AU754122B2 - Chlamydia proteins and their uses - Google Patents

Description

WO 99/53948 PCTIUS99/08744 CHLAMYDIA PROTEINS AND THEIR USES I. FIELD OF THE INVENTION The present invention relates to the detection of Chlamydia and to the diagnosis, treatment and prevention of Chlamydia infections in animals.

II. BACKGROUND Chlamydiae are obligate intracellular bacterial pathogens with a unique biphasic life cycle.

They appear as two distinct cellular types, a small dense cell or elementary body (EB) that is enclosed in a rigid bacterial cell wall, and a larger metabolically active reticulate body The EB is resistant to physical disruption and is infectious, whereas the RB is more fragile and only exists inside cells. The Chlamydia life cycle begins with the attachment of the EB form to the host cell which is followed by endocytosis into a nascent vacuole, also called an "inclusion membrane." After EB attachment and entry, replication of the EB form produces RB forms that continue to grow within the vacuole. By 72 hour post-infection, this growth phase is terminated when the RBs condense, and reorganize back to EBs. The lysis of the host cell results in release of EBs to infect new host cells. The difficulties in working with Chlamydiae center on the obligate intracellular requirement for growth and the fact that no adequate genetic engineering methods have been developed for this organism.

The genus Chlamydia includes two species that are primarily associated with human disease: C. trachomatis and C. pneumoniae. C. trachomatis causes trachoma, an eye disease that is the leading cause of preventable infectious blindness worldwide with an estimated 500 million cases of active trachoma worldwide. C. trachomatis also causes a sexually transmitted chlamydial disease which is very common worldwide. C. trachomatis also causes lymphogranuloma venereum, a debilitating systemic disease characterized by lymphatic gland swelling. The most serious sequelae of chlamydial genital infections of females include salpingitis, pelvic inflammatory disease, and ectopic pregnancy. In the US alone, it is estimated that over 4 million new sexually transmitted C. trachomatis infections occurred in 1990, leading to over four billion dollars in direct and indirect medical expenses. The World Health Organization estimates that 89 million new cases of genital Chlamydia occurred worldwide in 1995 (Peeling and Brunham, 1996).

C. pneumoniae causes respiratory diseases including so called walking pneumonia, a lowgrade disease such that the infected person frequently fails to obtain treatment and remains in the community as an active, infectious carrier. C. pneumoniae is currently of interest because of its strong epidemiological association with coronary artery disease, and there is also some evidence to link it with multiple sclerosis.

Of the other disease-causing species of Chlamydia, Chlamydia psittaci and Chlamydia pecorum are primarily pathogens of wild and domestic animals, but these species may infect humans accidentally. C. psittaci is acquired through respiratory droplet infection and is considered an occupational health hazard for bird fanciers and poultry workers.

There is tremendous interest in the identification of candidate antigens for protection against chlamydial disease. While a prior infection with C. trachomatis will protect against a subsequent challenge by the same strain, indicating a protective component that stimulates the host immune response, most serious chlamydial diseases are exacerbated by an overaggressive anti-chlamydial immune response. Antigens recognized in the context of an infection appear to elicit a protective response whereas immunization with purified, killed (EB form) Chlamydia results in an immunopathological response. Therefore for the purposes of vaccine development, one needs to find epitopes that confer protection, but do not contribute to pathology. It is an object of this invention to provide Chlamydia polypeptides for use as vaccines that induce a protective immune response without inducing the pathological response caused by the antigens associated with the EB form of Chlamydia. Such immunostimulatory peptides will be useful in the treatment, as well as in the diagnosis, detection and prevention of Chlamydial infections.

SUMMARY OF THE INVENTION According to a first embodiment of the present invention there is provided a vaccine preparation comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14. According to a second embodiment of the present invention there is provided a vaccine preparation comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8, or SEQ ID NO:14. According to a third embodiment of the present invention there is provided a method of preparing a vaccine comprising combining a pharmaceutically acceptable excipient with at least one purified peptide having an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

According to a fourth embodiment of the present invention there is provided a method of vaccination, comprising administering a vaccine preparation according to the first or second embodiment to a mammal.

According to a fifth embodiment of the present invention there is provided a composition for inducing an immune response in a subject, comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence Zs set forth in SEQ ID NO:8 or SEQ ID NO: 14.

[R:\LIBVV]02761.doc:ais According to a sixth embodiment of the present invention there is provided a composition for inducing an immune response comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

According to a seventh embodiment of the present invention there is provided a method of making a composition for inducing an immune response comprising combining a pharmaceutically acceptable excipient with at least one purified peptide having an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

According to an eighth embodiment of the present invention there is provided a method of inducing an immune response in a subject, comprising administering a composition according to the fifth or sixth embodiment to a mammal.

