systems include combinations of monophosphoryl lipid a and saponin derivatives, such as QS21 and 3D-MPL (WO 94/00153), or less responsive compositions in which QS21 is quenched with cholesterol (see WO 96/33739). Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly effective adjuvant comprising QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720(Seppic, France), SAF (Chiron, Calif., USA), ISCOMS (CSL), MF-59(Chiron), SBAS series adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixendart, Belgium), Detox (Corixa; Seattle, Wash.), RC-529 (Corixa; Seattle, Wash.) and other aminoglycoside 4-phosphate aminoalkyl esters (AGP), as described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entirety).

All vaccines provided herein can be prepared using well known methods that result in a combination of antigen, immunostimulant and suitable carrier or excipient. The compositions described herein can be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge, or gel (e.g., consisting of a polysaccharide) that effects slow release of the compound after administration). Such formulations can generally be prepared using well known techniques (see, e.g., Coombes et al, Vaccine 14:1429-1438, 1996) and can be administered, for example, orally, rectally or subcutaneously, or by implantation at the desired target site. Sustained release formulations may contain the polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained in a reservoir surrounded by a rate controlling membrane.

The carriers used in these formulations are biocompatible and may also be biodegradable; preferably the formulation provides a relatively constant level of active ingredient release. Such carriers include microparticles of lactic-glycolic acid copolymers, as well as polyacrylates, latexes, starches, celluloses, and dextrans. Other slow release carriers include supramolecular biovectors comprising a non-liquid hydrophilic core (e.g. cross-linked polysaccharides or oligosaccharides) and optionally an outer layer containing amphiphilic compounds such as phospholipids (see, e.g., U.S. patent 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound in the sustained release formulation depends on the site of implantation, the rate and expected duration of release and the nature of the disease to be treated or prevented.

A variety of delivery vehicles (vehicles) may be used in pharmaceutical compositions and vaccines to facilitate the generation of antigen-specific immune responses that target chlamydia-infected cells. Delivery vehicles include Antigen Presenting Cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that can be engineered to become potent APCs. These cells may, but need not, be genetically modified to increase the ability to present antigens, to enhance activation and/or maintenance of a T cell response, to have an anti-chlamydial effect per se, and/or to be immunocompatible with a recipient (i.e., a matched HLA haplotype). Typically, APCs can be isolated from any of a variety of biological fluids and organs, and can be autologous, allogeneic, syngeneic, or allogeneic cells.

Certain preferred embodiments of the invention use dendritic cells or progenitors thereof as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392: 245-. In general, dendritic cells can be identified by their typical shape (star-shaped in situ, with visible distinct cytoplasmic processes (dendrites) in vitro), ability to efficiently uptake, process and present antigen, and ability to activate naive T cell responses. Of course, dendritic cells can be engineered to express specific cell surface receptors or ligands that are not normally present on dendritic cells, either in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secretory vesicles of antigen-loaded dendritic cells (known as exocytic vesicles) may be used in the vaccine (see Zitvogel et al, Nature Med.4:594-600, 1998).

Dendritic cells and progenitor cells can be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, cord blood, or any other suitable tissue or body fluid. For example, dendritic cells can be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13, and/or TNF α to a culture of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow can be differentiated into dendritic cells by adding to the culture medium a combination of GM-CSF, IL-3, TNF α, CD40 ligand, LPS, flt3 ligand and/or other compounds that induce differentiation, maturation and proliferation of dendritic cells.

Dendritic cells can be conveniently divided into "immature" and "mature" cells, which allows two distinct phenotypes to be distinguished in a simple manner. However, this nomenclature should not be construed as excluding all possible intermediate differentiation stages. Immature dendritic cells can be characterized as APCs with high antigen uptake and processing capacity associated with high expression of Fc γ receptors and mannose receptors. The mature phenotype is typically characterized by low expression of these markers, but high expression of cell surface molecules responsible for T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1 BB).

The APC can generally be transfected with a polynucleotide encoding a Chlamydia protein (or portion or other variant thereof) such that the Chlamydia polypeptide or immunogenic portion thereof can be expressed on the cell surface. The transfection may be performed ex vivo, and the composition or vaccine comprising the transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets dendritic cells or other antigen presenting cells can be administered to the patient, resulting in transfection occurring in vivo. For example, dendritic cells can be transfected in vivo and ex vivo using any method well known in the art, such as the method described in WO97/24447, or the biolistic method described in Mahvi et al, Immunology and cell Biology75:456-460, 1997. Can be prepared by using a chlamydia polypeptide, DNA (naked or in a plasmid vector) or RNA; or by incubating dendritic cells or progenitor cells with recombinant bacteria or viruses expressing the antigen (e.g., vaccinia virus, fowlpox virus, adenovirus or lentiviral vectors) to load the dendritic cells with the antigen. Prior to loading, the polypeptide may be covalently conjugated to an immunological partner (e.g., a carrier molecule) that may provide T cell help. Alternatively, the dendritic cells can be pulsed with a non-conjugated immunological partner alone or in the presence of a polypeptide.

The route and frequency of administration, as well as the dosage of the pharmaceutical compositions and vaccines vary from individual to individual. In general, the pharmaceutical compositions and vaccines can be administered by injection (e.g., intradermally, intramuscularly, intravenously or subcutaneously), intranasally (e.g., by inhalation), or orally. The administration can be 1-3 times over a period of 1-36 weeks. Preferably, 3 administrations are performed, at 3-4 month intervals, after which booster inoculations may be given periodically. Different dosing regimens may be appropriate for different patients. A suitable amount to be administered is an amount of polypeptide or DNA which, when administered as described above, is capable of eliciting an immune response in the patient to be immunized which is sufficient to protect the patient from chlamydial infection for at least 1-2 years. Generally, the amount of polypeptide present in a dose (or the amount of polypeptide produced in situ from DNA in a dose) is from about 1pg to about 100mg/kg host, typically from about 10pg to about 1mg, preferably from about 100pg to about 1. mu.g. Suitable amounts for administration vary with the size of the patient's volume, but typically range from about 0.1mL to about 5 mL.

Although any suitable carrier known to those of ordinary skill in the art may be used in the pharmaceutical compositions of the present invention, the type of carrier depends on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above carriers or solid carriers may be employed, such as mannitol, lactose, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose and magnesium carbonate. Biodegradable microspheres (e.g., lactic acid-glycolic acid copolymer) can also be used as carriers for the pharmaceutical compositions of the present invention. Biodegradable microspheres for use are disclosed, for example, in U.S. Pat. nos. 4,897,268 and 5,075,109.

In general, an appropriate dosage and treatment regimen will provide an amount of active compound sufficient to result in a therapeutic and/or prophylactic benefit. This response can be monitored by determining the improved clinical outcome exhibited by treated patients compared to untreated patients. An increase in the preexisting immune response to the chlamydia protein is generally associated with improved clinical outcomes. Typically, these immune responses can be evaluated using standard proliferation, cytotoxicity, or cytokine assays, which can be performed using samples obtained from the patient before and after treatment.

In another aspect, the invention provides methods for diagnosing chlamydial infection using the polypeptides described above. In this aspect, the invention provides methods of detecting chlamydial infection in a biological sample using one or more of the above polypeptides, alone or in combination. For clarity, the term "polypeptide" is used in describing particular embodiments of the diagnostic methods of the present invention. However, it will be apparent to those skilled in the art that the fusion proteins of the present invention may also be used in these methods.

Herein, "biological sample" refers to a sample containing antibodies obtained from a patient. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient. The polypeptide is used in the assay to determine whether antibodies to the polypeptide are present in the sample by comparing predetermined cut-off values, as described below. The presence of this antibody indicates prior sensitization with a chlamydia antigen, which may be indicative of a chlamydia infection.

In embodiments where more than one polypeptide is used, the polypeptides used are preferably complementary (i.e., one polypeptide component is prone to detect an infection in a sample that is not detected by another polypeptide component). Typically, complementary polypeptides can be identified by evaluating serum samples obtained from a series of patients known to be infected with chlamydia using each polypeptide individually. After determining which samples can be detected as positive using each polypeptide (see below), a combination of two or more polypeptides can be formulated that will be capable of detecting infection in most or all of the test samples.

For the detection of antibodies in a sample using one or more polypeptides, various formats of assays are known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: ALABORT Manual, Cold Spring Harbor Laboratory, 1988 (incorporated herein by reference). In a preferred embodiment, the assay involves binding and isolating antibodies in a sample using polypeptides immobilized on a solid support. The bound antibody can then be detected using a detection reagent containing a reporter group. Suitable detection reagents include antibodies that bind to the antibody/polypeptide complex and free polypeptide labeled with a reporter group (e.g., in a semi-competitive assay). Alternatively, a competition assay may be utilized in which an antibody that binds to the polypeptide is labeled with a reporter group and allowed to bind to the immobilized antigen after the antigen and sample are incubated together. The degree of inhibition of binding of the labeled antibody and polypeptide by the components of the sample is indicative of the reactivity of the sample with the immobilized polypeptide.

The solid support may be any solid material known to one of ordinary skill in the art to which an antigen may be attached. For example, the solid support may be a test well in a microtiter plate, or nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, glass fibre, latex or a plastics material such as polystyrene or polyvinyl chloride. The support may also be a magnetic particle or fiber optic sensor such as those described in U.S. Pat. No. 5,359,681.

The polypeptides can be bound to the solid support using a variety of techniques known to those of ordinary skill in the art. In the context of the present invention, the term "binding" refers to non-covalent attachment such as adsorption and covalent attachment (which may be a direct bond between the antigen and a functional group on the support, or may be a bond through a cross-linking agent). Preferably by adsorption to a well or membrane of the microtiter plate. In these cases, adsorption may be achieved by contacting the polypeptide with the solid support in a suitable buffer for an appropriate period of time. The contact time varies with temperature but is typically between about 1 hour and 1 day. Generally, contacting one well of a plastic microtiter plate with an amount of polypeptide from about 10ng to about 1mg, preferably about 100ng, is sufficient to bind a sufficient amount of antigen.

Covalent attachment of the polypeptide to the solid support can generally be achieved by first reacting the support with a bifunctional reagent (i.e., a reagent that can react with a functional group, such as a hydroxyl or amino group, on the support and polypeptide). For example, the polypeptide may be bound to a support having a suitable polymer coating using benzoquinone or by condensation of aldehyde groups on the support with amines and active hydrogens on the polypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, A12-A13).

In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). The assay may be performed by first contacting the sample with the polypeptide antigen which has been immobilized on a solid support, typically a microtiter plate, so that anti-polypeptide antibodies in the sample can bind to the immobilized polypeptide. Unbound sample is then removed from the immobilized polypeptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection reagent remaining bound to the solid support is then determined using methods appropriate for the particular detection reagent.

More specifically, once the polypeptide is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as Bovine Serum Albumin (BSA) or Tween 20 ^TM(Sigma Chemical Co., St. Louis, Mo.) may be used. The immobilized polypeptide is then incubated with the sample and the antibody and antigen are allowed to bind. The sample may be diluted with a suitable diluent such as Phosphate Buffered Saline (PBS) prior to incubation. Generally, an appropriate contact time (i.e., incubation time) refers to a period of time sufficient to detect the presence of the antibody in the HGE-infected sample.Preferably, the contact time is long enough to achieve a level of binding that is at least 95% of the level of binding achieved when the bound and unbound antibodies are in equilibrium. It will be appreciated by those of ordinary skill in the art that the time necessary to reach equilibrium can be readily determined by testing the level of binding over a period of time. An incubation time of typically about 30 minutes at room temperature is sufficient.

This can then be done by using an appropriate buffer, e.g. containing 0.1% Tween 20^TMThe solid support was washed with PBS to remove unbound sample. Detection reagents can then be added to the solid support. Suitable detection reagents are any compounds that bind to the immobilized antibody-polypeptide complex and can be detected by any of a variety of methods known in the art. Preferably, the detection reagent contains a binding reagent (e.g., protein a, protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Preferred reporter groups include enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, chemiluminescent groups, fluorescent groups, and biotin. Conjugation of the binding agent and reporter group can be accomplished using standard methods known to those of ordinary skill in the art. Common binding agents conjugated to a variety of reporter groups can also be purchased from a number of commercial sources (e.g., Zymed Laboratories, San Francisco, calif., and Pierce, Rockford, IL).

The detection reagent and the immobilized antibody-polypeptide complex are then incubated together for an amount of time sufficient to detect the bound antibody. The appropriate length of time can generally be determined from the manufacturer's specifications, or by testing the level of binding over a period of time. Unbound detection reagent is then removed and the bound detection reagent is detected using the reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiography is generally appropriate. Spectroscopy can be used to detect dyes, chemiluminescent groups, and fluorescent groups. Biotin can be detected using avidin coupled to a different reporter group, usually a radioactive or fluorescent group or an enzyme. The enzyme reporter group can generally be detected by addition of a substrate (generally over a specified period of time) followed by analysis of the reaction product by spectroscopy or other methods.

To determine whether anti-chlamydia antibodies are present in a sample, the signal detected from the reporter group remaining bound to the solid support is typically compared to a signal corresponding to a predetermined cut-off value. In a preferred embodiment, the cut-off value is the average signal obtained when the immobilized antibody is incubated with a sample from an uninfected patient. Generally, samples that produce a signal of 3 standard deviations above the predetermined cutoff value are considered positive for chlamydial infection. In another preferred embodiment, the cut-off value is determined by the method of Sackett et al (Clinical epidemiology: Basic Science for Clinical Medicine), LittleBrown and Co., 1985, pp.106-107) using a Receiver Operator curve. Briefly, in this embodiment, the true positive rate (i.e., sensitivity) and false positive rate (100% specificity) for each possible cutoff value of the diagnostic test result are plotted, and the cutoff value can then be determined from the plot. The cut-off value closest to the top left on the graph (i.e., the value that includes the largest area) is the most accurate cut-off value, and samples that produce a signal above the cut-off value determined by the method may be considered positive. Alternatively, the cutoff value may be shifted to the left along the graph to minimize the false positive rate, or to the right to minimize the false negative rate. Generally, samples that produce a signal above the cut-off value determined by this method are considered positive for chlamydial infection.

In a related embodiment, the assay is performed on a rapid flow-through or strip test format in which the antigen is immobilized on a membrane such as nitrocellulose. In the flow-through test, antibodies in the sample bind to the immobilized polypeptides as the sample passes through the membrane. The detection reagent (e.g., protein a-colloidal gold) will then bind to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane. The bound detection reagent may then be detected as described above. In the strip test format, one end of the membrane to which the polypeptide is bound is immersed in a solution containing the sample. The sample migrates along the membrane through the zone containing the detection reagent and to the zone containing the immobilized polypeptide. Concentration of the detection reagent at the polypeptide location indicates the presence of anti-chlamydia antibodies in the sample. Typically, concentration of the detection reagent at the site produces a pattern, such as a line, that can be observed by the naked eye. The absence of this graphic indicates a negative result. Generally, the amount of polypeptide immobilized on the membrane is selected so that a visually recognizable pattern is produced at the location of the polypeptide when the biological sample contains a level of polypeptide sufficient to produce a positive signal in the ELISA discussed above. Preferably, the amount of polypeptide immobilized on the membrane is between about 25ng and about 1 μ g, more preferably between about 50ng and about 500 ng. These tests are typically able to be performed with very small amounts (e.g., one drop) of patient serum or blood.

Of course, there are many other assays that are suitable for use with the polypeptides of the invention. The above description is intended by way of example only. An example of an alternative assay protocol that may be used in these methods is Western blotting, in which proteins present in a biological sample are separated on a gel and then exposed to a binding agent. These techniques are well known to those skilled in the art.

The invention also provides agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to chlamydia proteins. An antibody or antigen-binding fragment thereof is considered herein to "specifically bind" to a chlamydia protein if it reacts at a detectable level with the chlamydia protein (in, for example, an ELISA) but does not detect a reaction with an unrelated protein under similar conditions. As used herein, "association" refers to the non-covalent attachment of two separate molecules such that a complex is formed. Binding capacity can be assessed, for example, by determining the binding constant at which the complex is formed. The binding constant is a value obtained when the concentration of the complex is divided by the product of the concentrations of the components. Generally, in the context of the present invention, when the complex formation binding constant is greater than about 10 ³L/mol, two compounds are considered to be "associated". The combinationConstants can be determined using methods well known in the art.

Using the representative assays provided herein, the binding agents are also able to distinguish whether a patient is infected with chlamydia. In other words, an antibody or other binding agent that binds to a chlamydia protein will produce a signal indicative of the presence of a chlamydia infection in at least about 20% of patients with the disease, while a signal indicative of the absence of the disease will be produced in at least about 90% of individuals who are not infected. To determine whether a binding agent meets this requirement, the presence of a polypeptide that binds to the binding agent in a biological sample (e.g., blood, serum, sputum, urine, and/or biopsy) from a chlamydia-infected or uninfected patient (as determined by standard clinical tests) can be determined as described herein. It is clear that the number of diseased and non-diseased samples to be analyzed should be statistically significant. Each binder should meet the above criteria; however, one of ordinary skill in the art will appreciate that multiple binders may be used in combination to increase sensitivity.

Any agent that meets the above requirements may be a binding agent. For example, the binding agent may be a ribosome, RNA molecule or polypeptide with or without a peptide component. In a preferred embodiment, the binding agent is an antibody or antigen-binding fragment thereof. Antibodies can be prepared using any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be prepared by cell culture techniques, including the preparation of monoclonal antibodies as described herein, or by transfecting an appropriate bacterial or mammalian cell host with an antibody gene, so as to allow for the production of recombinant antibodies. In one technique, an immunogen comprising a polypeptide of the invention is first injected into any one of a variety of mammals (e.g., a mouse, rat, rabbit, sheep, or goat). In this step, the polypeptide of the present invention may be used as an immunogen without modification. Or, especially for relatively short polypeptides, if the polypeptide and carrier protein, such as bovine serum albumin or keyhole limpet Hemocyanins, when linked together, can elicit excellent immune responses. The animal host is injected with the immunogen and one or more boosters are added, preferably according to a predetermined schedule, and then blood is periodically taken from the animal. Polyclonal antibodies specific for the polypeptide can then be purified from the antisera, for example by affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interest can be prepared, for example, using the techniques of Kohler and Milstein (Eur. J. Immunol.6:511-519, 1976), and modifications thereof. Briefly, these methods involve the preparation of immortalized cell lines capable of producing antibodies with the desired specificity (i.e., reactivity with the polypeptide of interest). These cell lines can be prepared, for example, from spleen cells obtained from animals immunized as described above. The spleen cells are then immortalized, for example, by fusion with a myeloma cell fusion partner, preferably a myeloma cell that is isogenic to the immunized animal. A variety of fusion techniques may be employed. For example, spleen cells and myeloma cells can be mixed with a non-ionic detergent for several minutes and then plated at low density on selective media that support hybrid cells but not myeloma cell growth. The preferred screening technique employs HAT (hypoxanthine, aminopterin, thymidine) screening. After a sufficient period of time, typically about 1-2 weeks, hybrid colonies can be observed. Single colonies were selected and their culture supernatants tested for binding activity to the polypeptides of the invention. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies can be isolated from the supernatant of the cultured hybridoma colonies. In addition, various techniques can be used to increase production, such as injecting the hybridoma cell line into the abdominal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibodies can then be harvested from ascites fluid or blood. Impurities can be removed from these antibodies by conventional techniques such as chromatography, gel filtration, precipitation and extraction. The polypeptides of the invention may be used in purification methods such as affinity chromatography steps.

In certain embodiments, it may be preferred to employ antigen-binding fragments of antibodies. These fragments include Fab fragments, which can be prepared using standard techniques. Briefly, immunoglobulins can be purified from rabbit serum by affinity chromatography on a protein A bead column (Harlow and Lane, antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988), followed by papain digestion to produce Fab and Fc fragments. The Fab and Fc fragments can be separated by affinity chromatography on a protein A bead column.

The monoclonal antibodies of the invention may be conjugated with one or more therapeutic agents. Suitable therapeutic agents in this regard include radionuclides, differentiation inducing agents, drugs, toxins and derivatives thereof. Preferred radionuclides include ⁹⁰Y、¹²³I、¹²⁵I、¹³¹I、¹⁸⁶Re、¹⁸⁸Re、²¹¹At and²¹²and (4) Bi. Preferred drugs include methotrexate, and analogs of pyrimidine and purine. Preferred differentiation inducers include phorbol ester and butyric acid. Preferred toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pseudomonas exotoxin, shigella toxin, and pokeweed antiviral protein.

The therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody, either directly or indirectly (i.e., through a linker group). When the agent and the antibody each have a substituent capable of reacting with each other, a direct reaction between them is possible. For example, a nucleophilic group on one, such as an amino or thiol group, can react with a carbonyl-containing group, such as an anhydride or acid halide, or an alkyl group containing a good leaving group (e.g., a halogen) on the other.

Alternatively, it may be desirable to couple the therapeutic agent and the antibody via a linker group. The linker group can act as a spacer, separating the antibody and agent to avoid interference with the binding properties. Linker groups may also be used to enhance the chemical reactivity of substituents on the agent or antibody, thereby increasing the coupling efficiency. The enhanced chemical reactivity may also facilitate applications of the agent, or functional groups on the agent, that would otherwise not be possible.

It will be apparent to those skilled in the art that there are a variety of homofunctional and heterofunctional bifunctional or multifunctional reagents, such as those described in the Pierce Chemicals company (Rockford, IL) catalog, that can be used as linker groups. Conjugation may be achieved, for example, by amino, carboxyl, sulfhydryl or oxidized sugar residues. There are many documents describing this methodology, such as U.S. Pat. No. 4,671,958 to Rodwell et al.

When the therapeutic agent is more effective when separated from the antibody portion of the immunoconjugate of the invention, it may be desirable to employ a linker group that is cleavable during or after internalization into the cell. Many different cleavable linker groups have been described. Mechanisms for intracellular release of therapeutic agents from these linker groups include cleavage by reduction of disulfide bonds (e.g., Spitler, U.S. Pat. No. 4,489,710), irradiation of photolabile bonds (e.g., Senter, et al, U.S. Pat. No. 4,625,014), hydrolysis of derivatized amino acid side chains (e.g., Kohn, et al, U.S. Pat. No. 4,638,045), serum complement-mediated hydrolysis (e.g., Rodwell, et al, U.S. Pat. No. 4,671,958), and acid-catalyzed hydrolysis (e.g., Blattler, et al, U.S. Pat. No. 4,569,789).

It may be desirable to conjugate more than one agent to the antibody. In one embodiment, multiple molecules of one agent are conjugated to one antibody molecule. In another embodiment, more than one type of agent may be conjugated to an antibody. Regardless of the specific embodiment, there are a variety of ways to prepare immunoconjugates with more than one agent. For example, more than one agent may be directly conjugated to an antibody molecule, or linkers providing multiple attachment sites may be employed. Alternatively, a carrier may be used.

The carrier may carry the agent in a variety of ways, including covalent bonding, either directly or through a linker group. Suitable carriers include proteins such as albumin (e.g., U.S. Pat. No. 4,507,234 to Kato et al), peptides, and polysaccharides such as glycosaminoglycans (e.g., U.S. Pat. No. 4,699,784 to Shih et al). The carrier may also carry the agent by non-covalent association or by inclusion, for example, in liposome vesicles (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers that are particularly useful for radionuclide agents include radiohalogenated small molecules and chelating compounds. Representative radiohalogenated small molecules and their synthesis are disclosed, for example, in U.S. patent 4,735,792. Radionuclide chelates may be formed from chelating compounds, including those containing nitrogen and sulfur atoms as donor atoms for binding metal or metal oxide radionuclides. For example, Davison et al, U.S. Pat. No. 4,673,562, discloses representative chelating compounds and their synthesis.

For these antibodies and immunoconjugates, a variety of routes of administration can be employed. Typically, administration can be intravenous, intramuscular, subcutaneous, or by an appropriate method in a site-specific area. It is clear that the precise dosage of antibody/immunoconjugate will vary with the antibody used, the antigen density, and the clearance rate of the antibody.

Antibodies can be used in diagnostic assays to detect the presence of chlamydia antigens using test methods similar to those described above and other techniques well known to those skilled in the art, thereby providing a means of detecting chlamydial infection in a patient.

The diagnostic reagents of the invention also comprise DNA sequences encoding one or more of the above polypeptides, or one or more portions thereof. For example, at least two oligonucleotide primers, at least one of which is specific for a DNA molecule encoding a polypeptide of the invention, can be used in a Polymerase Chain Reaction (PCR) -based assay to amplify chlamydia-specific cDNA derived from a biological sample. The presence of the amplified cDNA may then be detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes specific for DNA encoding a polypeptide of the invention can be used in hybridization assays to detect the presence of a polypeptide of the invention in a biological sample.

As used herein, the term "oligonucleotide primer/probe specific for a DNA molecule" refers to an oligonucleotide sequence that is at least about 80%, preferably at least about 90%, and more preferably at least about 95% identical to the DNA molecule in question. Oligonucleotide primers and/or probes that may be used in the diagnostic methods of the invention preferably have at least about 10-40 nucleotides. In a preferred embodiment, the oligonucleotide primer contains at least about 10 contiguous nucleotides of a DNA molecule encoding one of the polypeptides disclosed herein. Preferably, the oligonucleotide probe used in the diagnostic methods of the present invention comprises at least about 15 contiguous nucleotides of a DNA molecule encoding one of the polypeptides disclosed herein. Techniques for PCR-based assays and hybridization assays are well known in the art (see, e.g., Mullis et al, supra; Ehrlich, supra). Thus, a primer or probe can be used to detect a chlamydia-specific sequence in a biological sample. DNA probes or primers comprising the above-mentioned oligonucleotide sequences may be used alone or in combination with each other.

The following examples are offered by way of illustration and not by way of limitation.

Example 1

Isolation of DNA sequence encoding Chlamydia antigen

Chlamydia antigens of the invention which have been shown to induce proliferation of PBMCs and IFN-. gamma.in an immunoreactive T cell line were isolated by expression cloning of a genomic DNA library from Chlamydia trachomatis LGVII essentially as described by Sanderson et al (J.Exp.Med.1995, 182: 1751-.

Chlamydia-specific T cell lines were prepared by stimulating PBMCs from normal donors with an infectious history of the chlamydeless reproductive tract with the protoplasts (elementary bodies) of chlamydia trachomatis LGVII. This T cell line, termed TCL-8, was found to recognize monocyte-derived dendritic cells infected with Chlamydia trachomatis and Chlamydia pneumoniae.

A randomly sheared genomic library of Chlamydia trachomatis LGV II was constructed in lambda zAP (Stratagene, La Jolla, Calif.) and the amplified library was plated at a density of 30 clones/well in 96-well microtiter plates. The bacteria were induced to express the recombinant protein in the presence of 2mM IPTG for 3 hours, then pelleted and resuspended in 200. mu.l RPMI 10% FBS. 10 μ l of the induced bacterial suspension was transferred to a 96-well plate containing autologous monocyte-derived dendritic cells. After 2 hours induction, the dendritic cells were washed to remove free E.coli and then Chlamydia-specific T cells were added. Positive E.coli pools (pools) were identified by measuring IFN-. gamma.production and T cell proliferation in response to the E.coli pool.

4 positive pools were identified and resolved to yield 4 pure clones with insert sizes of 481bp, 183bp, 110bp and 1400bp, respectively (designated 1-B1-66, 4-D7-28, 3-G3-10 and 10-C10-31). The determined DNA sequences of 1-B1-66, 4-D7-28, 3-G3-10 and 10-C10-31 are provided in SEQ ID NOS: 1-4, respectively. Clone 1-B1-66 was located approximately in the 536690 region of the Chlamydia trachomatis genome (NCBI Chlamydia trachomatis database). In clone 1-B1-66, an Open Reading Frame (ORF) (nucleotide 115-375) encoding the previously identified 9kDa protein (Stephens et al, Genbank accession AE001320) was identified whose sequence is provided in SEQ ID NO: 5 in (c). Clone 4-D7-28 is a smaller region within the same ORF (amino acids 22 to 82 of 1-B1-66). Clone 3-G3-10 was located approximately in the 74559 region of the Chlamydia trachomatis genome. The insert was cloned in the antisense orientation with respect to its orientation in the genome. Clone 10-C10-31 contained an open reading frame corresponding to the sequence of the previously disclosed Chlamydia trachomatis S13 ribosomal protein (Gu, L.et al, J.bacteriology, 177: 2594-one 2601, 1995). The predicted protein sequences for 4-D7-28 and 10-C10-31 are provided in SEQ ID NOs: 6 and 12. The predicted protein sequence of 3-G3-10 is provided in SEQ ID NO: 7-11.

In a related series of screening studies, another T cell line was used to screen the genomic DNA library of the aforementioned Chlamydia trachomatis LGV II. A chlamydia-specific T cell line (TCT-1) was obtained from chlamydia genital tract infected patients by stimulating PBMCs of the patients with autologous monocyte-derived dendritic cells infected with chlamydia trachomatis LGV II protoplasts. One clone, 4C9-18(SEQ ID NO: 21), contained a 1256bp insert that elicited a specific immune response from the Chlamydia-specific T cell line TCT-1, as measured in a standard proliferation assay. Subsequent analysis showed that this clone contained 3 known sequences: lipoamide dehydrogenase (Genbank accession AE001326), disclosed in SEQ ID NO: 22, respectively; putative protein CT429(Genbank accession AE001316), disclosed in SEQ ID NO: 23, and (b); a portion of the open reading frame of ubiquinone methyltransferase CT428(Genbank accession AE001316), disclosed in SEQ ID NO: 24, respectively.

In a further study involving clone 4C9-18(SEQ ID NO: 21), the full-length amino acid sequence of the lipoamide dehydrogenase of Chlamydia trachomatis (LGV II) (SEQ ID NO: 22) was expressed in clone CtL2-LPDA-FL disclosed in SEQ ID NO: 90 (c).

To further characterize this open reading frame containing the T cell stimulatory epitope, a cDNA fragment bearing a cDNA sequence encoding a 6 × histidine tag at the amino terminus and containing nucleotides 1-695 of clone 4C9-18 was subcloned into the pET17b vector (Novagen, Madison, Wis.) in the NdeI/EcoRI site, designated clone 4C9-18#2 BL21pLys S (SEQ ID NO: 25, the corresponding amino acid sequence being provided in SEQ ID NO: 26), and transformed into E.coli. Selective induction of transformed E.coli with 2mM IPTG for 3 hours resulted in the expression of a 26kDa protein from clone 4C9-18#2 BL21pLysS, as confirmed by standard Coomassie blue stained SDS-PAGE. To determine the immunogenicity of the protein encoded by clone 4C9-18#2 BL21pLysS, E.coli expressing the 26kDa protein was grown at 1X 10⁴Monocyte-derived dendritic cells were titrated on and incubated for 2 hours. The dendritic cell culture was washed and 2.5X 10 added⁴T cells (TCT-1) and allowed to incubate for an additional 72 hours at which time the level of IFN-. gamma.in the culture supernatant was determined by ELISA. As shown in FIG. 1, the T cell line TCT-1 was found to be responsive to induced cultures (as measured by IFN-g), indicating a Chlamydia-specific T cell response against the thiooctanoyl dehydrogenase sequence. Similarly, the protein encoded by clone 4C9-18#2 BL21pLysS was shown to stimulate TCT- 1T cell line.

Subsequent studies to identify additional Chlamydia trachomatis antigens using the CD4+ T cell expression cloning technique described above yielded additional clones. The genomic library of Chlamydia trachomatis LGVII was screened using TCT-1 and TCL-8 Chlamydia specific T cell lines and TCP-21T cell line. The TCP-21T cell line is derived from a patient with a humoral immune response to chlamydia pneumoniae. TCT-1 cell lines identified 37 positive pools (pools), TCT-3 cell lines identified 41 positive pools, and TCP-21 cell lines identified 2 positive pools. The following 10 positive pools from which clones were derived. Clone 11-A3-93(SEQ ID NO: 64), identified by the TCP-21 cell line, is a 1339bp genomic fragment with homology to the HAD superfamily (CT 103). The second insert in the same clone has homology to the fab I gene (CT104) present on the complementary strand. Clone 11-C12-91(SEQ ID NO: 63), identified using the TCP-21 cell line, has a 269bp insert that is part of the OMP2 gene (CT443) and has homology to the Chlamydia pneumoniae 60kDa cysteine-rich outer membrane protein.

Clone 11-G10-46(SEQ ID NO: 62), identified using the TCT-3 cell line, contained a 688bp insert with homology to pseudoprotein CT 610. Clone 11-G1-34(SEQ ID NO: 61) identified using the TCT-3 cell line had two partial Open Reading Frames (ORFs), and the insert was 1215bp. in size, one ORF shares homology with the malate dehydrogenase gene (CT376) and the other ORF shares homology with the glycohydrolase gene (CT 042). Clone 11-H3-68(SEQ ID NO: 60) identified using the TCT-3 cell line had two ORFs and a total insert size of 1180 bp. One part of the ORF encodes the plasmid-encoded PGP6-D virulence protein, while the other ORF is the complete ORF of the L1 ribosomal gene (CT 318). Clone 11-H4-28(SEQ ID NO: 59), identified using TCT-3, has an insert size of 552bp and is part of the ORF of the dnaK gene (CT 396). Clone 12-B3-95(SEQ ID NO: 58), identified using TCT-1, has a 463bp insert that is part of the lipoamide dehydrogenase gene (CT557) ORF. Clones 15-G1-89 and 12-B3-95 were identical (SEQ ID NOS: 55 and 58, respectively), were identified using the TCT-1 cell line, had an insert size of 463bp, and were part of the lipoamide dehydrogenase gene (CT557) ORF. Clone 12-G3-83(SEQ ID NO: 57), identified using the TCT-1 cell line, had a 1537bp insert and had a partial ORF of the putative protein CT 622.

Clone 23-G7-68(SEQ ID NO: 79), identified using the TCT-3 cell line, contained a 950bp insert and contained a small portion of the L11 ribosomal ORF, the entire ORF of the L1 ribosomal protein and a portion of the ORF of the L10 ribosomal protein. In addition, the clones identified patient cell lines CT4, CT5, CT11, CT12 and CHHO 37. Clone 22-F8-91(SEQ ID NO: 80), identified using the TCT-1 cell line, contained a 395bp insert that contained a partial pmpC ORF on the complementary strand of the clone. Clone 21-E8-95(SEQ ID NO: 81) identified using the TCT-3 cell line contained a 2,085bp insert containing a partial CT613 ORF, the complete ORF of CT612, the complete ORF of CT611 and the partial ORF of CT 610. Clone 19-F12-57(SEQ ID NO: 82), identified using the TCT-3 cell line, contained a 405bp insert containing a partial CT858 ORF and a small portion of recAORF. Clone 19-F12-53(SEQ ID NO: 83), identified using the TCT-3 cell line, contained a 379bp insert, which is part of the ORF of CT455 encoding glutamyl tRNA synthetase. Clone 19-A5-54(SEQ ID NO: 84), identified using the TCT-3 cell line, contained a 715bp insert that was part of ORF3 of the cryptic plasmid (the complementary strand of this clone). Clone 17-E1-72(SEQ ID NO: 85), identified using the TCT-1 cell line, contained a 476bp insert that was part of the ORFs for Opp _2 and pmpD. The pmpD region of this clone was covered by the pmpD region of clone 15-H2-76. Clone 17-C1-77(SEQ ID NO: 86), identified using patient cell lines CT3, CT1, CT4, and CT12, contained a 1551bp insert that was part of the CT857 ORF and part of the CT858 ORF. Clone 15-H2-76(SEQ ID NO: 87), identified using the TCT-1 cell line, contained a3,031 bp insert containing the majority of the pmpD ORF, a portion of the CT089 ORF, and a portion of the ORF of SycE. Clone 15-A3-26(SEQ ID NO: 88) contained a 976bp insert containing a partial ORF of CT 858. Clone 17-G4-36, (SEQ ID NO: 267) identified using patient cell lines CL8, TCT-10, CT1, CT5, CT13 and CHHO37 contained a 680bp insert that is in frame with the β -gal in this plasmid and homologous to a portion of the ORF of the DNA-directed RNA polymerase β subunit (CT 315 in SerD).

Several of the above clones share homology with various polymorphic membrane proteins. The genomic sequence of C.trachomatis contains a family of 9 polymorphic membrane protein genes (referred to as pmp). These genes are referred to as pmpA, pmpB, pmpC, pmpD, pmpE, pmpF, pmpG, pmpH and pmpI. These gene-expressed proteins are believed to be of biological importance in generating a protective immune response against chlamydial infection. In particular, pmpC, pmpD, pmpE, and pmpI contain predictable signal peptides, suggesting that they are outer membrane proteins and therefore potential immune targets.