According to a ninth embodiment of the present invention there is provided a vaccine preparation comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO: 14 when used to vaccinate a mammal. According to a tenth embodiment of the present invention there is provided a vaccine preparation comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8, or SEQ ID NO:14 when used to vaccinate a mammal.

According to an eleventh embodiment of the present invention there is provided a composition when used for inducing an immune response in a subject, said composition comprising at least one purified peptide comprising at least 5 contiguous amino acids of ooo an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14. :.0 According to a twelfth embodiment of the present invention there is provided a '0, composition when used for inducing an immune response in a subject, said composition comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

According to a thirteenth embodiment of the present invention there is provided use of at least one purified peptide comprising at least 5 contiguous amino acids of amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 for the manufacture of a vaccine preparation for use in vaccinating a mammal.

According to a fourteenth embodiment of the present invention there is provided use of at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 for the manufacture of a vaccine preparation for use in k vaccinating a mammal.

[R:\LIBVV]02761.doc:ais According to a fifteenth embodiment of the present invention there is provided use of a composition comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 for the manufacture of a medicament for inducing an immune response in a subject.

According to a sixteenth embodiment of the present invention there is provided use of a composition comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 for the manufacture of a medicament for inducing an immune response in a subject.

Also disclosed herein is the use of Chlamydia proteins that show enhanced expression in the reticulate body (RB) stage relative to the elementary body (EB) stage of the Chlamydia life cycle. These proteins are not present at detectable levels in the EB form using current immunological techniques and are thus said to be"infection-specific".

Certain of these infection-specific proteins are found in the inclusion membrane of the infected cell, and so have been termed "Inc"proteins. These include the IncA, IncB, and IncC proteins of Chlamydia as described in the present disclosure. The genes that encode the IncA, IncB and IncC proteins are referred to as incA, incB and incC respectively.

Other proteins of Chlamydia described herein have also been shown by the inventors to be infection-specific, but are not known to be incorporated into the inclusion membrane; these include the p242, TroA, and TroB proteins. The TroA and TroB proteins have been so named because they resemble the Tro proteins of Treponema pallidum, which are thought to form part of an ABC transport system.

The inventors have shown that the infection-specific Chlamydia proteins of the disclosure are recognized by convalescent antisera antisera taken from an animal that has recovered from a Chlamydia infection) but are not recognized by antisera against the killed EB form of Chlamydia. Thus, the proteins are expressed only during active chlamydial infection and are therefore useful as protective antigens. These infectionspecific proteins may be used to confer a protective immune response without inducing a pathological effect. Additionally, immuno fluorescence microscopy and immunoblotting with antisera demonstrated that the infection-specific proteins are present in Chlamydiainfected HeLa cells, but are undetectable in purified EBs and absent in uninfected HeLa cells.

Immunofluorescense microscopy reveals that IncA, IncB and IncC are localized to the inclusion membrane of infected HeLa cells. Reverse-transcription polymerase chain reactions (RT-PCR), northern hybridization data, and restriction analysis revealed that the SincB and incC genes are closely linked and transcribed in an operon. RT-PCR, restriction [R:\LIBVV]02761 .doc:ais analysis and sequential Southern hybridizations of incA then incC to the same filter provided evidence that incA is separated from the incB and incC operon by about 110 kb.

The C. trachomatis Tro genes are not closely linked with the p242 gene.

Also disclosed herein are the nucleotide and amino acid sequences for certain infection-specific proteins from Chlamydia. These proteins are p242, TroA, and TroB from C. trachomatis, and the IncB, and IncC proteins from C. psittaci. Fragments of these proteins may be used in a vaccine preparation or may be used in a method of detecting Chlamydia antibodies. Such fragments may be, for example, 5, 10, 15, 20, or 30 contiguous amino acids in length. They may even encompass the entire protein.

More specifically, herein disclosed are the purified infection-specific proteins having amino acid sequences as shown in SEQ ID NOS: 2, 4, 6, 10, and 12, amino acid sequences that differ from such sequences by one or more conservative amino acid substitutions, and amino acid sequences that show at least 75 sequence identity with such amino acid sequences. Also disclosed herein are isolated nucleic acid molecules that encode a protein as described in the above paragraph, including isolated nucleic acid molecules with nucleotide sequences as shown in SEQ ID NOS: 1, 3, 5, 9, and 11. Also disclosed herein is a vaccine or immunostimulatory preparation directed against the reticulate body (RB) form of Chlamydia comprising one or more purified infection specific peptides (or portions or fragments thereof, or peptides showing sequence similarity to a portion of such a peptide). Such peptide fragments may be, for example, 5, 10, 15, 20, 25, or 30 contiguous amino acids in length, of the sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 18. Peptides used in such a vaccine may even encompass the entire purified peptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 18, a peptide that differs from such a peptide by one or more conservative amino acid substitutions, or a peptide having at least 75 sequence identity with such a peptide.