Based on the C.trachomatis LGVII serovariant sequence, primer pairs were designed to PCR amplify a full-length fragment of pmpC, pmpD, pmpE, pmpG, pmpH and pmpI. The resulting fragment was subcloned into the DNA vaccinia vector JA4304 or JAL (which is JA4304 with a modified linker) (SmithKine beeecham, london, uk). Specifically, pmpC was subcloned into the JAL vector using 5 'oligonucleotide GAT AGG CGC GCC GCA ATC ATG AAA TTT ATG TCA GCT ACT GCT G and 3' oligonucleotide CAG AAC GCG TTT AGA ATG TCA TAC GAG CAC CGC A (provided in SEQ ID NOS: 197 and 198, respectively). After inserting a short nucleotide sequence GCAATC (SEQ ID NO: 199) upstream of the ATG to generate a Kozak-like sequence, the gene was PCR-amplified under conditions well known in the art and ligated to the 5 'ASCI/3' MluI site of the JAL vector. The resulting expression vector contained a full-length pmpC gene (SEQ ID NO: 173) containing 5325 nucleotides with a putative signal sequence, which encodes a 187kD protein (SEQ ID NO: 179). The pmpD gene was PCR amplified using the following oligonucleotides: 5 'oligo TGC AAT CAT GAG TTC GCA GAA AGA TAT AAA AAG C (SEQ ID NO: 200) and 3' oligo CAG AGC TAG CTT AAA AGA TCA ATC GCA ATCCAG TAT TC (SEQ ID NO: 201), and this gene was then subcloned into JA4303 vaccinia vector. This gene was ligated into the 5 'blunt HII I/3' MluI site of the JA4304 vaccinia vector using standard techniques well known in the art. CAATC (SEQ ID NO: 202) was inserted upstream of the ATG to generate a Kozak-like sequence. This clone was unique because blunt end ligation caused the last threonine at the HindIII site and the last glycine of the Kozak-like sequence to be lost. The insert, the 4593bp nucleotide fragment (SEQ ID NO: 172), is a pmpD full-length gene containing a putative signal sequence, encoding a 161kD protein (SEQ ID NO: 178). The pmpE was subcloned into the JA4304 vector using 5 'oligonucleotide TGC AAT CAT GAA AAA AGC GTT TTT CTT TTT C (SEQ ID NO: 203), and 3' oligonucleotide CAG AAC GCG TCT AGA ATC GCA GAG CAATTT C (SEQ ID NO: 204). After PCR amplification, the gene was ligated into the 5 'blunt HIII/3' MluI site of JA 4303. To facilitate this, a short nucleotide sequence TGCAATC (SEQ ID NO: 293) was added upstream of the start codon to construct a Kozak-like sequence and to reconstruct a HindIII site. This insert is the full-length pmpE gene (SEQ ID NO: 171) containing the putative signal sequence. The pmpE gene encodes the 105kD protein (SEQ ID NO: 177). The pmpG gene was PCR amplified using 5 'oligo GTG CAA TCA TGA TTC CTCAAG GAA TTT ACG (SEQ ID NO: 205), and 3' oligo CAG AAC GCG TTTAGA ACC GGA CTT TAC TTC C (SEQ ID NO: 206) and subcloned into the JA4303 vector. A similar cloning strategy was used for the pmpI and pmpK genes. In addition, primer pairs were designed to amplify full-length or overlapping fragments of the pmp gene by PCR, then subcloned in pET17b vector (Novagen, Madison, Wis.) for protein expression, then transfected into E.coli BL21 pLysS for expression, and subsequently purified using histidine-nickel affinity chromatography methodology supplied by Novagen. To facilitate protein expression, several of the genes encoding recombinant proteins, described below, lack native signal sequences. Full-length protein expression of pmpC was achieved by expressing two overlapping fragments representing the amino and carboxy termini. The pmpC amino-terminal portion lacking the signal sequence (SEQ ID NO: 187, with the corresponding amino acid sequence provided in SEQ ID NO: 195) was subcloned into the 5 'NdeI/3' KPN cloning site of the vector using 5 'oligonucleotide CAG ACA TAT GCATCA CCA TCA CCA TCA CGA GGC GAG CTC GAT CCA AGA TC (SEQ ID NO: 207), and 3' oligonucleotide CAG AGG TAC CTC AGA TAG CAC TCT CTC CTA TTAAAG TAG G (SEQ ID NO: 208). The following primers were used: 5 'oligonucleotide CAG AGC TAG CAT GCA TCA CCA TCACCA TCA CGT TAA GAT TGA GAA CTT CTC TGG C (SEQ ID NO: 209), and 3' oligonucleotide CAG AGG TAC CTT AGA ATG TCA TAC GAG CAC CGC AG (SEQ ID NO: 210), the carboxy-terminal portion of pmpC, the carboxy-terminal fragment of the gene (SEQ ID NO: 186, with the corresponding amino acid sequence provided in SEQ ID NO: 194), was subcloned into the 5 'NheI/3' KPN cloning site of the expression vector. pmpD was also expressed as two overlapping proteins. The amino-terminal portion of pmpD lacking the signal sequence (SEQ ID NO: 185, corresponding amino acid sequence provided in SEQ ID NO: 193) contains the start codon of pET17b and is expressed as an 80kD protein. For protein expression and purification purposes, a 6 histidine tag was attached after the start codon and fused at the 28 th amino acid (84 th nucleotide) position of the gene. The following primers, 5 'oligonucleotide CAG ACA TAT GCA TCACCA TCA CCA TCA CGG GTT AGC (SEQ ID NO: 211), and 3' oligonucleotide CAG AGG TAC CTC AGC TCC TCC AGC ACA CTC TCT TC (SEQ ID NO: 212), were used in order to splice into the 5 'NdeI/3' KPN cloning site of the vector. The pmpD-carboxy terminal portion (SEQ ID NO: 184) is expressed as a 92kD protein (SEQ ID NO: 192). For expression and subsequent purification, additional methionine, alanine and serine were included, representing the start codon and the first two amino acids of the pET17b vector. The methionine, alanine and serine downstream 6 histidine tag was fused at the position of 691 amino acid (2073 nucleotide) in the gene. The insert was subcloned into the 5 'NheI/3' KPN cloning site of the expression vector using 5 'oligonucleotide CAG AGC TAG CCA TCA CCA TCA CCA TCA CGGTGC TAT TTC TTG CTT ACG TGG (SEQ ID NO: 213) and 3' oligonucleotide CAGAGG TAC TTn AAA AGA TCA ATC GCA ATC CAG TAT TCG (SEQ ID NO: 214). The pmpE is expressed as a 106kD protein (SEQ ID NO: 183, corresponding amino acid sequence provided in SEQ ID NO: 191). This pmpE insert also lacks the native signal sequence. The gene was PCR amplified using the following oligonucleotide primers under conditions well known in the art: 5 'oligonucleotide CAG AGGATC CAC ATC ACC ATC ACC ATC ACG GAC TAG CTA GAG AGG TTC (SEQ ID NO: 215), and 3' oligonucleotide CAG AGA ATT CCT AGA ATC GCA GAG CAATTT C (SEQ ID NO: 216), and ligated the amplified insert into the 5 'BamHI/3' EcoRI site of JA 4303. SEQ ID NO: 217 was inserted upstream of the start codon to construct a Kozak-like sequence and to reconstruct a HindIII site. The expressed protein contained the start codon from the pET17b expression vector and 21 amino acids downstream, i.e., MASMTGGQQMGRDSSLVPSSDP (SEQ ID NO: 218). In addition, a 6 histidine tag was included upstream of the above sequence, fused to the 28 th amino acid (nucleotide 84) of the gene, eliminating the putative signal peptide. SEQ ID NO: 183 and SEQ ID NO: 191 do not include these additional sequences. The pmpG gene (SEQ ID NO: 182, corresponding amino acid sequence provided in SEQ ID NO: 190) was PCR amplified using the following oligonucleotide primers under conditions well known in the art: 5 'oligonucleotide CAG AGG TAC CGC ATC ACC ATC ACC ATC ACA TGA TTC CTC AAGGAA TTT ACG (SEQ ID NO: 219), and 3' oligonucleotide CAG AGC GGC CGC TTAGAA CCG GAC TTT ACT TCC (SEQ ID NO: 220), which were then ligated to the 5 'KPN/3' NotI cloning site of the expression vector. The expressed protein contained an additional amino acid sequence at the amino terminus, MASMTGGQQNGRDSSLVPHHHHHH (SEQ ID NO: 221), which included the start codon and other sequences from the pET17b expression vector. The pmpI gene (SEQ ID NO: 181, corresponding amino acid sequence provided in SEQ ID NO: 189) was PCR amplified using oligonucleotide primers as follows under conditions well known in the art: 5 'oligonucleotide CAG AGCTAG CCA TCA CCA TCA CCA TCA CCT CTT TGG CCA GGA TCC C (SEQ ID NO: 222), and 3' oligonucleotide CAG AAC TAG TCT AGA ACC TGT AAG TGG TCC (SEQ ID NO: 223), which were ligated to the 5 'NheI/3' SpeI cloning site of the expression vector. The 95kD expression protein contains the start codon from the pET17b vector and additional alanine and serine at the amino terminus of the protein. In addition, a 6 histidine tag was fused to the 21 st amino acid position of the gene, eliminating the putative signal peptide.

Clone 14-H1-4(SEQ ID NO: 56) identified using the TCT-3 cell line contained the TSA gene, the complete ORF of thiol-specific antioxidant-CT 603 (the ORF of CT603 is a homologue of CPn0778 from Chlamydia pneumoniae). The TSA open reading frame in clone 14-H1-4 was amplified so that the expressed protein had an additional methionine and 6 × histidine tag (amino terminus). The amplified insert was subcloned into the pET17b vector at the Nde/EcoRI site. After induction of the clone with IPTG, the 22.6kDa protein was purified by Ni-NTA agarose affinity chromatography. Determination of the amino acid sequence of the 195 amino acid ORF of clone 14-H1-4 encoding the TSA gene is provided in SEQ ID NO: 65 (c). Further analysis yielded a full-length clone of the TSA gene, designated CTL2-TSA-FL, the full-length amino acid sequence being provided in SEQ ID NO: 92.

In other studies, 10 additional clones were identified by TCT-1 and TCT-3T cell lines as described above. The clones identified for the TCT-1 cell line were: 16-D4-22, 17-C5-19, 18-C5-2, 20-G3-45, and 21-C7-66; the clones identified for the TCT-3 cell line were: 17-C10-31, 17-E2-9, 22-A1-49 and 22-B3-53. Clone 21-G12-60 was recognized by TCT-1 and TCT-3T cell lines. In addition, clone 20-G3-45 contained sequences specific to pmpB and was identified for patient lines CT1 and CT 4. Clone 16-D4-22(SEQ ID NO: 119), identified using the TCT-1 cell line, contained a 953bp insert containing two genes, part of the open reading frame 3(ORF3) and ORF4 of the Chlamydia trachomatis plasmid, for growth in mammalian cells. Clone 17-C5-19(SEQ ID NO: 118) contained a 951bp insert containing a partial ORF of DT431 encoding clpP _1 protease and a partial ORF of CT430 (diaminopimelate epimerase). Clone 18-C5-2(SEQ ID NO: 117), identified using the TCT-1 cell line, has a 446bp insert, which is a partial ORF of the S1 ribosomal protein. Clone 20-G3-45(SEQ ID NO: 116), identified using the TCT-1 cell line, contained a 437bp insert that was part of the pmpB gene (CT 413). Clone 21-C7-8(SEQ ID NO: 115) identified using the TCT-1 cell line contained a 995bp insert encoding a portion of a dnaK-like protein. The insert of this clone did not overlap with the insert of TCT-3 clone 11-H4-28(SEQ ID NO: 59), which proved to be part of the dnaK gene CT 396. Clone 17-C10-31(SEQ ID NO: 114) identified using the TCT-3 cell line contained a 976bp insert. This clone contained a partial ORF of CT858 (protease containing IRBP and DHR domains). Clone 17-E2-9(SEQ ID NO: 113) the partial ORFs for two genes CT611 and CT610, spanning an insert of 1142 bp. Clone 22-A1-49(SEQ ID NO: 112), identified using the TCT-3 cell line, also contained two genes in the 698bp insert. Part of the ORF of CT660(DNA gyrase { gyrA _2}) was present on the upper strand, while the complete ORF of pseudoprotein CT659 was present on the complementary strand. Clone 22-B3-53(SEQ ID NO: 111), identified using the TCT-1 cell line, had a 267bp insert encoding a partial ORF of GroEL (CT 110). Clone 21-G12-60(SEQ ID NO: 110) identified by the TCT-1 and TCT-3 cell lines contained a 1461bp insert containing a partial ORF for the pseudoproteins CT875, CT229 and CT 228.

Additional chlamydia antigens were obtained by screening expression libraries of the chlamydia trachomatis (LGV II serovar) genome in lambda Screen-1 vector (Novagen, Madison, WI) with pooled sera of several chlamydia infected patients according to techniques well known in the art. The following immunoreactive clones were identified and the inserts containing the chlamydia gene were sequenced: CTL2#1(SEQ ID NO: 71); CTL2#2(SEQ ID NO: 70); CTL2#3-5 '(SEQ ID NO: 72, representing the first determined genomic sequence at the 5' end); CTL2#3-3 '(SEQ ID NO: 73, representing the second determined genomic sequence at the 3' end); CTL2#4(SEQ ID NO: 53); CTL2#5(SEQ ID NO: 69); CTL2#6(SEQ ID NO: 68); CTL2#7(SEQ ID NO: 67); CTL2#8b (SEQ ID NO: 54); CTL2#9(SEQ ID NO: 66); CTL2#10-5 '(SEQ ID NO: 74, representing the first determined genomic sequence at the 5' end); CTL2#10-3 '(SEQ ID NO: 75, representing the second determined genomic sequence at the 3' end); CTL2#11-5 '(SEQ ID NO: 45, representing the first determined genomic sequence at the 5' end); CTL2#11-3 '(SEQ ID NO: 44, representing the second determined genomic sequence at the 3' end); CTL2#12(SEQ ID NO: 46); CTL2# 16-5' (SEQ ID NO: 47); CTL2#18-5 '(SEQ ID NO: 49, representing the first determined genomic sequence at the 5' end); CTL2#18-3 '(SEQ ID NO: 48, representing the second determined genomic sequence at the 3' end); CTL2#19-5 '(SEQ ID NO: 76, representing the determined genomic sequence of the 5' end); CTL2#21(SEQ ID NO: 50); CTL2#23(SEQ ID NO: 51; and CTL2#24(SEQ ID NO: 52).

Other Chlamydia trachomatis antigens were identified by serological expression cloning. These studies used pooled sera from several chlamydia infected individuals, as described above, but using IgA and IgM antibodies as secondary antibodies in addition to IgG. Clones screened by this method have increased the identification of antigens recognized by early immune responses to chlamydia infection (i.e., mucosal humoral immune responses). We characterized the following immunoreactive clones and sequenced the inserts containing the chlamydia genes: CTL2gam-1(SEQ ID NO: 290), CTL2gam-2(SEQ ID NO: 289), CTL2gam-5(SEQ ID NO: 288), CTL2gam-6-3 '(SEQ ID NO: 287, representing the second determined genomic sequence of the 3' terminus), CTL2gam-6-5 '(SEQ ID NO: 286, representing the first determined genomic sequence of the 5' terminus), CTL2gam-8(SEQ ID NO: 285), CTL2gam-10(SEQ ID NO: 284), CTL2gam-13(SEQ ID NO: 283), CTL2gam-15-3 '(SEQ ID NO: 282, representing the second determined genomic sequence of the 3' terminus), CTL2gam-15-5 '(SEQ ID NO: 281, representing the first determined genomic sequence of the 5' terminus), CTL2gam-17(SEQ ID NO: 280), CTL2gam-18(SEQ ID NO: 279), CTL2gam-21(SEQ ID NO: 278), CTL2gam-23(SEQ ID NO: 277), CTL2gam-24(SEQ ID NO: 276), CTL2gam-26(SEQ ID NO: 275), CTL2gam-27(SEQ ID NO: 274), CTL2gam-28(SEQ ID NO: 273), CTL2gam-30-3 '(SEQ ID NO: 272, representing the second determined genomic sequence at the 3' end) and CTL2gam-30-5 '(SEQ ID NO: 271, representing the first determined genomic sequence at the 5' end).

Example 2

Chlamydia trachomatis antigen-induced T cell proliferation and interferon-gamma production

The ability of recombinant chlamydia trachomatis antigen to induce T cell proliferation and interferon gamma production was determined as follows.

The protein was induced by IPTG and purified using Ni-NTA agarose affinity chromatography (Webb et al, J.immunology 157: 5034-. The purified polypeptides are then screened for the ability to induce T cell proliferation in a preparation of PBMCs. PBMCs from C.trachomatis patients and PBMCs from normal donors whose T cells are known to proliferate in response to Chlamydia antigens were cultured in medium containing RPMI1640 supplemented with 10% confluent human serum and 50. mu.g/ml gentamicin. Purified polypeptide was added in duplicate at a concentration of 0.5 to 10. mu.g/ml. After 6 days of incubation in a volume of 200. mu.l in a 96-well round bottom plate, 50. mu.l of medium was removed from each well for determining the level of IFN-. gamma.as described below. The plates were then pulsed with 1. mu. Ci/well tritiated thymidine for an additional 18 hours, the cells were harvested and tritium uptake was measured using a gas (gas) scintillation counter. Components that resulted in cell proliferation 3-fold greater than that observed in cells cultured in medium alone were considered positive in duplicate experiments.

IFN-. gamma.was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with mouse monoclonal antibodies against human IFN-. gamma. (PharMingen, San Diego, Calif.) in PBS for 4 hours at room temperature. The wells were then blocked with PBS containing 5% (w/v) dry skim milk for 1 hour at room temperature. The plates were washed 6 times in PBS/0.2% Tween-20, and then 1:2 diluted samples in culture medium were incubated overnight at room temperature in ELISA plates. Plates were washed again and polyclonal rabbit anti-human IFN-. gamma.serum was added to each well at a 1:3000 dilution in PBS/10% normal goat serum. The plates were then incubated at room temperature for 2 hours, washed and added to a 1:2000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (Sigma chemical so, st. After another two hour incubation at room temperature, the plates were washed and TMB substrate was added. After 20 minutes the reaction was stopped with 1N sulfuric acid. The optical density was measured at 450nm using 570nm as a reference wavelength. Fractions that resulted in an OD that was two-fold greater than the average OD plus 3 standard deviations of cells cultured in medium alone were considered positive in both replicates.

Using the methodology described above, recombinant 1B1-66 protein (SEQ ID NO: 5) was found as well as the proteins corresponding to SEQ ID NO: two synthetic peptides at amino acid residues 48-67 (SEQ ID NO: 13; referred to as 1-B1-66/48-67) and 58-77 (SEQ ID NO: 14; referred to as 1B1-66/58-77) can induce a proliferative response and IFN- γ production in a Chlamydia-specific T cell line used for screening of a genomic library of Chlamydia trachomatis LGV II.

Further studies identified a chlamydia trachomatis specific T cell epitope in the ribosomal S13 protein. Two T cell epitopes were identified in ribosomal S13 protein (rS13) using a Chlamydia-specific T cell line from donor CL-8 (T cell line TCL-8EB/DC) using standard epitope mapping techniques well known in the art. FIG. 8 shows that the first peptide rS131-20(SEQ ID NO: 106) is 100% identical to the corresponding C.pneumoniae sequence, explaining the cross-reactivity of this T cell line with rS13 of recombinant C.trachomatis and C.pneumoniae. The response to the second peptide rS1356-75(SEQ ID NO: 108) is specific for Chlamydia trachomatis, indicating that the rS13 response in this healthy asymptomatic donor is caused by exposure to Chlamydia trachomatis rather than Chlamydia pneumoniae or any other microbial infection.

Clone 11-C12-91(SEQ ID NO: 63), identified using the TCP-21 cell line, had a 269bp insert that was part of the OMP2 gene (CT443) and had homology to the Chlamydia pneumoniae 60kDa cysteine-rich outer membrane protein (called OMCB), as described in example 1. To further define reactive epitopes, epitope mapping was performed using a series of overlapping peptides and the immunoassay methods previously described. In short, in the presence of 1X 10 ⁴2.5X 10 stimulation of monocyte-derived dendritic cells with non-infectious protoplasts derived from Chlamydia trachomatis and Chlamydia pneumoniae or with peptides derived from the OMCB protein sequence of Chlamydia trachomatis or Chlamydia pneumoniae (0.1. mu.g/ml)⁴TCP-21T cells to determine proliferative responses. The TCP-21T cell responded to epitopes CT-OMCB # 167-. Notably, the TCP-21T cell line also produced a proliferative response to the homologous C.pneumoniae peptide CP-OMCB #171-186(SEQ ID NO: 253) that was the same or higher than the response to C.trachomatis peptide. Amino acid substitutions at positions 2 (i.e., Asp for Glu) and 4 (i.e., Cys for Ser) did not alter the proliferative response of T cells, thereby indicating that the epitope is a cross-reactive epitope between chlamydia trachomatis and chlamydia pneumoniae.

To further elucidate the above epitope, another T cell line, TCT-3, was used in epitope mapping experiments. Immunoassays were performed as described above except that only peptides from chlamydia trachomatis were tested. The T cells generated proliferative responses in response to the two peptides CT-OMCB #152-171 and CT-OMCB #157-176 (SEQ ID NOS: 246 and 247, respectively), thereby identifying another immunogenic epitope in this cysteine-rich outer membrane protein of Chlamydia trachomatis.

Clone 14H1-4(SEQ ID NO: 56, corresponding full-length amino acid sequence provided in SEQ ID NO: 92) was identified using the TCT-3 cell line in the CD4T cell expression cloning system described earlier, which was demonstrated to contain the complete ORF of the thiol-specific antioxidant gene (CT603) (designated TSA). Epitope mapping immunoassay assays were performed as described above to further define the epitope. The TCT-3T cells showed strong proliferative responses to the overlapping peptides CT-TSA #96-115, CT-TSA #101-120 and CT-TSA #106-125 (SEQ ID NO: 254-256, respectively), thus demonstrating immunoreactive epitopes in the thiol-specific antioxidant gene of the Chlamydia trachomatis serovar LGV II.

Example 3

Preparation of synthetic Polypeptides

Peptides can be synthesized using FMOC chemistry and HPTU (O-benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate) activation on a Millipore 9050 peptide synthesizer. A Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a means of conjugating or labeling the peptide. Cleavage of the peptide from the solid support can be performed using the following cleavage mixture: trifluoroacetic acid, ethanedithiol, thioanisole, water and phenol (40:1:2:2: 3). After 2 hours of cleavage, the peptide can be precipitated in glacial methyl-tert-butyl ether. The peptide precipitate can then be dissolved in water containing 0.1% trifluoroacetic acid (TFA), then lyophilized, and purified by reverse phase HPLC with C18. Peptides can be washed using a gradient of 0-60% acetonitrile in water (containing 0.1% TFA). After lyophilization of the purified fractions, the peptides can be characterized using electrospray mass spectrometry and amino acid analysis.

Example 4

Isolation and characterization of DNA encoding Chlamydia antigens Using retroviral expression vector System and subsequent immunological analysis

A genomic library of C.trachomatis LGV II was constructed by limited digestion using BamHI, BglII, BstYi and MboI restriction enzymes. The restriction digested fragment was then ligated into the BamHI site of retrovirus pBIB-KS1, 2, 3. This set of vectors was modified to contain a Kosak translation start site and a termination site such that it would allow the expression of proteins from short DNA genome segments, as shown in FIG. 2. A DNA library of 80 clones was prepared and used to transfect the retroviral packaging cell line Phoenix-Ampho, see Pear, W.S., Scott, M.L., and Nolan, G.P., and to prepare high-titer helper-free retroviruses by transient transfection, Methods in Molecular Medicine: Gene therapy protocols, Humana Press, Totowa, N.J., pp.41-57. The chlamydia library was then transduced in retroviral form into P815 cells expressing H2-Ld, which were then used as target cells to stimulate antigen-specific T cell lines.

Repeated stimulation of Chlamydia-specific mice H2 with irradiated C.trachomatis infected J774 cells and irradiated syngeneic spleen cells was performed multiple times as described by Starnbach, M., J.Immunol.153:5183, 1994 ^dRestriction of the CD8+ T cell line for expansion in cultureThe cell line is expanded. The chlamydia-specific T cell line was used to screen the aforementioned chlamydia genomic library expressed by retroviral transduced P815 cells. Positive DNA pools were identified by detecting IFN-. gamma.production using the Elispot assay (see Lalvani et al, J. Experimental Medicine 186:859-865, 1997).

Two positive pools, designated 2C7 and 2E10, were identified in the IFN- γ Elispot assay. Individual clones were screened for their ability to elicit IFN- γ production from a chlamydia-specific CTL cell line by stable transductance of P815 cells from the 2C7 pool by limiting dilution cloning. 4 positive clones were selected by this screening method, designated 2C7-8, 2C7-9, 2C7-19 and 2C 7-21. Similarly, the positive pool 2E10 was also screened, resulting in another positive clone containing 3 inserts. These 3 inserts are the CT016, tRNA synthetase and clpX gene fragments (SEQ ID NO: 268-270, respectively).

Transgenic DNA from these 4 positive 2C7 clones was PCR amplified using pBIB-KS specific primers to selectively amplify Chlamydia DNA inserts. The amplified insert was gel purified and sequenced. An immunoreactive clone, 2C7-8(SEQ ID NO: 15, corresponding predicted amino acid sequence provided in SEQ ID NO: 32) is a 160bp fragment homologous to nucleotide 597304 and 597145 of Chlamydia trachomatis serotype D (NCBI, BLASTN search; SEQ ID NO: 33, predicted amino acid sequence provided in SEQ ID NO: 34). The sequence mapping of clone 2C7-8, located within two putative open reading frames in the highly homologous regions described immediately above, specifically, one of which consisted of a 298 amino acid fragment (SEQ ID NO: 16, with the predicted amino acid sequence provided in SEQ ID NO: 17), was demonstrated to exhibit immunological activity.

A full-length clone of this 298 amino acid fragment (designated as CT529 and/or Cap1 gene) was obtained from the serovariant L2 by PCR amplification using 5 '-ttttgaagcaggtaggtgaatatg (forward) (SEQ ID NO: 159) and 5' -ttaagaaattaaaaaatccctta (reverse) (SEQ ID NO: 160) primers and using purified Chlamydia trachomatis L2 genomic DNA as template. The PCR product was gel purified, cloned into pCRBlunt (Invitrogen, Carlsbad, Calif.) for sequencing, and subcloned into the EcoRI site of pBIB-KMS (a derivative of pBIB-KS) for expression. The chlamydia pneumoniae homolog of CT529 is provided in seq id NO: 291, the corresponding amino acid sequence is provided in SEQ ID NO: 292, respectively.

Full length DNA encoding various CT529 serovariants is essentially as follows Denamur, e., c.sayada, a.souriau, j.orafia, a.rodolakis and j.eion.1991.j.gen.microbiol.137: 2525 the total amount of 10A was determined by PCR⁵IFU is amplified from bacterial lysates. The following serovars were amplified as described: ba (SEQ ID NO: 134, corresponding predicted amino acid sequence shown in SEQ ID NO: 135); e (BOUR) and E (MTW447) (SEQ ID NO: 122, corresponding predicted amino acid sequence see SEQ ID NO: 123); f (NI1) (SEQ ID NO: 128, corresponding predicted amino acid sequence shown in SEQ ID NO: 129); g; (SEQ ID NO: 126, corresponding predicted amino acid sequence is shown in SEQ ID NO: 127); ia (SEQ ID NO: 124, corresponding predicted amino acid sequence shown in SEQ ID NO: 125); l1(SEQ ID NO: 130, corresponding predicted amino acid sequence shown in SEQ ID NO: 131); l3(SEQ ID NO: 132, corresponding predicted amino acid sequence shown in SEQ ID NO: 133); i (SEQ ID NO: 263, corresponding predicted amino acid sequence is shown in SEQ ID NO: 264); k (SEQ ID NO: 265, corresponding predicted amino acid sequence shown in SEQ ID NO: 266); and MoPn (SEQ ID NO: 136, corresponding predicted amino acid sequence see SEQ ID NO: 137). PCR reactions were performed using the Advantage Genomic PCR kit (Clontech, Palo Alto, Calif.) and primers specific for serovar L2 DNA (outside the ORF). The remaining primer sequences were 5 ' -ggtataatatctctctaaattttg (forward-SEQ ID NO: 161) and 5 ' -agataaaaaaggctgtttc ' (reverse-SEQ ID NO: 162), except that MoPn requires 5 ' -ttttgaagcaggtaggtgaatatg (forward-SEQ ID NO: 163) and 5 ' -tttacaataagaaaagctaagcactttgt (reverse-SEQ ID NO: 164). The PCR amplified DNA was purified using the qiaquick PCR purification kit (Qiagen, Valencia, Calif.) and cloned in PCR2.1(Invitrogen, Carlsbad, Calif.) for sequencing.

Inserts from PCR-amplified immunoreactive clones were sequenced on an automated sequencer (ABI 377) using both pBIB-KS specific forward primer 5 '-ccttacacagtcctgctgac (SEQ ID NO: 165) and reverse primer 3' -gtttccgggccctcacattg (SEQ ID NO: 166). The DNA of the PCRBlunt clone encoding CT529 serovariant L2 and the pcrr 2.1 clone DNA encoding CT529 serovariants Ba, E (bour), E (MTW447), F (NI1), G, Ia, K, L1, L3 and MoPn were sequenced using the T7 promoter primer and the universal M13 forward and M13 reverse primers.

To determine whether these two putative open reading frames (SEQ ID NOS: 16 and 20) encode proteins with relevant immunological functions, overlapping peptides (17-20 amino acids in length) spanning these two open reading frames were synthesized as described in example 3. Determination of peptide pulsed H2 using standard chromium release assay^dPercent specific lysis of restricted target cells. In this test, 100. mu. Ci was used⁵¹Cr labelling of P815 cells at 37 ℃ with or without 1. mu.g/ml of the indicated peptide (H2)^d) Aliquots were aliquoted for 1 hour. After incubation, the labeled P815 cells were washed to remove excess⁵¹Cr and peptide, cells were then plated in duplicate in microplates at a concentration of 1,000 cells/well. Effector CTLs (Chlamydia-specific CD 8T cells) were added at the indicated effector to target cell ratio. After 4 hours incubation, the supernatant was harvested and purified by washing the supernatant ⁵¹The gamma count of Cr was measured. Two overlapping peptides from the 298 amino acid open reading frame specifically stimulated CTL lines. Synthesis of SEQ ID NO: 138-156, which is a translation of the CT529 open reading frame (Cap1 gene) of serovar D and the L2 homolog of the 216 amino acid open reading frame. As shown in FIG. 3, the peptides CtC7.8-12(SEQ ID NO: 18, also known as Cap1#132-147, SEQ ID NO: 139) and CtC7.8-13(SEQ ID NO: 19, also known as Cap1#138-155, SEQ ID NO: 140) were able to cause specific lysis of 38 to 52% at a ratio of effector cells to target cells of 10:1, respectively. Notably, the overlap of the two peptides contained a predicted H2^d(K^dAnd L^d) A binding peptide. A10 amino acid peptide corresponding to this overlapping sequence (SEQ ID NO: 31) was synthesized and found to be responsible for the resistance in the elispot assayStrong immune response of chlamydia CTL cell line. Significantly, recent Genbank database searches revealed no previous description of the protein of the gene. Thus, clone 2C7-8(SEQ ID NO: 15) encoding the putative open reading frame defines a gene comprising a Chlamydia antigen capable of stimulating antigen-specific CD8+ T cells in an MHC-I restricted manner, indicating that the antigen can be used in the development of a vaccine against Chlamydia.

To validate these results and to further map the epitope, truncated peptides (SEQ ID NO: 138-156) were prepared and tested for T cell recognition in the IFN-g ELISPOT assay. Truncation of Ser139(Cap1#140-147, SEQ ID NO: 146) or Leu147(Cap1#138-146, SEQ ID NO: 147) abolished T cell recognition. These results indicate that the 9-residue peptide Cap1#139-147(SFIGGITYL, SEQ ID NO: 145) is the minimal epitope recognized by Chlamydia-specific T cells.

Sequence alignment of Cap1(CT529) of selected Chlamydia trachomatis serum variants (SEQ ID NOs: 121, 123, 125, 127, 129, 131, 133, 135, 137 and 139) showed that one amino acid difference appeared at position 2 of the proposed epitope. The homologous serovar D peptide is SIIGGITYL (SEQ ID NO: 168). Comparing SFIGGITYL and SIIGGITYL makes cells a target for chlamydia-specific T cell recognition. Serial dilutions of each peptide were incubated with P815 cells, as described above⁵¹T cells were tested for their recognition in the Cr release assay. Chlamydia-specific T cells recognize the lowest concentration of 1nM of serovar L2 peptide and the lowest concentration of 10nM of serovar D peptide.

Further studies showed that Cap1#139-147 specific T cell clones recognized Chlamydia trachomatis infected cells. To verify Cap1 _139-147Whether presented on the surface of Chlamydia-infected cells, Balb-3T3 (H-2) was infected with Chlamydia trachomatis serovar L2^d) Cells were tested to determine whether these cells could be identified by the Cap1#139-147 epitope (SEQ ID NO: 145) specific CD8+ T cell clones. Cap1#139-147 epitope specific T cell clones were obtained from the 69T cell line at limiting dilution. The T cell gramThe clones specifically recognized chlamydia infected cells. In these experiments, the target cells were C.trachomatis-infected (positive control) or uninfected Balb/3T3 cells, which showed specific lysis of 45%, 36% and 30% at 30:1, 10:1 and 3:1 effector cells to target cell ratios, respectively; or Cap1#139-147 epitope (SEQ ID NO: 145) coated or untreated P815 cells, which showed specific lysis of 83%, 75% and 58% at 30:1, 10:1 and 3:1 effector cells to target cell ratios, respectively (negative controls had less than 5% lysis in all cases). This data suggests that the epitope is presented during infection.

In vivo studies showed that Cap1#139-147 epitope specific T cells were primed during mouse infection with Chlamydia trachomatis. To determine whether Chlamydia trachomatis infection elicits a Cap1#139-147 epitope specific T cell response, 10 was used ⁸Mice were injected intraperitoneally with IFU trachoma chlamydia serum variant L2. Two weeks after injection, mice were sacrificed and splenocytes were stimulated on irradiated isogenic splenocytes pulsed with the Cap1#139-147 epitope peptide. After 5 days of stimulation, at standard⁵¹Cultures were used in a Cr release assay to determine the presence of Cap1#139-147 epitope specific T cells in culture. Specifically, splenocytes from mice immunized with the Chlamydia trachomatis serovar L2 or control mice injected with PBS after 5 days of culture with syngeneic splenocytes coated with the Cap1#139-147 peptide and CD8+ T cells capable of specifically recognizing the Cap1#139-147 epitope gave 73%, 60% and 32% specific lysis at the ratios of 30:1, 10:1 and 3:1 effector and target cells, respectively. Control mice gave a percent lysis of approximately 10% at a ratio of 30:1 effector cells to target cells and steadily decreased as the ratio of effector cells to target cells decreased. The target cells were P815 cells coated with the Cap1#139-147 peptide or untreated. These data suggest that Cap1#139-147 peptide-specific T cells were primed during mouse infection with Chlamydia trachomatis.

Ct529 mapping

It was shown by studies that Ct529 (referred to herein as Cap-1) localizes on the inclusion membrane (inclusion membrane) of C.trachomatis infected cells, independent of the protosome or reticulum body. As described above, Cap-1 was identified as a Chlamydia-derived product that stimulated CD8+ CTL. These CTLs are protective in mouse infection models, thus making Cap-1 a good vaccine candidate. Moreover, since these CTLs are MHC-1 restricted, the Cap-1 gene must be accessible to the cytoplasm of infected cells, which may be a unique feature of the specific Chlamydia gene product. Therefore, determining the cellular location of the gene product is useful for characterizing Cap-1 as a vaccine candidate. To detect the intracellular localization of Cap-1, Chlamydia-infected McCoy cells were stained with rabbit polyclonal antibodies against a recombinant polypeptide comprising 125 amino acids from the N-terminus of Cap-1 (SEQ ID NO: 305, the amino acid sequence comprising the N-terminal 6-His tag is provided in SEQ ID NO: 304).

Rabbit anti-Cap-1 polyclonal antibodies were obtained by hyperimmunizing rabbits with recombinant polypeptide rCt529c1-125(SEQ ID NO: 305), which includes the N-terminal portion of Cap-1. Recombinant rCt529c1-125 protein was obtained from E.coli transformed with pET expression plasmid (see above) containing nucleotides 1-375 encoding the 1-125 amino acids of the N-terminus of Cap-1. Recombinant proteins were purified by Ni-NTA using techniques well known in the art. For positive control antisera, polyclonal antisera to antigenic spheroids were prepared by immunizing rabbits with purified chlamydia trachomatis protoplasts (Biodesign, Sacco, Maine). Pre-immune sera from rabbits immunized with Cap-1 polypeptide were used as negative controls.

Grown on a cover glass and covered with a protective layer of 10⁶Immunocytochemistry experiments were performed on McCoy cell monolayers inoculated with chlamydia trachomatis serovar L2 or chlamydia psittaci (c.psitacci) strain 6BC at a concentration of IFU (Inclusion Forming Units)/ml. After 2 hours, the medium was aspirated and replaced with RP-10 medium freshly supplemented with cycloheximide (1.0. mu.g/ml). At 7% CO₂The infected cells were incubated for 24 hours and then fixed by aspirating the medium, washing the cells once with PBS and then methanol for 5 minutes. For antigen staining, the fixed cell monolayer was washed with PBS and incubated with 1:100 dilution of specific or control antisera at 37 deg.C For 2 hours. Cells were washed with PBS and incubated with Fluorescein Isothiocyanate (FITC) -labeled anti-rabbit IgG (KPL, Gaithersburg) for 1 hour, followed by staining with Evans blue (Evans blue) (0.05%) in PBS. Fluorescence was observed with a 100 Xobjective (Zeiss epifluorescence microscope) and photographed (Nikon UFX-11A camera).

The results of this study showed that Cap-1 is located on the inclusion body membrane of C.trachomatis infected cells. The Cap-1 specific antibody labels the inclusion body membranes of Chlamydia trachomatis infected cells, but does not label Chlamydia protozoa protsomes contained in these inclusion bodies or released during the fixation process. In contrast, antibodies against the protosomes clearly mark the bacterial body, which includes not only the protosomes in the inclusion bodies but also the protosomes released during the immobilization process. The specificity of anti-Cap-1 antibodies can be demonstrated by the fact that the antibodies do not stain C.psittaci infected cells. The specificity of this Cap-1 marker can also be demonstrated by the lack of reactivity in preimmune serum. These results suggest that Cap-1 is released from bacteria and binds to the Chlamydia inclusion body membrane. Thus, Cap-1 is a gene product that can be used to stimulate CD8+ T cells in the development of vaccines against chlamydial infection.

Two additional series of studies further illustrate the importance of the Cap-1 gene as a potential CTL antigen in vaccines against Chlamydia infection. First, CTL specific for the MHC-I epitope (SEQ ID NO: 144) of the peptide Chlamydia trachomatis Cap-1CT529 #138-147 was confirmed to be sensitized with high frequency during natural infection. Specifically, with 10⁶Chlamydia trachomatis serovar L2 was inoculated into Balb/C mice. After 2 weeks, spleens were harvested and the number of IFN-. gamma.secreting cells in response to Cap-1#138-147 peptide pulsed antigen presenting cells was quantified by Elispot analysis. In two experiments, 10⁵The number of IFN- γ secreting cells in individual splenocytes was approximately 1% of total CD8+ T cells. This high frequency of CD8+ CTL responses to the MHC-1 epitope (Cap-1CT529 #138-147 peptide) suggests that Cap-1 is highly immunogenic in infection.

The results of the second series of studies showed that the Cap-1 protein reached the cytoplasm of the host cells almost immediately after infection. This was confirmed by the time course of presentation of the Cap-1CT529 #138-147 peptide. Briefly, 3T3 cells were infected with the Chlamydia trachomatis serum variant L2 for various lengths of time and then tested for recognition by the Cap-1CT529 #138-147 peptide-specific CTL. The results showed that chlamydia trachomatis infected 3T3 cells were targeted for recognition by antigen-specific CTLs after only 2 hours of infection. These results suggest that Cap-1 is an early protein synthesized during the development of Chlamydia trachomatis protosomes into the dictyoid. In vaccines against chlamydial infection, a CD8+ CTL immune response against the gene product expressed early in the infection may be particularly effective.

Example 5

Generation of antibody and T cell responses in mice immunized with Chlamydia antigens

We performed immunogenicity studies to determine the antibody and CD4+ T cell responses of mice immunized with purified SWIB or S13 protein formulated with Montanide adjuvant, or with pcDNA-3 expression vectors (DNA-based immunization) containing the DNA sequences of SWIB or S13. SWIB is also known as clone 1-B1-66(SEQ ID NO: 1, corresponding amino acid sequence provided in SEQ ID NO: 5), while the S13 ribosomal protein is also known as clone 10-C10-31(SEQ ID NO: 4, corresponding amino acid sequence provided in SEQ ID NO: 12). In the first experiment, several groups of 3C 57BL/6 mice were immunized twice and their antibody and CD4+ T cell responses were monitored. DNA immunization was performed intradermally at the base of the tail and peptide immunization was administered by the subcutaneous route. Criteria for splenocytes from immunized mice³The results of the H-incorporation assay showed that the group immunized with the purified recombinant SWIB polypeptide (SEQ ID NO: 5) produced a strong proliferative response. As previously described, further analysis using cytokine induction assays demonstrated that groups immunized with SWIB polypeptide produced detectable IFN-. gamma.and IL-4 responses. ELISA-based assays were subsequently performed to determine the predominant antibody isotype response in the experimental groups immunized with SWIB polypeptides. FIG. 4 illustrates that the SWIB immunization group gave a humoral response dominated by IgG 1.

In a second experiment, C3H mice were immunized 3 times with 10 μ g of purified SWIB protein (also known as clone 1-B1-66, SEQ ID NO: 5) in PBS or Montanide at 3 week intervals and cells were harvested two weeks after the third immunization. Antibody titers against SWIB protein were determined by standard ELISA-based techniques well known in the art, demonstrating that SWIB protein formulated in Montanide induces a strong humoral immune response. T cell proliferative responses were measured by XTT-based assays (Scudiero et al, Cancer Research, 1988, 48: 4827). As shown in figure 5, splenocytes from mice immunized with SWIB polypeptide plus Montanide elicited an antigen-specific proliferative response. In addition, the ability of splenocytes from immunized animals to secrete IFN- γ in response to soluble recombinant SWIB polypeptides was determined using the cytokine induction assay previously described. Splenocytes from all animals in the group of animals immunized with SWIB polypeptide formulated with Montanide adjuvant secreted IFN- γ in response to SWIB chlamydia antigen exposure, indicating a chlamydia-specific immune response.

In another experiment, C3H mice received 10 μ g of purified SWIB or S13 protein (Chlamydia trachomatis, SWIB protein, clone 1-B1-66, SEQ ID NO: 5, and S13 protein, clone 10-C10-31, SEQ ID NO: 4) formulated in SBAS2 adjuvant (SmithKline Beecham, London, UK) at the base of the tail at three different time points. Measurement of antigen-specific antibody titers by ELISA showed that both polypeptides induced a strong IgG response with titers ranging from 1X 10 ^-4To 1X 10^-5. The IgG1 and IgG2a components of the response were present in fairly equivalent amounts. Antigen-specific T cell proliferative response to SWIB (by criteria performed on splenocytes isolated from immunized mice³H incorporation assay) was very strong (50,000 cpm higher than negative control), while the response to S13 was even stronger (100,000 cpm higher than negative control). IFN γ production was analyzed by standard ELISA techniques from supernatants from proliferating cultures. Repeated in vitro stimulation of this culture with S13 induced high levels of IFN γ production, approximately 25ng/ml versus 2ng/ml for the negative control. Repeated stimulation with the SWIB protein also induced IFN γ, but to a lesser extent.

In a related experiment, C3H mice were in triplicateAt different time points, 10. mu.g of purified SWIB or S13 protein (Chlamydia trachomatis, SWIB protein, clone 1-B1-66, SEQ ID NO: 5, and S13 protein, clone 10-C10-31, SEQ ID NO: 4) mixed with 10. mu.g cholera toxin was received for immunization. Mucosal immunization was performed by intranasal vaccination. Antigen-specific antibody responses were determined using standard ELISA techniques. Antigen-specific IgG antibodies with a titer of 1X 10 were present in the blood of SWIB-immunized mice^-3To 1X 10^-4But not in animals immunized with S13. The antigen specific T cell response of isolated splenocytes, measured as IFN γ production, gave results similar to those obtained with the systemic immunization described immediately above.

To determine the immunogenicity of the CT529 serovariant LGVIICTL epitope, defined by the CT 52910 mer consensus peptide (CSFIGGITYL-SEQ ID NO: 31) identified as an H2-Kd restricted CTL epitope, an animal study was performed. BALB/c mice (3 mice per group) were immunized 3 times with 25 μ g of peptide in combination with different adjuvants. Peptides from the SKB adjuvant system SBAS-2 '', SBAS-7(SmithKline Beecham, London, UK) or Montanide were administered on the basal system of the tail. Peptides mixed with 10 μ g Cholera Toxin (CT) were also administered intranasally. Natural mice (nasal mice) were used as controls. 4 weeks after 3 rd immunization, LPS blasts (LPS-blasts) pulsed with 10. mu.g/ml CT 52910 residue consensus peptide at 3 different effector to LPS blasts ratios (6, 1.5 and 0.4) at 1X 10⁶Spleen cells were repeatedly stimulated at individual cells/ml. After 2 repeated stimulations, the ability of the effector cells to lyse peptide-pulsed P815 cells was tested using a standard chromium release assay. Irrelevant peptides from chicken egg ovalbumin were used as negative controls. The results demonstrate that a significant immune response was elicited against this CT 52910 residue consensus peptide, and that antigen-specific T cells capable of lysing the peptide-pulsed target were elicited in response to immunization with this peptide. Specifically, antigen-specific lytic activity was found in the SBAS-7 and CT adjuvant groups, whereas Montanide and SBAS-2 "failed to aid in this CTL epitope immunization.

Example 6

Expression and characterization of Chlamydia pneumoniae genes

The human T cell line TCL-8 described in example 1 recognizes monocyte-derived dendritic cells infected with chlamydia trachomatis and chlamydia pneumoniae, suggesting that chlamydia trachomatis and chlamydia pneumoniae may encode cross-reactive T cell epitopes. To isolate the Chlamydia pneumoniae genes homologous to Chlamydia trachomatis LGVII clone 1B1-66 (also known as SWIB, SEQ ID NO: 1) and clone 10C10-31 (also known as S13 ribosomal protein, SEQ ID NO: 4), HeLa229 cells were infected with the Chlamydia pneumoniae TWAR strain (CDC/CWL-029). After 3 days of incubation, chlamydia pneumoniae infected HeLa cells were harvested, washed and resuspended in 200 μ l water, and then heated in a boiling water bath for 20 minutes. 10. mu.l of the disrupted cell suspension was used as a template for PCR.

Chlamydia pneumoniae specific primers for clones 1B1-66 and 10C10-31 were designed so that the 5 'end had a 6 × histidine tag and an NdeI site inserted, and the 3' end had a stop codon and included a BamHI site (FIG. 6). The PCR products are amplified and sequenced by standard techniques well known in the art. The chlamydia pneumoniae-specific PCR product was cloned into the expression vector pET17B (Novagen, Madison, WI) and transfected into e.coli BL21 pLysS for expression and subsequent purification using histidine-nickel affinity chromatography methodology (provided by Novagen). Two proteins of C.pneumoniae were thus produced, a 10-11kDa protein, designated CpSWIB (SEQ ID NO: 27, and SEQ ID NO: 78 with a 6 XHis tag, respectively, the corresponding amino acid sequences being provided in SEQ ID NO: 28), and a 15kDa protein, designated CpS13(SEQ ID NO: 29, and SEQ ID NO: 77 with a 6 XHis tag, the corresponding amino acid sequences being provided in SEQ ID NO: 30 and 91, respectively).

Example 7

Induction of T cell proliferation and interferon-gamma production by Chlamydia pneumoniae antigens

The ability of recombinant chlamydia pneumoniae antigens to induce T cell proliferation and interferon- γ production was determined as follows.

The protein was induced by IPTG and purified by Ni-NTA agarose affinity chromatography (Webb et al, J.Immuno logy 157: 5034-5041, 1996). The purified polypeptides are then screened for the ability to induce T cell proliferation in a preparation of PBMCs. PBMCs from patients with Chlamydia pneumoniae and from normal donors whose T cells are known to proliferate in response to Chlamydia antigens were cultured in medium containing RPMI1640 supplemented with 10% confluent human serum and 50. mu.g/ml gentamicin. Purified polypeptide was added in duplicate at a concentration of 0.5 to 10. mu.g/ml. After 6 days of incubation in a volume of 200. mu.l in a 96-well round bottom plate, 50. mu.l of medium was removed from each well for determining the level of IFN-. gamma.as described below. The plates were then pulsed with 1. mu. Ci/well tritiated thymidine for an additional 18 hours, the cells were harvested and tritium uptake was measured using a gas scintillation counter. Components that resulted in cell proliferation in duplicate experiments that was more than 3 times greater than that observed in cells cultured in medium alone were considered positive.

IFN-. gamma.was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with mouse monoclonal antibodies against human IFN-. gamma. (PharMingen, San Diego, Calif.) in PBS for 4 hours at room temperature. The wells were then blocked with PBS containing 5% (w/v) dry skim milk for 1 hour at room temperature. The plates were washed 6 times in PBS/0.2% Tween-20, and the 1:2 diluted samples in medium were incubated overnight at room temperature in ELISA plates. Plates were washed again and polyclonal rabbit anti-human IFN-. gamma.serum was added to each well at a 1:3000 dilution in PBS/10% normal goat serum. The plates were then incubated at room temperature for 2 hours, washed and added to a 1:2000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (Sigma Chemical So., St. Louis, Mo.) in PBS/5% dry skim milk. After another two hour incubation at room temperature, the plates were washed and TMB substrate was added. After 20 minutes the reaction was stopped with 1N sulfuric acid. The optical density was measured at 450nm using 570nm as a reference wavelength. Fractions that resulted in an OD that was two times greater than the average OD of cells cultured in medium alone plus 3 standard deviations in both replicate experiments were considered positive.

Human anti-chlamydia T cells capable of cross-reacting with chlamydia trachomatis and chlamydia pneumoniae The line (TCL-8) was used to determine whether the expressed proteins described in the above examples (i.e., CpSWIB, SEQ ID NO: 27, and SEQ ID NO: 78 with a 6 XHis tag, the corresponding amino acid sequences being provided in SEQ ID NO: 28; and the 15kDa protein designated CpS13, SEQ ID NO: 29, and SEQ ID NO: 77 with a 6 XHis tag, the corresponding amino acid sequences being provided in SEQ ID NO: 30 and 91, respectively) have a T cell epitope common to Chlamydia trachomatis and Chlamydia pneumoniae. In short, at 1X 10⁴Coli expressing chlamydia antigens were titrated on monocyte derived dendritic cells. After 2 hours, the dendritic cell cultures were washed and 2.5X 10 added⁴T cells (TCL-8) were incubated for a further 72 hours. The amount of IFN-. gamma.in the culture supernatant was then determined by ELISA. As shown in fig. 7A and 7B, the TCL-8T cell line specifically recognized the S13 ribosomal protein of both chlamydia trachomatis and chlamydia pneumoniae, as demonstrated by the antigen-specific induction of IFN- γ, whereas the T cell line only recognized the SWIB protein from chlamydia trachomatis. To validate these results, T cell epitopes of chlamydia trachomatis SWIB were identified by epitope mapping using target cells pulsed with a series of overlapping peptides and T cell line TCL-8. The 3H-thymidine incorporation assay showed that the peptide designated C.t. SWIB52-67 (SEQ ID NO: 39) caused the strongest proliferation of the TCL-8 cell line. topoisomerase-SWIB fusions corresponding to the SWIB sequence of Chlamydia pneumoniae (SEQ ID NO: 40), Chlamydia pneumoniae (SEQ ID NO: 43) and Chlamydia trachomatis (SEQ ID NO: 42), and homologous peptides of the human SWI domain (SEQ ID NO: 41) were synthesized and tested in the above assay. The T cell line TCL-8 recognizes only SEQ ID NO: 39 and does not recognize the corresponding chlamydia pneumoniae peptide (SEQ ID NO: 40) or other corresponding peptides described above (SEQ ID NO: 41-43).

Chlamydia-specific T cell lines were generated from donor CP-21 with positive serum titers against C.pneumoniae by stimulation of donor PBMC with monocyte-derived dendritic cells infected with C.trachomatis or C.pneumoniae, respectively. T cells generated against chlamydia pneumoniae respond to recombinant chlamydia pneumoniae SWIB but not chlamydia trachomatis SWIB, whereas T cells generated against chlamydia trachomatis do not respond to either chlamydia trachomatis or chlamydia pneumoniae SWIB (see fig. 9). The chlamydia pneumoniae SWIB-specific immune response of donor CP-21 confirms the presence of chlamydia pneumoniae infection and indicates that chlamydia pneumoniae SWIB-specific T cells are elicited during in vivo chlamydia pneumoniae infection.

Epitope mapping of the T cell response to C.pneumoniae SWIB shows that the Cp-SWIB specific T cell response overlaps the peptides Cp-SWIB32-51(SEQ ID NO: 101) and Cp-SWIB37-56(SEQ ID NO: 102), indicating the C.pneumoniae SWIB specific T cell epitope Cp-SWIB37-51(SEQ ID NO: 100).

In other experiments, T cell lines were prepared from donor CP1 (also a chlamydia pneumoniae seropositive donor) by stimulating PBMCs with non-infectious protoplasts from chlamydia trachomatis and chlamydia pneumoniae, respectively. Specifically, in the presence of 1 × 10 ⁴Monocyte-derived dendritic cells and noninfectious protozoa derived from Chlamydia trachomatis and Chlamydia pneumoniae, or 2.5X 10 stimulated in the presence of recombinant Chlamydia trachomatis or Chlamydia pneumoniae SWIB protein⁴T cells to determine proliferative responses. T cell responses against SWIB are similar to the data obtained using the CP-21 derived T cell line in the following respects: the C.pneumoniae T cell line is responsible for the C.pneumoniae SWIB but not C.trachomatis SWIB. Furthermore, the C.trachomatis T cell line did not proliferate in response to C.trachomatis or C.pneumoniae SWIB, but did proliferate in response to CT and CP protoplasts. Clone 11-C12-91(SEQ ID NO: 63), identified using the TCP-21 cell line, had a 269bp insert that was part of the OMP2 gene (CT443) and had homology to the Chlamydia pneumoniae 60kDa cysteine-rich outer membrane protein (called OMCB), as described in example 1. To further determine the reactive epitope, epitope mapping was performed using a series of overlapping peptides and the immunoassay method described previously. In short, at 1X 10⁴2.5X 10 stimulation of monocyte-derived dendritic cells with peptides (0.1. mu.g/ml) derived from non-infectious protoplasts of Chlamydia trachomatis and Chlamydia pneumoniae or from the protein sequence of the OMCB protein of Chlamydia trachomatis or Chlamydia pneumoniae in the presence of 2.5X 10 ⁴Individual TCP-21T cells to determine proliferative responses. TCP-21T cell responds to the epitopes CT-OMCB # 167-. Notably, the TCP-21T cell line also produced a proliferative response in response to the homologous C.pneumoniae peptide CP-OMCB #171-186(SEQ ID NO: 253) to the same or greater extent than in response to C.trachomatis peptide. Neither amino acid substitutions at positions 2 (i.e., Asp to Glu) nor 4 (i.e., Cys to Ser) altered the proliferative response of T cells, thereby indicating that the epitope is a cross-reactive epitope between chlamydia trachomatis and chlamydia pneumoniae.

Example 8

Immune response of human PBMC and T cell lines to Chlamydia antigens

The examples provided herein suggest that a healthy donor population exists among the general population that has been infected with Chlamydia trachomatis and that has developed a protective immune response that controls the infection with Chlamydia trachomatis. These donors are clinically asymptomatic and seronegative for chlamydia trachomatis. To characterize the immune response of normal donors to chlamydia antigens identified by CD4 expressing clones, PBMCs obtained from 12 healthy donors were tested for response to a panel of recombinant chlamydia antigens (including chlamydia trachomatis SWIB, chlamydia pneumoniae SWIB and chlamydia trachomatis S13, chlamydia pneumoniae S13). The data are summarized in table I below. All donors were chlamydia trachomatis serum negative, whereas 6/12 had positive chlamydia pneumoniae titers. Using a stimulation index of >4 as a positive response, 11/12 subjects responded to chlamydia trachomatis protosomes and 12/12 responded to chlamydia pneumoniae protosomes. One donor, AD104, responded to recombinant chlamydia pneumoniae S13 protein but not recombinant chlamydia trachomatis S13 protein, indicating that this is a chlamydia pneumoniae-specific response. 3 of the 12 donors had a specific response to the C.trachomatis SWIB but not C.pneumoniae SWIB, confirming a C.trachomatis infection. Chlamydia trachomatis and chlamydia pneumoniae S13 elicited a response in the 8/12 donor, suggesting a chlamydial infection. These illustrations demonstrate the ability of SWIB and S13 to elicit T cell responses in PBMCs of normal subjects.

TABLE I

Immune response to Chlamydia in normal subjects

Donor	Sex	Chlamydia IgG titer	CTEB	CPEB	CTSwib	CPSwib	CTS13	CPS13	CTIpdA	CTTSA
											AD100	For male	Negative of	++	+++	+	-	++	++	-	n.t.
AD104	Woman	Negative of	+++	++	-	-	-	++	-	n.t.
											AD108	For male	CP1:256	++	++	+	+/-	+	+	+	n.t.
AD112	Woman	Negative of	++	++	+	-	+	-	+/-	n.t.
											AD120	For male	Negative of	-	+	-	-	-	-	-	n.t.
AD124	Woman	CP1:128	++	++	-	-	-	-	-	n.t.
											AD128	For male	CP1:512	+	++	-	-	++	+	++	-
AD132	Woman	Negative of	++	++	-	-	+	+	-	-
											AD136	Woman	CP1:128	+	++	-	-	+/-	-	-	-
AD140	For male	CP1:256	++	++	-	-	+	+	-	-
											AD142	Woman	CP1:512	++	++	-	-	+	+	+	-
AD146	Woman	Negative of	++	++	-	-	++	+	+	-

CT ═ chlamydia trachomatis; CP is chlamydia pneumoniae; EB ═ chlamydia protosome; swib ═ recombinant chlamydia Swib protein; s13 ═ recombinant chlamydia S13 protein; lpdA ═ recombinant chlamydia lpdA protein; TSA is a recombinant chlamydia TSA protein. Values represent results from standard proliferation assays. By incubating with 1X 10 of the antigen(s) previously incubated with the respective recombinant antigen or protosome (EB)⁴Single monocyte-derived dendritic cell stimulation of 3X 10⁵PBMC, measuring proliferative responses. After 6 days, the medicine is taken³The test results were harvested 18 hours after the last pulse of H-thymidine.

And (3) SI: stimulation index

+/-： SI～4

+： SI>4

++： SI10-30

+++： SI>30

In the first series of experiments, T cell lines were prepared from healthy female individuals (CT-10) who had been exposed to C.trachomatis from the reproductive tract by stimulating T cells with C.trachomatis LGV II protoplasts as described previously. Although the subject had been exposed to Chlamydia trachomatis, she had not experienced seroconversion nor had clinical symptoms, suggesting that donor CT-10 may have developed a protective immune response against Chlamydia trachomatis. As shown in FIG. 10, a primary Chlamydia-specific T cell line derived from donor CT-10 was responsive to recombinant Chlamydia trachomatis SWIB, but not Chlamydia pneumoniae SWIB protein, confirming that CT-10 was exposed to Chlamydia trachomatis. Epitope mapping of the T cell response to C.trachomatis SWIB showed that this donor responded to the same epitope Ct-SWIB 52-67(SEQ ID NO: 39) as T cell line TCL-8, as shown in FIG. 11.

Additional T cell lines were generated as described above for different patients with chlamydia trachomatis. The clinical profile of the patients and the proliferative response to various chlamydia trachomatis and chlamydia pneumoniae protozoa and recombinant proteins are summarized in table II below:

NGU ═ non-gonococcal urethritis; BV-bacterial vaginosis; CT ═ chlamydia trachomatis; CP is chlamydia pneumoniae; EB ═ chlamydia protosome; swib ═ recombinant chlamydia Swib protein; s13 ═ recombinant chlamydia S13 protein; lpdA ═ recombinant chlamydia lpdA protein; TSA is a recombinant chlamydia TSA protein. Values represent results from standard proliferation assays. By stimulating 3X 10 with individual recombinant antigens or protosomes (EBs)⁵PBMC, measuring proliferative responses. After 6 days, the medicine is taken³The test results were harvested 18 hours after the last pulse of H-thymidine.

And (3) SI: stimulation index

+/-： SI～4

+： SI>4

++： SI10-30

+++： SI>30

Using these asymptomatic (as defined above) subjects and chlamydia trachomatis patients (summarized in tables I and II), the immune response of PBMCs derived from both groups was studied comprehensively. Briefly, PBMCs from C.pneumoniae patients as well as from normal donors were cultured in medium containing RPMI 1640 supplemented with 10% confluent human serum and 50. mu.g/ml gentamicin. Purified polypeptides, a panel of recombinant chlamydia antigens, including chlamydia trachomatis and chlamydia pneumoniae SWIB and S13, and chlamydia trachomatis lpdA and TSA, were added in duplicate at concentrations of 0.5 to 10 μ g/ml. After 6 days of incubation in a volume of 200. mu.l in a 96-well round bottom plate, 50. mu.l of medium was removed from each well for determining the level of IFN-. gamma.as described below. The plates were then pulsed with 1. mu. Ci/well tritiated thymidine for an additional 18 hours, the cells were harvested and tritium uptake was measured using a gas scintillation counter. Components that resulted in cell proliferation that was 3-fold higher than that observed in cells cultured in medium alone were considered positive in duplicate experiments.

The proliferative response to recombinant chlamydia antigens demonstrated that most asymptomatic donors and chlamydia trachomatis patients recognized the chlamydia trachomatis S13 antigen (8/12), most chlamydia trachomatis patients recognized the chlamydia pneumoniae S13 antigen (8/12), and the asymptomatic of 4/12 also recognized the chlamydia pneumoniae S13 antigen. Furthermore, 6 of 12 chlamydia trachomatis patients and 4 of 12 asymptomatic donors gave a proliferative response to the chlamydia trachomatis lpdA antigen. These results indicate that the chlamydia trachomatis and chlamydia pneumoniae S13 antigens, the chlamydia trachomatis Swib antigen and the chlamydia trachomatis lpdA antigen are all recognised by asymptomatic donors, indicating that these antigens are recognised and elicit an immune response against them during exposure to chlamydia. This suggests that these antigens may have a role in conferring protective immunity to a human host. Furthermore, the chlamydia trachomatis and chlamydia pneumoniae S13 antigens were equally well recognized in patients with chlamydia trachomatis, thus suggesting that chlamydia trachomatis and chlamydia pneumoniae may have a common epitope in the S13 protein. Table III summarizes the results of these studies.

TABLE III

Antigens	Normal donor	C.t. patients
			C.t.-Swib	3/12	0/12
C.p.-Swib	0/12	0/12
			C.t.-S13	8/12	8/12
C.p.-S13	4/12	8/12
			lpdA	4/12	6/12
TSA	0/12	2/12

We have initiated a series of studies to determine the cellular immune response of short-term T cell lines generated from asymptomatic donors and patients with chlamydia trachomatis. Cellular immune responses were measured by standard proliferation assays and IFN- γ (see example 7). In particular, most antigens take the form of a single E.coli clone expressing the Chlamydia antigen, but some recombinant proteins have also been used in these experiments. At 1X 10 ⁴These monocaryotic E.coli clones were titrated (titer) onto monocyte-derived dendritic cells, and after 2 hours, the cultures were washed and 2.5X 10 cells were added⁴And (4) T cells. Assays Using recombinant proteins were performed as described aboveThe method is carried out. Proliferation was determined 4 days later by the last 18 hours of a standard 3H-thymidine pulse. IFN- γ induction was measured in culture supernatants harvested 4 days later using standard ELISA assays as described above. The results show that all chlamydia trachomatis antigens tested, except c.t.swib, elicited a proliferative response in one or more different T cell lines derived from patients with chlamydia trachomatis. In addition, the following chlamydia genes also elicited a proliferative response from chlamydia trachomatis patients and asymptomatic donors: CT622, groEL, pmpD, CT610, and rS 13.

The 12G3-83 clone contains sequences for CT734 and CT764 in addition to CT622, and therefore these gene sequences may also have immunoreactive epitopes. Similarly, clone 21G12-60 contained the sequences of the pseudoprotein genes CT229 and CT228 in addition to CT 875; and 15H2-76 also contained sequences from CT812 and CT088 and having homology to the sycE gene. Clone 11H3-61 also contained sequences with homology to PGP6-D virulence protein.

TABLE IV

Cloning	C.t. antigen (putative))	TCL from asymptomatic donors	TCL from c.t. patients	SEQ ID NO：
					1B1-66 (Escherichia coli)	Swib	2/2	0/4	5
1B1-66 (protein)	Swib	2/2	0/4	5
					12G3-83 (Escherichia coli)	CT622	2/2	4/4	57
22B3-53 (Escherichia coli)	groEL	1/2	4/4	111
					22B3-53 (protein)	groEL	1/2	4/4	111
15H2-76 (Escherichia coli)	PmpD	1/2	3/4	87
					11H3-61 (Escherichia coli)	rL1	0/2	3/4	60
14H1-4 (Escherichia coli)	TSA	0/2	3/4	56
					14H1-4 (protein)	TSA	0/2	3/4	56
11G10-46 (Escherichia coli)	CT610	1/2	1/4	62
					10C10-17 (Escherichia coli)	rS13	1/2	1/4	62
10C10-17 (protein)	rS13	1/2	1/4	62
					21G12-60 (Escherichia coli)	CT875	0/2	2/4	110
11H4-32 (Escherichia coli)	dnaK	0/2	2/4	59
					21C7-8 (Escherichia coli)	dnaK	0/2	2/4	115
17C10-31 (Escherichia coli)	CT858	0/2	2/4	114

Example 9

Protection studies using chlamydia antigens

1.SWIB

Protection studies were performed in mice to determine whether immunization with a chlamydia antigen could affect reproductive tract disease caused by chlamydial vaccination. Two models were used: an intravaginal inoculation model using a human isolate containing a Chlamydia psittaci strain (MTW 447); an intrauterine vaccination model involving a human isolate identified as chlamydia trachomatis serovar F (strain NI 1). Both strains cause inflammation of the upper reproductive tract, which resembles endometritis and salpingitis caused by chlamydia trachomatis in women. In the first experiment, C3H mice (4 mice per group) were immunized 3 times with 100. mu.g of pcDNA-3 expression vector containing C.trachomatis SWIBDNA (SEQ ID NO: 1, corresponding amino acid sequence provided in SEQ ID NO: 5). For systemic immunization, vaccination was performed at the base of the tail. At 2 weeks after the last immunization, the animals were treated with progesterone and infected vaginally or by injection of the inoculum in the uterus. 2 weeks after infection, mice were sacrificed and the reproductive tract was sectioned, stained and histopathologically examined. The level of inflammation was scored (from + (very mild) to + + +++ (very severe)). The individual tubal/ovarian scores were summed and then divided by the number of organs examined to give an average score for inflammation for the group. In contrast to the uterine vaccination model, in which animals immunized with the empty vector negative control exhibited consistent inflammation with an ovarian/oviduct mean inflammation score of 6.12, the fraction of the DNA immunization group was 2.62. In the vaginal vaccination and ascending infection models, mice immunized with the negative control had an ovarian/oviduct mean inflammation score of 8.37 versus 5.00 for the DNA immunization group. Moreover, in the latter model, the vaccinated mice showed no signs of tubal occlusion, while the negative control vaccinated group had inflammatory cells in the tubal lumen.

In a second experiment, C3H mice (4 mice per group) were immunized 3 times with 50 μ g of pcDNA-3 expression vector containing the C.trachomatis SWIB DNA (SEQ ID NO: 1, corresponding amino acid sequence provided in SEQ ID NO: 5) encapsulated in poly (Lactide co-Glycolide, PLG); immunization was performed intraperitoneally. At 2 weeks after the last immunization, the animals were treated with progesterone and infected by vaginal inoculation with chlamydia psittaci. 2 weeks after infection, mice were sacrificed and the reproductive tract was sectioned, stained and histopathologically examined. The level of inflammation was scored as described above. The individual tubal/ovarian scores were summed and then divided by the number of organs examined to give an average score for inflammation for the group. Negative control immunized mice receiving PLG-encapsulated empty vector showed consistent inflammation with an average ovarian/oviduct inflammation score of 7.28 versus 5.71 for groups immunized with PLG-encapsulated DNA. Inflammation in the peritoneum was 1.75 for the vaccinated group and 3.75 for the control.

In a third experiment, C3H mice (4 mice per group) were immunized 3 times with 10. mu.g of purified recombinant protein SWIB (SEQ ID NO: 1, corresponding amino acid sequence provided in SEQ ID NO: 5) or S13(SEQ ID NO: 4, corresponding amino acid sequence provided in SEQ ID NO: 12) mixed with Cholera Toxin (CT); the formulation was administered intranasally in a volume of 20 μ l after anesthesia. 2 weeks after the last immunization, animals were treated with progesterone and infected by vaginal inoculation with Chlamydia psittaci or by injection of Chlamydia trachomatis serovar F in the uterus. 2 weeks after infection, mice were sacrificed and the reproductive tract was sectioned, stained and histopathologically examined. The degree of inflammation was scored as described above. The individual tubal/ovarian scores were summed and then divided by the number of organs examined to give an average score for inflammation for the group. In the uterine vaccination model, mice immunized with a negative control receiving cholera toxin alone exhibited an average ovarian/oviduct inflammation score of 4.25 (only 2 mice analyzed; the other 2 mice died), relative to the group immunized with S13 plus cholera toxin scoring 5.00 and the group immunized with SWIB plus cholera toxin scoring 1.00. Untreated infected animals had an average ovarian/oviduct inflammation score of 7. In the vaginal vaccination and ascending infection models, negative control immunized mice showed an average ovarian/oviduct inflammation score of 7.37, versus 6.75 for the group immunized with S13 plus cholera toxin and 5.37 for the group immunized with SWIB plus cholera toxin. Untreated infected animals had an average ovarian/oviduct inflammation score of 8.

The 3 experiments above suggest that SWIB-specific protection can be obtained. This protection was more pronounced in the allogenic infection model, but it also existed in the xenogenic challenge infection with C.psittaci.

2.CT529/Cap1

CT529/Cap1 was previously identified as a Chlamydia product that stimulated CD8+ CTL. In this example, we sought to demonstrate that immunization with Cap1 would be protective in animal models of chlamydial infection.

To prepare recombinant vaccinia virus for delivery of Cap1 immunogenic fragments, PCR was used^TMA DNA fragment containing the modified Kozak sequence and 319-530 bp of the cap1 gene (CT529) was amplified from the Chlamydia trachomatis L2 genomic DNA and ligated into pSC11ss (Earl PL, Koenig S, Moss B (1991) biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: analysis by expression of truncated proteins and proteins with deletions by recombinant vaccinia virus, J.Virol.65: 31-41). The DNA was digested with SalI and StuI. The portion of the Cap1 gene ligated into pSC11ss encodes amino acids 107-176 of the Cap1 protein, which contains the CTL epitope identified earlier as amino acids 139-147. The obtained plasmid was used for transfectionCV-1 cells (ATCC # CCL-70; Jensen FC et al (1964) were infected with Rous sarcoma virus in human and simian tissue cultures, Proc. Natl. Acad. Sci. USA 52:53-59), and subsequently re-infected with wild-type vaccinia virus. Homologous recombination of wild-type virus and plasmid DNA yields recombinant vaccinia virus, which is selected as described earlier (Chakrabarti et al, mol.cell.biol.1985, 5 (12): 3403-9) based on β -galactosidase expression and inactivation of thymidine kinase. Recombinant virus was plaque purified 3 times and titrated after growth on human TK-143B cells. The virus preparation was treated with an equal volume of 0.25mg/ml trypsin for 30 minutes at 37 ℃. It was then diluted in PBS and subsequently used to immunize mice. A group of 5 mice was used as all experimental and control groups. The data given below are representative of three independent experiments.

By 10⁶Recombinant vaccinia virus immunized a group of mice intraperitoneally (i.p.) and then allowed to recover for 3 weeks. A negative control group was immunized with buffer alone or wild-type vaccinia virus. As a positive control group, use 10⁶i.f.u. chlamydia trachomatis intravenously (i.v.) infected mouse groups. It has been previously demonstrated that the number of organisms given to the positive control group can be cleared within 2 weeks. After 3 weeks, use 10 weeks⁶i.f.u. chlamydia trachomatis intravenously challenged animals of each group. 3 days after challenge, animals were sacrificed and the i.f.u. number per spleen was determined.

Animals immunized with Cap 1-expressing vaccinia virus (7.1X 10)⁴) With buffer (1.8X 10)⁵) Or wild type vaccinia (1.9X 10)⁵) Compared with the immunized control group, the average number of organisms found in the spleen is 2.6 times less (p)<0.01; wilcoxon's rank sum test). Animals in the positive group contained organisms per spleen (2.4X 10)³) 77 times lower than that of negative control group (P)<0.01; wilcoxon's rank sum test). These data indicate that immunization with an immunogenic fragment of Cap1 can provide a statistically significant level of protection against chlamydia trachomatis infection.

Example 10

Pmp/Ra12 fusion protein

A variety of Pmp/Ra12 fusion constructs were prepared by first synthesizing a PCR fragment of the Pmp gene using primers containing NotI restriction sites. Each PCR fragment was then ligated into the NotI restriction site of pCRX 1. The pCRX1 vector contains the 6HisRa12 portion of the fusion. The Ra12 portion of the fusion construct encodes a polypeptide corresponding to amino acid residues 192-323 of Mycobacterium tuberculosis MTB32A (described in U.S. patent application 60/158,585, the disclosure of which is incorporated herein by reference). The correct orientation of each insert was determined by its restriction map and its sequence was verified. For PmpA, PmpB, PmpC, PmpF, and PmpH, a number of fusion constructs were prepared, as further described below:

PmpA fusion protein

PmpA is a 107kD protein containing 982 amino acids, cloned from serum variant E. The PmpA protein was divided into 2 overlapping fragments, PmpA (N-terminal) and (C-terminal) portions.

PmpA (N-terminal) was amplified with the following sense and antisense primers: the primers were GAGAGCGGCCGCTCATGTTTATAACAAAGGAACTTATG (SEQ ID NO: 306) GAGAGCGGCCGCTTACTTAGGTGAGAAGAAGGGAGTTTC (SEQ ID NO: 307), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 308, encoding a 66kD protein (619 amino acids) expressing the amino acid segment from 1 to 473 of PmpA. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 309.

PmpA (C-terminal) was amplified with the following sense and antisense primers: the primers were GAGAGCGGCCGCTCCATTCTATTCATTTCTTTGATCCTG (SEQ ID NO: 310) GAGAGCGGCCGCTTAGAAGCCAACATAGCCTCC (SEQ ID NO: 311), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 312 encoding the 74kD protein (691 amino acids) expressing the 438 nd and 982 nd amino acid segment of PmpA. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 313.

pmpf fusion protein

PmpF is a 112kD protein containing 1034 amino acids, cloned from serum variant E. The PmpF protein was divided into 2 overlapping fragments, the PmpF (N-terminal) and (C-terminal) portions.

PmpF (N-terminal) was amplified with sense and antisense primers as follows: the primers were GAGAGCGGCCGCTCATGATTAAAAGAACTTCTCTATCC (SEQ ID NO: 314) GAGAGCGGCCGCTTATAATTCTGCATCATCTTCTATGGC (SEQ ID NO: 315), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 316, encoding a 69kD protein (646 amino acids) expressing the amino acid segment from position 1 to 499 of PmpF. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 317.

PmpF (C-terminal) was amplified with sense and antisense primers as follows: the primers were GAGAGCGGCCGCTCGACATACGAACTCTGATGGG (SEQ ID NO: 318) GAGAGCGGCCGCTTAAAAGACCAGAGCTCCTCC (SEQ ID NO: 319), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 320 encoding a 77kD protein (715 amino acids) expressing the 466-1034 amino acid segment of PmpF. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 321.

PmDH fusion protein

PmpH is a 108kD protein containing 1016 amino acids, cloned from serum variant E. The PmpH protein was divided into 2 overlapping fragments, the PmpH (N-terminal) and (C-terminal) fractions.

The PmpH (N-terminal) was amplified with sense and antisense primers as follows: the primers were GAGAGCGGCCGCTCATGCCTTTTTCTTTGAGATCTAC (SEQ ID NO: 322) GAGAGCGGCCGCTTACACAGATCCATTACCGGACTG (SEQ ID NO: 323), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 324, encoding a 64kD protein (631 amino acids) expressing the amino acid segment from position 1 to 484 of PmpH. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 325. donor system CHH037 was found to be reactive to this protein.

The PmpH (C-terminal) was amplified with sense and antisense primers as follows: the primers were GAGAGCGGCCGCTCGATCCTGTAGTACAAAATAATTCAGC (SEQ ID NO: 326) GAGAGCGGCCGCTTAAAAGATTCTATTCAAGCC (SEQ ID NO: 327), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 328 which encodes a 77kD protein (715 amino acids) expressing the 449-1016 amino acid segment of PmpH. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 329. patient line CT12 was found to be responsive to this protein.

Pmpb fusion proteins

PmpB is a 183kD protein containing 1750 amino acids cloned from serum variant E. The PmpB protein was divided into 4 overlapping fragments, PmpB (1), (2), (3) and (4).

PmpB (1) was amplified with the following sense and antisense primers: the primers were GAGAGCGGCCGCTCATGAAATGGCTGTCAGCTACTGCG (SEQ ID NO: 330) GAGAGCGGCCGCTTACTTAATGCGAATTTCTTCAAG (SEQ ID NO: 331), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 332 and encodes a 53kD protein (518 amino acids) expressing the amino acid segment from position 1 to 372 of PmpB. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 333.

PmpB (2) was amplified with the following sense and antisense primers: the primers were GAGAGCGGCCGCTCGGTGACCTCTCAATTCAATCTTC (SEQ ID NO: 334) GAGAGCGGCCGCTTAGTTCTCTGTTACAGATAAGGAGAC (SEQ ID NO: 335), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 336 encoding a 60kD protein (585 amino acids) expressing the amino acid segment at position 330 and 767 of PmpB. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 337. cell lines derived from patient lines CT1, CT3, CT4 responded to the recombinant pmpB protein.

PmpB (3) was amplified with the following sense and antisense primers: the primers were GAGAGCGGCCGCTCGACCAACTGAATATCTCTGAGAAC (SEQ ID NO: 338) GAGCGGCCGCTTAAGAGACTACGTGGAGTTCTG (SEQ ID NO: 339), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 340, encoding a 67kD protein (654 amino acids) expressing the amino acid segment at position 732-1236 of PmpB. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 341.

PmpB (4) was amplified with the following sense and antisense primers: the primers were GAGAGCGGCCGCTCGGAACTATTGTGTTCTCTTCTG (SEQ ID NO: 342) GAGAGCGGCCGCTTAGAAGATCATGCGAGCACCGC (SEQ ID NO: 343), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 344 encoding a 76kD protein (700 amino acids) expressing the 1160-1750 amino acid segment of PmpB. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 345.

pmpc fusion proteins

PmpC is a 187kD protein containing 1774 amino acids cloned from serovariant E/L2. The PmpC protein was divided into 3 overlapping fragments, PmpC (1), (2) and (3).

PmpC (1) was amplified with sense and antisense primers as follows: the primers were GAGAGCGGCCGCTCATGAAATTTATGTCAGCTACTGC (SEQ ID NO: 346) GAGAGCGGCCGCTTACCCTGTAATTCCAGTGATGGTC (SEQ ID NO: 347), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 348, encoding a 51kD protein (487 amino acids) expressing the amino acid segment at positions 1-340 of PmpC. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 349.

PmpC (2) was amplified with sense and antisense primers as follows: the primers were GAGAGCGGCCGCTCGATACACAAGTATCAGAATCACC (SEQ ID NO: 350) GAGAGCGGCCGCTTAAGAGGACGATGAGACACTCTCG (SEQ ID NO: 351), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 352, encoding a 60kD protein (583 amino acids) expressing the 741 nd amino acid segment of PmpC at position 305-. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 353.

PmpC (3) was amplified with sense and antisense primers as follows: the primers were GAGAGCGGCCGCTCGATCAATCTAACGAAAACACAGACG (SEQ ID NO: 354) GAGAGCGGCCGCTTAGACCAAAGCTCCATCAGCAAC (SEQ ID NO: 355), respectively. The resulting fusion construct has the amino acid sequence of SEQ ID NO: 356 encoding a 70kD protein (683 amino acids) expressing the amino acid segment 714-1250 of PmpC. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 357.

example 11

Immunogenicity of CT622

Chlamydia-specific T cell lines were prepared from two chlamydial infected patients and these cell lines were designated CT1 and CT 13. These T cell lines were either prepared against monocyte-derived dendritic cells infected with Chlamydia trachomatis serotype E72 hours (CT1-ERB) or against killed serovar E protoplasts (EB) (CT 13-EEB). Once prepared, these cell lines were tested in proliferation assays against the recombinant chlamydia-specific protein CT 622. Proliferation assays were performed by: at 1X 10 ⁴Stimulation of 2.5X 10 with recombinant CT antigen (2. mu.g/ml) or Chlamydia EB (1. mu.g/ml) in the Presence of monocytic derived dendritic cells⁴And (4) T cells. In this test, the cells were incubated for 4 days with³The last 18 hours of the H-thymidine pulse.

The cell line CT1-ERB exhibited a significantly higher proliferative response than the media control when stimulated with CT622, CT875, and CT EB. The cell line CT13-EEB exhibited a significantly higher proliferative response than the media control when stimulated with CT622, CT875, and CT EB (see fig. 12).

Example 12

Cloning and expression of the full-length Chlamydia trachomatis genes CT611, ORF3 and OppA1

The clones containing the genes CT611, ORF-3 and OppA1 were subjected to recombinant protein expression in the full-length development reading frame. The clones containing the gene of interest were CtL2-8(SEQ ID NO: 285) encoding 4 ORFs (CT474, CT473, CT060 and CT139), CtL2-10(SEQ ID NO: 284) encoding ORFs of CT610 and CT611, and clones 16CtL2-16(SEQ ID NO: 47), 16-D4-22(SEQ ID NO: 119) and 19-A5-54(SEQ ID NO: 84), each containing a sequence related to ORF-3. The sequences in CtL2-10(Ct-610) and CtL2-16(ORF-3) were also independently identified by T cell expression cloning methods. Clone CtL2-8 was further studied because it stimulated both T cell lines prepared against serovar E to produce a proliferative response and IFN- γ production.

Cloning and expression of cloned sequences:

CtL2-10 was found to encode 2 Open Reading Frames (ORFs), CT610 and CT611, which were found to be arranged adjacent to each other in the genomic clone. The full-length ORF of CT610 (containing the PQQ synthesis domain) has been previously expressed and demonstrated to stimulate a proliferative response in anti-chlamydia producing T cell lines. To determine whether the second ORF (CT611) was also recognized by T cells, the full-length sequence of CT611 was PCR amplified and engineered for protein expression. The nucleotide sequence of which is disclosed in SEQ ID NO: 361, the corresponding amino acid sequence is disclosed in SEQ ID NO: 365.

The second serological clone CtL2-8 was found to contain 4 ORFs (CT474, CT473, CT060 and CT 139). Overlapping peptides of the three smallest predicted ORFs (CT474, CT473 and CT060) did not stimulate the proliferative response of the T cell lines. This suggests that the immunostimulatory antigen is located in the 4 th ORF (CT 139). The ORF for CT139 is approximately 450 nucleotides. The full-length nucleotide sequence is disclosed in SEQ ID NO: 359, and the full-length amino acid sequence is disclosed in SEQ ID NO: 363 (f). Amino acid comparisons with GenBank revealed that it is an oligopeptide-binding protein (oppA1) and that it belongs to the peptide ABC transporter family. The protein is 462 amino acids long, has a predicted size of 48.3kDa, and appears to contain 2 transmembrane domains.

To express the full-length sequence of oppA1, primers were designed to specifically amplify the sequence starting from amino acid position 22 (lacking the first transmembrane domain), the nucleotide sequence of which is disclosed in SEQ ID NO: 358, whose amino acid sequence discloses SEQ ID NO: 362 (c). This sequence has been shown to express the protein in E.coli.

In addition, full-length cloning and recombinant protein expression of ORF-3 was obtained. The nucleotide and amino acid sequences are disclosed in SEQ ID NO: 360 and 364.

Example 13

Recombinant chlamydia antigens recognized by T cell lines

Patient T cell lines were generated from the following donors: CT1, CT2, CT3, CT4, CT5, CT6, CT7, CT8, CT9, CT10, CT11, CT12, CT13, CT14, CT15 and CT16, some of which are discussed above. Their details are summarized in table V.

NGU ═ non-gonococcal urethritis; BV-bacterial vaginosis; CT ═ chlamydia trachomatis; cp is chlamydia pneumoniae; eb is chlamydia protosome; HPV ═ human papilloma virus; dx-diagnosis; PID is pelvic inflammatory disease; LCR ═ ligase chain reaction.

PBMCs from a series of donors were collected and T cell lines were prepared from subpopulations thereof. The details of 3 such T cell lines are summarized in the table below.

Donor CHH011 is a 49-year-old healthy female donor who is chlamydia trachomatis serum negative. PBMC produce greater amounts of IFN- γ in response to chlamydia trachomatis protoplasts than in response to chlamydia pneumoniae protoplasts, indicating a chlamydia trachomatis-specific response. Donor choo 37 was a 22 year old healthy female donor who was chlamydia trachomatis serum negative. PBMC produced greater amounts of IFN- γ in response to chlamydia trachomatis protoplasts than in response to chlamydia pneumoniae protoplasts, suggesting that this is a chlamydia trachomatis specific response. CHHO42 is a 25 year old healthy female donor with an IgG titer of 1:16 against Chlamydia pneumoniae. PBMC produced greater amounts of IFN- γ in response to chlamydia trachomatis protoplasts than in response to chlamydia pneumoniae protoplasts, suggesting that this is a chlamydia trachomatis specific response.

Recombinant proteins of several chlamydia trachomatis genes were prepared as described above. The sequence of MOMP is derived from serovariant F. The genes CT875, CT622, pmp-B-2, pmpA and CT529 were derived from serovar E, while the sequences of the gro-EL, Swib, pmpD, pmpG, TSA, CT610, pmpC, pmpE, S13, lpdA, pmpI and pmpH-C genes were derived from LII.

Several of the above patient and donor lines were tested against these recombinant chlamydia proteins. Table IV summarizes the results of T cell responses to these recombinant chlamydia proteins.

Although the present invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, modifications and changes may be made without departing from the scope of the invention, which is intended to be limited only by the scope of the appended claims.

Sequence listing

<110>Corixa Corporation

Fling，Steven P.

Skeiky，Yasir A.W.

Probst，Peter

Bhatia，Ajay

<120> Compounds and methods for treating and diagnosing chlamydial infections

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<141>2001-07-20

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Chlamydia trachomatis (213)

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Chlamydia trachomatis (213)

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Chlamydia trachomatis (213)

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Chlamydia trachomatis (213)

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Chlamydia trachomatis (213)

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Chlamydia trachomatis (213)

<400>7

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Chlamydia trachomatis (213)

<400>8

<210>9

<211>5

<212>PRT

Chlamydia trachomatis (213)

<400>9

<210>10

<211>11

<212>PRT

Chlamydia trachomatis (213)

<400>10

<210>11

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<212>PRT

Chlamydia trachomatis (213)

<400>11

<210>12

<211>122

<212>PRT

Chlamydia trachomatis (213)

<400>12

<210>13

<211>20

<212>PRT

Chlamydia trachomatis (213)

<400>13

<210>14

<211>20

<212>PRT

Chlamydia trachomatis (213)

<400>14

<210>15

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Chlamydia trachomatis (213)

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<210>16

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<212>DNA

Chlamydia trachomatis (213)

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<212>PRT

Chlamydia trachomatis (213)

<400>17

<210>18

<211>18

<212>PRT

Chlamydia trachomatis (213)

<400>18

<210>19

<211>18

<212>PRT

Chlamydia trachomatis (213)

<400>19

<210>20

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<212>PRT

Chlamydia trachomatis (213)

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<210>21

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Chlamydia trachomatis (213)

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<210>22

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Chlamydia trachomatis (213)

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Chlamydia trachomatis (213)

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Chlamydia trachomatis (213)

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Chlamydia trachomatis (213)

<400>25

<210>26

<211>231

<212>PRT

Chlamydia trachomatis (213)

<400>26

<210>27

<211>264

<212>DNA

213 Chlamydia pneumoniae (Chlamydia pneumoniae)

<400>27

<210>28

<211>87

<212>PRT

Chlamydia pneumoniae of <213>

<400>28

<210>29

<211>369

<212>DNA

Chlamydia pneumoniae of <213>

<400>29

<210>30

<211>122

<212>PRT

Chlamydia pneumoniae of <213>

<400>30

<210>31

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>31

<210>32

<211>53

<212>PRT

Chlamydia trachomatis (213)

<400>32

<210>33

<211>161

<212>DNA

Chlamydia trachomatis (213)

<400>33

<210>34

<211>53

<212>PRT

Chlamydia trachomatis (213)

<400>34

<210>35

<211>55

<212>DNA

Chlamydia pneumoniae of <213>

<400>35

<210>36

<211>33

<212>DNA

Chlamydia pneumoniae of <213>

<400>36

<210>37

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<212>DNA

Chlamydia pneumoniae of <213>

<400>37

<210>38

<211>30

<212>DNA

Chlamydia pneumoniae of <213>

<400>38

<210>39

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>39

<210>40

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>40

<210>41

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>41

<210>42

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>42

<210>43

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>43

<210>44

<211>509

<212>DNA

<213> Chlamydia (Chlamydia)

<400>44

<210>45

<211>481

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>23

<223> n ═ A, T, C or G

<400>45

<210>46

<211>427

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>20

<223> n ═ A, T, C or G

<400>46

<210>47

<211>600

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>522

<223> n ═ A, T, C or G

<400>47

<210>48

<211>600

<212>DNA

<213> genus Chlamydia

<400>48

<210>49

<211>600

<212>DNA

<213> genus Chlamydia

<400>49

<210>50

<211>406

<212>DNA

<213> genus Chlamydia

<400>50

<210>51

<211>602

<212>DNA

<213> genus Chlamydia

<400>51

<210>52

<211>145

<212>DNA

<213> genus Chlamydia

<400>52

<210>53

<211>450

<212>DNA

<213> genus Chlamydia

<400>53

<210>54

<211>716

<212>DNA

213 genus chlamydia

<400>54

<210>55

<211>463

<212>DNA

Chlamydia trachomatis (213)

<400>55

<210>56

<211>829

<212>DNA

Chlamydia trachomatis (213)

<400>56

<210>57

<211>1537

<212>DNA

Chlamydia trachomatis (213)

<400>57

<210>58

<211>463

<212>DNA

Chlamydia trachomatis (213)

<400>58

<210>59

<211>552

<212>DNA

Chlamydia trachomatis (213)

<400>59

<210>60

<211>1180

<212>DNA

Chlamydia trachomatis (213)

<400>60

<210>61

<211>1215

<212>DNA

Chlamydia trachomatis (213)

<400>61

<210>62

<211>688

<212>DNA

Chlamydia trachomatis (213)

<400>62

<210>63

<211>269

<212>DNA

Chlamydia trachomatis (213)

<400>63

<210>64

<211>1339

<212>DNA

Chlamydia trachomatis (213)

<400>64

<210>65

<211>195

<212>PRT

Chlamydia trachomatis (213)

<400>65

<210>66

<211>520

<212>DNA

<213> genus Chlamydia

<400>66

<210>67

<211>276

<212>DNA

<213> genus Chlamydia

<400>67

<210>68

<211>248

<212>DNA

<213> genus Chlamydia

<400>68

<210>69

<211>715

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>34

<223> n ═ A, T, C or G

<400>69

<210>70

<211>323

<212>DNA

<213> genus Chlamydia

<400>70

<210>71

<211>715

<212>DNA

<213> genus Chlamydia

<400>71

<210>72

<211>641

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>550，559，575，583，634，638

<223> n ═ A, T, C or G

<400>72

<210>73

<211>584

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>460，523，541，546

<223> n ═ A, T, C or G

<400>73

<210>74

<211>465

<212>DNA

<213> genus Chlamydia

<400>74

<210>75

<211>545

<212>DNA

<213> genus Chlamydia

<400>75

<210>76

<211>797

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>788，789

<223> n ═ A, T, C or G

<400>76

<210>77

<211>399

<212>DNA

<213> genus Chlamydia

<400>77

<210>78

<211>285

<212>DNA

<213> genus Chlamydia

<400>78

<210>79

<211>950

<212>DNA

<213> genus Chlamydia

<400>79

<210>80

<211>395

<212>DNA

<213> genus Chlamydia

<400>80

<210>81

<211>2085

<212>DNA

<213> genus Chlamydia

<400>81

<210>82

<211>405

<212>DNA

<213> genus Chlamydia

<400>82

<210>83

<211>379

<212>DNA

<213> genus Chlamydia

<400>83

<210>84

<211>715

<212>DNA

<213> genus Chlamydia

<400>84

<210>85

<211>476

<212>DNA

<213> genus Chlamydia

<400>85

<210>86

<211>1551

<212>DNA

<213> genus Chlamydia

<400>86

<210>87

<211>3031

<212>DNA

<213> genus Chlamydia

<400>87

<210>88

<211>976

<212>DNA

<213> genus Chlamydia

<400>88

<210>89

<211>94

<212>PRT

<213> genus Chlamydia

<400>89

<210>90

<211>474

<212>PRT

<213> genus Chlamydia

<400>90

<210>91

<211>129

<212>PRT

<213> genus Chlamydia

<400>91

<210>92

<211>202

<212>PRT

<213> genus Chlamydia

<400>92

<210>93

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>93

<210>94

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>94

<210>95

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>95

<210>96

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>96

<210>97

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>97

<210>98

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>98

<210>99

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>99

<210>100

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>100

<210>101

<211>20

<212>PRT

<213>Artificial Sequence

<220>

<223>Made in alab

<400>101

<210>102

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>102

<210>103

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>103

<210>104

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>104

<210>105

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>105

<210>106

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>106

<210>107

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>107

<210>108

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>108

<210>109

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>109

<210>110

<211>1461

<212>DNA

<213> genus Chlamydia

<400>110

<210>111

<211>267

<212>DNA

<213> genus Chlamydia

<400>111

<210>112

<211>698

<212>DNA

<213> genus Chlamydia

<400>112

<210>113

<211>1142

<212>DNA

<213> genus Chlamydia

<400>113

<210>114

<211>976

<212>DNA

<213> genus Chlamydia

<400>114

<210>115

<211>995

<212>DNA

<213> genus Chlamydia

<400>115

<210>116

<211>437

<212>DNA

<213> genus Chlamydia

<400>116

<210>117

<211>446

<212>DNA

<213> genus Chlamydia

<400>117

<210>118

<211>951

<212>DNA

<213> genus Chlamydia

<400>118

<210>119

<211>953

<212>DNA

<213> genus Chlamydia

<400>119

<210>120

<211>897

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>395

<223> n ═ A, T, C or G

<400>120

<210>121

<211>298

<212>PRT

<213> genus Chlamydia

<400>121

<210>122

<211>897

<212>DNA

<213> genus Chlamydia

<400>122

<210>123

<211>298

<212>PRT

<213> genus Chlamydia

<400>123

<210>124

<211>897

<212>DNA

<213> genus Chlamydia

<400>124

<210>125

<211>298

<212>PRT

<213> genus Chlamydia

<400>125

<210>126

<211>897

<212>DNA

<213> genus Chlamydia

<400>126

<210>127

<211>298

<212>PRT

<213> genus Chlamydia

<400>127

<210>128

<211>897

<212>DNA

<213> genus Chlamydia

<400>128

<210>129

<211>298

<212>PRT

<213> genus Chlamydia

<400>129

<210>130

<211>897

<212>DNA

<213> genus Chlamydia

<400>130

<210>131

<211>298

<212>PRT

<213> genus Chlamydia

<400>131

<210>132

<211>897

<212>DNA

<213> genus Chlamydia

<400>132

<210>133

<211>298

<212>PRT

<213> genus Chlamydia

<400>133

<210>134

<211>897

<212>DNA

<213> genus Chlamydia

<400>134

<210>135

<211>298

<212>PRT

<213> genus Chlamydia

<400>135

<210>136

<211>882

<212>DNA

<213> genus Chlamydia

<400>136

<210>137

<211>293

<212>PRT

<213> genus Chlamydia

<400>137

<210>138

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>138

<210>139

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>139

<210>140

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>140

<210>141

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>141

<210>142

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>142

<210>143

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>143

<210>144

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>144

<210>145

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>145

<210>146

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>146

<210>147

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>147

<210>148

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>148

<210>149

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>149

<210>150

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>150

<210>151

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>151

<210>152

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>152

<210>153

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>153

<210>154

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>154

<210>155

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>155

<210>156

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>156

<210>157

<211>53

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>157

<210>158

<211>52

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>158

<210>159

<211>24

<212>DNA

<213> genus Chlamydia

<400>159

<210>160

<211>24

<212>DNA

<213> genus Chlamydia

<400>160

<210>161

<211>24

<212>DNA

<213> genus Chlamydia

<400>161

<210>162

<211>19

<212>DNA

<213> genus Chlamydia

<400>162

<210>163

<211>24

<212>DNA

<213> genus Chlamydia

<400>163

<210>164

<211>29

<212>DNA

<213> genus Chlamydia

<400>164

<210>165

<211>20

<212>DNA

<213> genus Chlamydia

<400>165

<210>166

<211>20

<212>DNA

<213> genus Chlamydia

<400>166

<210>167

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>167

<210>168

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>168

<210>169

<211>2643

<212>DNA

<213> genus Chlamydia

<400>169

<210>170

<211>2949

<212>DNA

<213> genus Chlamydia

<400>170

<210>171

<211>2895

<212>DNA

<213> genus Chlamydia

<400>171

<210>172

<211>4593

<212>DNA

<213> genus Chlamydia

<400>172

<210>173

<211>5331

<212>DNA

<213> genus Chlamydia

<400>173

<210>174

<211>5265

<212>DNA

<213> genus Chlamydia

<400>174

<210>175

<211>880

<212>PRT

<213> genus Chlamydia

<220>

<221>VARIANT

<222>336

<223> Xaa ═ any amino acid

<400>175

<210>176

<211>982

<212>PRT

<213> genus Chlamydia

<220>

<221>VARIANT

<222>981

<223> Xaa ═ any amino acid

<400>176

<210>177

<211>964

<212>PRT

<213> genus Chlamydia

<400>177

<210>178

<211>1530

<212>PRT

<213> genus Chlamydia

<400>178

<210>179

<211>1776

<212>PRT

<213> genus Chlamydia

<400>179

<210>180

<211>1752

<212>PRT

<213> genus Chlamydia

<400>180

<210>181

<211>2601

<212>DNA

<213> genus Chlamydia

<400>181

<210>182

<211>3021

<212>DNA

<213> genus Chlamydia

<400>182

<210>183

<211>2934

<212>DNA

<213> genus Chlamydia

<400>183

<210>184

<211>2547

<212>DNA

<213> genus Chlamydia

<400>184

<210>185

<211>2337

<212>DNA

<213> genus Chlamydia

<400>185

<210>186

<211>2847

<212>DNA

<213> genus Chlamydia

<400>186

<210>187

<211>2466

<212>DNA

<213> genus Chlamydia

<400>187

<210>188

<211>1578

<212>DNA

<213> genus Chlamydia

<400>188

<210>189

<211>866

<212>PRT

<213> genus Chlamydia

<220>

<221>VARIANT

<222>220，242，425，448，453，455

<223> Xaa ═ any amino acid

<400>189

<210>190

<211>1006

<212>PRT

<213> genus Chlamydia

<400>190

<210>191

<211>977

<212>PRT

<213> genus Chlamydia

<400>191

<210>192

<211>848

<212>PRT

<213> genus Chlamydia

<400>192

<210>193

<211>778

<212>PRT

<213> genus Chlamydia

<400>193

<210>194

<211>948

<212>PRT

<213> genus Chlamydia

<400>194

<210>195

<211>821

<212>PRT

<213> genus Chlamydia

<400>195

<210>196

<211>525

<212>PRT

<213> genus Chlamydia

<400>196

<210>197

<211>43

<212>DNA

<213> genus Chlamydia

<400>197

<210>198

<211>34

<212>DNA

<213> genus Chlamydia

<400>198

<210>199

<211>6

<212>DNA

<213> genus Chlamydia

<400>199

<210>200

<211>34

<212>DNA

<213> genus Chlamydia

<400>200

<210>201

<211>38

<212>DNA

<213> genus Chlamydia

<400>201

<210>202

<211>5

<212>DNA

<213> genus Chlamydia

<400>202

<210>203

<211>31

<212>DNA

<213> genus Chlamydia

<400>203

<210>204

<211>31

<212>DNA

<213> genus Chlamydia

<400>204

<210>205

<211>30

<212>DNA

<213> genus Chlamydia

<400>205

<210>206

<211>31

<212>DNA

<213> genus Chlamydia

<400>206

<210>207

<211>50

<212>DNA

<213> genus Chlamydia

<400>207

<210>208

<211>40

<212>DNA

<213> genus Chlamydia

<400>208

<210>209

<211>55

<212>DNA

<213> genus Chlamydia

<400>209

<210>210

<211>35

<212>DNA

<213> genus Chlamydia

<400>210

<210>211

<211>36

<212>DNA

<213> genus Chlamydia

<400>211

<210>212

<211>35

<212>DNA

<213> genus Chlamydia

<400>212

<210>213

<211>51

<212>DNA

<213> genus Chlamydia

<400>213

<210>214

<211>38

<212>DNA

<213> genus Chlamydia

<400>214

<210>215

<211>48

<212>DNA

<213> genus Chlamydia

<400>215

<210>216

<211>31

<212>DNA

<213> genus Chlamydia

<400>216

<210>217

<211>7

<212>DNA

<213> genus Chlamydia

<400>217

<210>218

<211>22

<212>PRT

<213> genus Chlamydia

<400>218

<210>219

<211>51

<212>DNA

<213> genus Chlamydia

<400>219

<210>220

<211>33

<212>DNA

<213> genus Chlamydia

<400>220

<210>221

<211>24

<212>PRT

<213> genus Chlamydia

<400>221

<210>222

<211>46

<212>DNA

<213> genus Chlamydia

<400>222

<210>223

<211>30

<212>DNA

<213> genus Chlamydia

<400>223

<210>224

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>224

<210>225

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>225

<210>226

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>226

<210>227

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>227

<210>228

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>228

<210>229

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>229

<210>230

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>230

<210>231

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>231

<210>232

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>232

<210>233

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>233

<210>234

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>234

<210>235

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>235

<210>236

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>236

<210>237

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>237

<210>238

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>238

<210>239

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>239

<210>240

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>240

<210>241

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>241

<210>242

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>242

<210>243

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>243

<210>244

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>244

<210>245

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>245

<210>246

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>246

<210>247

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>247

<210>248

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>248

<210>249

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>249

<210>250

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>250

<210>251

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>251

<210>252

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>252

<210>253

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>253

<210>254

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>254

<210>255

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>255

<210>256

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>256

<210>257

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>257

<210>258

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>258

<210>259

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>259

<210>260

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>260

<210>261

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>261

<210>262

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> laboratory system

<400>262

<210>263

<211>897

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>604

<223> n ═ A, T, C or G

<400>263

<210>264

<211>298

<212>PRT

<213> genus Chlamydia

<220>

<221>VARIANT

<222>202

<223> Xaa ═ any amino acid

<400>264

<210>265

<211>897

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>604

<223> n ═ A, T, C or G

<400>265

<210>266

<211>298

<212>PRT

<213> genus Chlamydia

<220>

<221>VARIANT

<222>202

<223> Xaa ═ any amino acid

<400>266

<210>267

<211>680

<212>DNA

<213> genus Chlamydia

<400>267

<210>268

<211>359

<212>DNA

<213> genus Chlamydia

<400>268

<210>269

<211>124

<212>DNA

<213> genus Chlamydia

<400>269

<210>270

<211>219

<212>DNA

<213> genus Chlamydia

<400>270

<210>271

<211>511

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>447

<223> n ═ A, T, C or G

<400>271

<210>272

<211>598

<212>DNA

<213> genus Chlamydia

<400>272

<210>273

<211>126

<212>DNA

<213> genus Chlamydia

<400>273

<210>274

<211>264

<212>DNA

<213> genus Chlamydia

<400>274

<210>275

<211>359

<212>DNA

<213> genus Chlamydia

<400>275

<210>276

<211>357

<212>DNA

<213> genus Chlamydia

<400>276

<210>277

<211>505

<212>DNA

<213> genus Chlamydia

<400>277

<210>278

<211>407

<212>DNA

<213> genus Chlamydia

<400>278

<210>279

<211>351

<212>DNA

<213> genus Chlamydia

<400>279

<210>280

<211>522

<212>DNA

<213> genus Chlamydia

<400>280

<210>281

<211>577

<212>DNA

<213> genus Chlamydia

<400>281

<210>282

<211>607

<212>DNA

<213> genus Chlamydia

<400>282

<210>283

<211>1077

<212>DNA

<213> genus Chlamydia

<400>283

<210>284

<211>407

<212>DNA

<213> genus Chlamydia

<400>284

<210>285

<211>802

<212>DNA

<213> genus Chlamydia

<400>285

<210>286

<211>588

<212>DNA

<213> genus Chlamydia

<400>286

<210>287

<211>489

<212>DNA

<213> genus Chlamydia

<220>

<221>misc_feature

<222>488

<223> n ═ A, T, C or G

<400>287

<210>288

<211>191

<212>DNA

<213> genus Chlamydia

<400>288

<210>289

<211>515

<212>DNA

<213> genus Chlamydia

<400>289

<210>290

<211>522

<212>DNA

<213> genus Chlamydia

<400>290

<210>291

<211>1002

<212>DNA

<213> genus Chlamydia

<400>291

<210>292

<211>333

<212>PRT

<213> genus Chlamydia

<400>292

<210>293

<211>7

<212>DNA

<213> genus Chlamydia

<400>293

<210>294

<211>196

<212>PRT

<213> genus Chlamydia

<400>294

<210>295

<211>181

<212>PRT

<213> genus Chlamydia

<400>295

<210>296

<211>124

<212>PRT

<213> genus Chlamydia

<400>296

<210>297

<211>488

<212>PRT

<213> genus Chlamydia

<400>297

<210>298

<211>140

<212>PRT

<213> genus Chlamydia

<400>298

<210>299

<211>361

<212>PRT

<213> genus Chlamydia

<400>299

<210>300

<211>207

<212>PRT

<213> genus Chlamydia

<400>300

<210>301

<211>183

<212>PRT

<213> genus Chlamydia

<400>301

<210>302

<211>232

<212>PRT

<213> genus Chlamydia

<400>302

<210>303

<211>238

<212>PRT

<213> genus Chlamydia

<400>303

<210>304

<211>133

<212>PRT

<213> genus Chlamydia

<400>304

<210>305

<211>125

<212>PRT

<213> genus Chlamydia

<400>305

<210>306

<211>38

<212>DNA

Chlamydia trachomatis (213)

<400>306

<210>307

<211>39

<212>DNA

Chlamydia trachomatis (213)

<400>307

<210>308

<211>1860

<212>DNA

Chlamydia trachomatis (213)

<400>308

<210>309

<211>619

<212>PRT

Chlamydia trachomatis (213)

<400>309

<210>310

<211>39

<212>DNA

Chlamydia trachomatis (213)

<400>310

<210>311

<211>33

<212>DNA

Chlamydia trachomatis (213)

<400>311

<210>312

<211>2076

<212>DNA

Chlamydia trachomatis (213)

<400>312

<210>313

<211>691

<212>PRT

Chlamydia trachomatis (213)

<400>313

<210>314

<211>38

<212>DNA

Chlamydia trachomatis (213)

<400>314

<210>315

<211>36

<212>DNA

Chlamydia trachomatis (213)

<400>315

<210>316

<211>1941

<212>DNA

Chlamydia trachomatis (213)

<400>316

<210>317

<211>646

<212>PRT

Chlamydia trachomatis (213)

<400>317

<210>318

<211>34

<212>DNA

Chlamydia trachomatis (213)

<400>318

<210>319

<211>33

<212>DNA

Chlamydia trachomatis (213)

<400>319

<210>320

<211>2148

<212>DNA

Chlamydia trachomatis (213)

<400>320

<210>321

<211>715

<212>PRT

Chlamydia trachomatis (213)

<400>321

<210>322

<211>37

<212>DNA

Chlamydia trachomatis (213)

<400>322

<210>323

<211>36

<212>DNA

Chlamydia trachomatis (213)

<400>323

<210>324

<211>1896

<212>DNA

Chlamydia trachomatis (213)

<400>324

<210>325

<211>631

<212>PRT

Chlamydia trachomatis (213)

<400>325

<210>326

<211>40

<212>DNA

Chlamydia trachomatis (213)

<400>326

<210>327

<211>33

<212>DNA

Chlamydia trachomatis (213)

<400>327

<210>328

<211>2148

<212>DNA

Chlamydia trachomatis (213)

<400>328

<210>329

<211>715

<212>PRT

Chlamydia trachomatis (213)

<400>329

<210>330

<211>38

<212>DNA

Chlamydia trachomatis (213)

<400>330

<210>331

<211>34

<212>DNA

Chlamydia trachomatis (213)

<400>331

<210>332

<211>1557

<212>DNA

Chlamydia trachomatis (213)

<400>332

<210>333

<211>518

<212>PRT

Chlamydia trachomatis (213)

<400>333

<210>334

<211>37

<212>DNA

Chlamydia trachomatis (213)

<400>334

<210>335

<211>39

<212>DNA

<213> Chlamydia trachomatis

<400>335

<210>336

<211>1758

<212>DNA

Chlamydia trachomatis (213)

<400>336

<210>337

<211>585

<212>PRT

Chlamydia trachomatis (213)

<400>337

<210>338

<211>38

<212>DNA

Chlamydia trachomatis (213)

<400>338

<210>339

<211>35

<212>DNA

Chlamydia trachomatis (213)

<400>339

<210>340

<211>1965

<212>DNA

Chlamydia trachomatis (213)

<400>340

<210>341

<211>654

<212>PRT

Chlamydia trachomatis (213)

<400>341

<210>342

<211>36

<212>DNA

Chlamydia trachomatis (213)

<400>342

<210>343

<211>35

<212>DNA

Chlamydia trachomatis (213)

<400>343

<210>344

<211>2103

<212>DNA

Chlamydia trachomatis (213)

<400>344

<210>345

<211>700

<212>PRT

Chlamydia trachomatis (213)

<400>345

<210>346

<211>37

<212>DNA

Chlamydia trachomatis (213)

<400>346

<210>347

<211>37

<212>DNA

Chlamydia trachomatis (213)

<400>347

<210>348

<211>1464

<212>DNA

Chlamydia trachomatis (213)

<400>348

<210>349

<211>487

<212>PRT

Chlamydia trachomatis (213)

<400>349

<210>350

<211>37

<212>DNA

Chlamydia trachomatis (213)

<400>350

<210>351

<211>37

<212>DNA

Chlamydia trachomatis (213)

<400>351

<210>352

<211>1752

<212>DNA

Chlamydia trachomatis (213)

<400>352

<210>353

<211>583

<212>PRT

Chlamydia trachomatis (213)

<400>353

<210>354

<211>39

<212>DNA

Chlamydia trachomatis (213)

<400>354

<210>355

<211>36

<212>DNA

Chlamydia trachomatis (213)

<400>355

<210>356

<211>2052

<212>DNA

Chlamydia trachomatis (213)

<400>356

<210>357

<211>683

<212>PRT

Chlamydia trachomatis (213)

<400>357

<210>358

<211>1248

<212>DNA

213 genus chlamydia

<400>358

<210>359

<211>1311

<212>DNA

213 genus chlamydia

<400>359

<210>360

<211>813

<212>DNA

213 genus chlamydia

<400>360

<210>361

<211>750

<212>DNA

213 genus chlamydia

<400>361

<210>362

<211>412

<212>PRT

213 genus chlamydia

<400>362

<210>363

<211>433

<212>PRT

213 genus chlamydia

<400>363

<210>364

<211>264

<212>PRT

213 genus chlamydia

<400>364

<210>365

<211>249

<212>PRT

213 genus chlamydia

<400>365

<210>366

<211>2418

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>366

<210>367

<211>888

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>367

<210>368

<211>237

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>368

<210>369

<211>1437

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>369

<210>370

<211>774

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>370

<210>371

<211>576

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>371

<210>372

<211>699

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>372

<210>373

<211>369

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>373

<210>374

<211>5172

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>374

<210>375

<211>5172

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>375

<210>376

<211>3759

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>376

<210>377

<211>675

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>377

<210>378

<211>1671

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>378

<210>379

<211>1386

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>379

<210>380

<211>1635

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>380

<2110>381

<211>1995

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>381

<210>382

<211>987

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>382

<210>383

<211>654

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>383

<210>384

<211>813

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>384

<210>385

<211>1956

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>385

<210>386

<211>805

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>386

<210>387

<211>295

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>387

<210>388

<211>78

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>388

<210>389

<211>478

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>389

<210>390

<211>257

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>390

<210>391

<211>191

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>391

<210>392

<211>232

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>392

<210>393

<211>122

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>393

<210>394

<211>1723

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>394

<210>395

<211>1723

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>395

<210>396

<211>1252

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>396

<210>397

<211>224

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>397

<210>398

<211>556

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>398

<210>399

<211>461

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>399

<210>400

<211>544

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>400

<210>401

<211>664

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>401

<210>402

<211>328

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>402

<210>403

<211>217

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>403

<210>404

<211>270

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>404

<210>405

<211>651

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>405

<210>406

<211>1074

<212>DNA

Chlamydia trachomatis serotype D

<400>406

<210>407

<211>1827

<212>DNA

Chlamydia trachomatis serotype D

<400>407

<210>408

<211>804

<212>DNA

Chlamydia trachomatis serotype D

<400>408

<210>409

<211>663

<212>DNA

Chlamydia trachomatis serotype D

<400>409

<210>410

<211>1470

<212>DNA

Chlamydia trachomatis serotype D

<400>410

<210>411

<211>234

<212>DNA

Chlamydia trachomatis serotype D

<400>411

<210>412

<211>1941

<212>DNA

Chlamydia trachomatis serotype D

<400>412

<210>413

<211>693

<212>DNA

Chlamydia trachomatis serotype D

<400>413

<210>414

<211>1599

<212>DNA

Chlamydia trachomatis serotype D

<400>414

<210>415

<211>1395

<212>DNA

Chlamydia trachomatis serotype D

<400>415

<210>416

<211>366

<212>DNA

Chlamydia trachomatis serotype D

<400>416

<210>417

<211>1659

<212>DNA

Chlamydia trachomatis serotype D

<400>417

<210>418

<211>576

<212>DNA

Chlamydia trachomatis serotype D

<400>418

<210>419

<211>825

<212>DNA

Chlamydia trachomatis serotype D

<400>419

<210>420

<211>5310

<212>DNA

Chlamydia trachomatis serotype D

<400>420

<210>421

<211>5253

<212>DNA

Chlamydia trachomatis serotype D

<400>421

<210>422

<211>1980

<212>DNA

Chlamydia trachomatis serotype D

<400>422

<210>423

<211>978

<212>DNA

Chlamydia trachomatis serotype D

<400>423

<210>424

<211>696

<212>DNA

Chlamydia trachomatis serotype D

<400>424

<210>425

<211>3756

<212>DNA

Chlamydia trachomatis serotype D

<400>425

<210>426

<211>894

<212>DNA

Chlamydia trachomatis serotype D

<400>426

<210>427

<211>894

<212>DNA

Chlamydia trachomatis serotype D

<400>427

<210>428

<211>459

<212>DNA

Chlamydia trachomatis serotype D

<400>428

<210>429

<211>1707

<212>DNA

Chlamydia trachomatis serotype D

<400>429

<210>430

<211>1998

<212>DNA

Chlamydia trachomatis serotype D

<400>430

<210>431

<211>609

<212>PRT

Chlamydia trachomatis serotype D

<400>431

<210>432

<211>268

<212>PRT

Chlamydia trachomatis serotype D

<400>432

<210>433

<211>221

<212>PRT

Chlamydia trachomatis serotype D

<400>433

<210>434

<211>490

<212>PRT

Chlamydia trachomatis serotype D

<400>434

<210>435

<211>78

<212>PRT

Chlamydia trachomatis serotype D

<400>435

<210>436

<211>647

<212>PRT

Chlamydia trachomatis serotype D

<400>436

<210>437

<211>231

<212>PRT

Chlamydia trachomatis serotype D

<400>437

<210>438

<211>533

<212>PRT

Chlamydia trachomatis serotype D

<400>438

<210>439

<211>465

<212>PRT

Chlamydia trachomatis serotype D

<400>439

<210>440

<211>122

<212>PRT

Chlamydia trachomatis serotype D

<400>440

<210>441

<211>553

<212>PRT

Chlamydia trachomatis serotype D

<400>441

<210>442

<211>192

<212>PRT

Chlamydia trachomatis serotype D

<400>442

<210>443

<211>275

<212>PRT

Chlamydia trachomatis serotype D

<400>443

<210>444

<211>1770

<212>PRT

Chlamydia trachomatis serotype D

<400>444

<210>445

<211>1751

<212>PRT

Chlamydia trachomatis serotype D

<400>445

<210>446

<211>660

<212>PRT

Chlamydia trachomatis serotype D

<400>446

<210>447

<211>326

<212>PRT

Chlamydia trachomatis serotype D

<400>447

<210>448

<211>232

<212>PRT

Chlamydia trachomatis serotype D

<400>448

<210>449

<211>1252

<212>PRT

Chlamydia trachomatis serotype D

<400>449

<210>450

<211>298

<212>PRT

Chlamydia trachomatis serotype D

<400>450

<210>451

<211>298

<212>PRT

Chlamydia trachomatis serotype D

<400>451

<210>452

<211>153

<212>PRT

Chlamydia trachomatis serotype D

<400>452

<210>453

<211>569

<212>PRT

Chlamydia trachomatis serotype D

<400>453

<210>454

<211>666

<212>PRT

Chlamydia trachomatis serotype D

<400>454

<210>455

<211>882

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>455

<210>456

<211>1185

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>456

<210>457

<211>1656

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>457

<210>458

<211>294

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>458

<210>459

<211>618

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>459

<210>460

<211>1809

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>460

<210>461

<211>975

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>461

<210>462

<211>1980

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>462

<210>463

<211>1236

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>463

<210>464

<211>1215

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>464

<210>465

<211>1632

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>465

<210>466

<211>312

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>466

<210>467

<211>1089

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>467

<210>468

<211>1308

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>468

<210>469

<211>1749

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>469

<210>470

<211>516

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>470

<210>471

<211>1083

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>471

<210>472

<211>1200

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>472

<210>473

<211>675

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>473

<210>474

<211>741

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>474

<210>475

<211>1062

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>475

<210>476

<211>561

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>476

<210>477

<211>3135

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>477

<210>478

<211>1041

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>478

<210>479

<211>984

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>479

<210>480

<211>444

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>480

<210>481

<211>1581

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>481

<210>482

<211>1908

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>482

<210>483

<211>945

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>483

<210>484

<211>3723

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>484

<210>485

<211>1731

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>485

<210>486

<211>4224

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>486

<210>487

<211>804

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>487

<210>488

<211>306

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>488

<210>489

<211>806

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>489

<210>490

<211>293

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>490

<210>491

<211>394

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>491

<210>492

<211>560

<212>PRT

Chlamydia pneumoniae of < 213 >

<220>

<221>VARIANT

<222>553，554，555，556，558，559，560

<223> Xaa ═ any amino acid

<400>492

<210>493

<211>97

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>493

<210>494

<211>205

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>494

<210>495

<211>602

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>495

<210>496

<211>324

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>496

<210>497

<211>659

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>497

<210>498

<211>411

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>498

<210>499

<211>404

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>499

<210>500

<211>543

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>500

<210>501

<211>103

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>501

<210>502

<211>362

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>502

<210>503

<211>582

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>503

<210>504

<211>435

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>504

<210>505

<211>171

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>505

<210>506

<211>360

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>506

<210>507

<211>399

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>507

<210>508

<211>224

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>508

<210>509

<211>246

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>509

<210>510

<211>353

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>510

<210>511

<211>186

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>511

<210>512

<211>276

<212>PRT

Chlamydia pneumoniae of < 213 >

<220>

<221>VARIANT

<222>269，270，271，272，274，275，276

<223> Xaa ═ any amino acid

<400>512

<210>513

<211>1044

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>513

<210>514

<211>346

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>514

<210>515

<211>327

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>515

<210>516

<211>101

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>516

<210>517

<211>261

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>517

<210>518

<211>526

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>518

<210>519

<211>147

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>519

<210>520

<211>635

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>520

<210>521

<211>314

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>521

<210>522

<211>1240

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>522

<210>523

<211>576

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>523

<210>524

<211>439

<212>PRT

Chlamydia pneumoniae of < 213 >

<220>

<221>VARIANT

<222>428，429，430，431，432，433，434，435，437，438，439

<223> Xaa ═ any amino acid

<400>524

<210>525

<211>867

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>525

<210>526

<211>1182

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>526

<210>527

<211>1650

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>527

<210>528

<211>300

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>528

<210>529

<211>615

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>529

<210>530

<211>1806

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>530

<210>531

<211>972

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>531

<210>532

<211>1938

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>532

<210>533

<211>1242

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>533

<210>534

<211>1212

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>534

<210>535

<211>1617

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>535

<210>536

<211>312

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>536

<210>537

<211>1008

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>537

<210>538

<211>1278

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>538

<210>539

<211>1815

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>539

<210>540

<211>519

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>540

<210>541

<211>1062

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>541

<210>542

<211>1263

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>542

<210>543

<211>693

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>543

<210>544

<211>729

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>544

<210>545

<211>1149

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>545

<210>546

<211>579

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>546

<210>547

<211>3159

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>547

<210>548

<211>1038

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>548

<210>549

<211>978

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>549

<210>550

<211>438

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>550

<210>551

<211>1581

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>551

<210>552

<211>1950

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>552

<210>553

<211>939

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>553

<210>554

<211>3711

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>554

<210>555

<211>1689

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>555

<210>556

<211>5253

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>556

<210>557

<211>792

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>557

<210>558

<211>306

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>558

<210>559

<211>729

<212>DNA

Chlamydia trachomatis D serovar < 213 >

<400>559

<210>560

<211>289

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>560

<210>561

<211>394

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>561

<210>562

<211>550

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>562

<210>563

<211>100

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>563

<210>564

<211>205

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>564

<210>565

<211>602

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>565

<210>566

<211>324

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>566

<210>567

<211>646

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>567

<210>568

<211>414

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>568

<210>569

<211>404

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>569

<210>570

<211>539

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>570

<210>571

<211>104

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>571

<210>572

<211>336

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>572

<210>573

<211>426

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>573

<210>574

<211>605

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>574

<210>575

<211>173

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>575

<210>576

<211>354

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>576

<210>577

<211>421

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>577

<210>578

<211>231

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>578

<210>579

<211>243

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>579

<210>580

<211>383

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>580

<210>581

<211>193

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>581

<210>582

<211>264

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>582

<210>583

<211>1053

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>583

<210>584

<211>346

<211>346

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>584

<210>585

<211>326

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>585

<210>586

<211>102

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>586

<210>587

<210>587

<211>243

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>587

<210>588

<211>527

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>588

<210>589

<211>146

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>589

<210>590

<211>650

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>590

<210>591

<211>313

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>591

<210>592

<211>1237

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>592

<210>593

<211>563

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>593

<210>594

<211>1751

<212>PRT

Chlamydia trachomatis D serovar < 213 >

<400>594

<210>595

<211>900

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>595

<210>596

<211>1743

<212>DNA

Chlamydia pneumoniae of < 213 >

<400>596

<210>597

<211>299

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>597

<210>598

<211>580

<212>PRT

Chlamydia pneumoniae of < 213 >

<400>598

<210>599

<211>358

<212>PRT

Chlamydia trachomatis serotype D

<400>599

Claims (10)

1. A composition for eliciting an immune response in an animal comprising an isolated polynucleotide sequence encoding an immunogenic chlamydia antigen protein comprising the sequence of SEQ ID NO:431 or a variant thereof having at least 90% sequence identity to the sequence of SEQ ID NO:431 as determined by comparing the two optimally aligned sequences over the full length comparison window of the sequences of SEQ ID NO: 431.

2. The composition according to claim 1, wherein said polynucleotide comprises the sequence of SEQ ID NO 407.

3. A composition for eliciting an immune response in an animal comprising an isolated immunogenic chlamydia antigen protein comprising the sequence of SEQ ID NO:431 or a variant thereof having at least 90% sequence identity to the sequence of SEQ ID NO:431 as determined by comparing the two optimally aligned sequences over a comparison window of the full length of the sequence of SEQ ID NO: 431.

4. The composition of claim 3, wherein the protein comprises a sequence having at least 99% identity to SEQ ID NO. 431 as determined by comparing the two optimally aligned sequences over a comparison window of the full length of the SEQ ID NO. 431 sequence.

5. The composition of claim 4, wherein the protein comprises the sequence of SEQ ID NO 431.

6. The composition of claim 5, wherein said protein consists of the sequence of SEQ ID NO 431.

7. The composition of any one of claims 1-6, which is a vaccine composition further comprising an immunostimulant.

8. The composition of any one of claims 1-6, which is a pharmaceutical composition further comprising a physiologically acceptable carrier.

9. Use of a composition according to any one of claims 1 to 6 for the preparation of a vaccine for stimulating protective immunity against chlamydial infection in a patient.

10. Use of a composition according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment of chlamydial infection in a patient.