Such vaccine preparations may contain one or more pharmaceutically acceptable excipients, adjuvants or diluents.

Further disclosed are methods for making a vaccine, comprising combining a pharmaceutically acceptable excipient with a peptide described herein. Also disclosed is a method of vaccination comprising administering a vaccine as described herein to a mammal.

Further disclosed is a method for the diagnostic use of the disclosed purified k infection-specific peptides, for instance by use in a diagnostic assay to detect the presence [R:\LIBVV]02761.doc:ais of infection-specific antibodies in a medical specimen, in which antibodies bind to the Chlamydia peptide and indicate that the subject from which the specimen was removed was previously exposed to Chlamydia. Such a method may comprise: supplying a biological sample, such as blood from an animal, that is suspected to contain infectionspecific anti-Chlamydia antibody, (ii) contacting the sample with at least one infectionspecific Chlamydia peptide described herein, such that a reaction between the peptide and the infection-specific anti-Chlamydia antibody gives rise to a detectable effect, such as a chromogenic conversion; and (iii) detecting this detectable effect.

Also disclosed herein is a method of using antibodies that bind specifically with the disclosed proteins for detection of infection-specific Chlamydia antigen, indicating the presence of Chlamydia in the RB stage as distinct from the EB stage. For instance, the relevant infection-specific antibodies may be used to provide specific binding in an Enzyme Linked Immunosorbant Assay (ELISA) or other immunological assay wherein the antibody Fc portion is linked to a chromogenic, fluorescent or radioactive molecule and the Fab portion specifically interacts with, and binds to, an infection-specific protein. Such a method may comprise: supplying a biological sample from an animal suspected to contain an infection-specific Chlamydia antigen, and (ii) contacting the sample with at least one infection-specific anti-Chlamydia antibody, such that a reaction between the antibody and the infection-specific Chlamydia protein gives rise to a detectable effect; and (iii) detecting this detectable effect. Also disclosed herein is the use of probes and primers derived from the nucleotide sequences that encode infection-specific peptides, to detect the presence of Chlamydia nucleic acids in medical specimens. Such probes and primers may be nucleotide o fragments, of, for example, 15, 20, 25, 30 or 40 contiguous nucleotides of the sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, or 17.

Also disclosed herein is a method of treating a Chlamydia infection by directing a therapeutic agent against a specific target, where the target is chosen from an infection specific protein of Chlamydia, a gene that encodes an infection-specific protein of Chlamydia, and an RNA transcript that encodes an infection-specific protein of Chlamydia, wherein the therapeutic agent interacts with said target to affect a reduction in pathology.

These and other aspects of the invention will become more apparent from the following description.

[R:\LIBVV]02761.doc:ais SEQUENCE LISTING SEQ ID NO: 1 shows a nucleic acid sequence encoding the p242 C. trachomatis protein, with deduced primary amino acid sequence also shown.

SEQ ID NO: 2 shows the amino acid sequence of the p242 C. trachomatis protein.

SEQ ID NO: 3 shows a nucleic acid sequence encoding the TroA C. trachomatis protein, with deduced primary amino acid sequence also shown.

SEQ ID NO: 4 shows the amino acid sequence of the TroA C. trachomatis protein.

SEQ ID NO: 5 shows a nucleic acid sequence encoding the TroB C. trachomatis protein, with deduced primary amino acid sequence also shown.

J [R:\LIBVV]02761.doc:ais WO 99/53948 PCT/US99/08744 SEQ ID NO:6 shows the amino acid sequence of the TroB C. trachomatis protein.

SEQ ID NO:7 shows a nucleic acid sequence encoding the IncA C. psittaci protein, with deduced primary amino acid sequence also shown.

SEQ ID NO:8 shows the amino acid sequence of the IncA C. psittaci protein.

SEQ ID NO:9 shows a nucleic acid sequence encoding the IncB C. psittaci protein, with deduced primary amino acid sequence also shown.

SEQ ID NO: 10 shows the amino acid sequence of the IncB C. psitlaci protein.

SEQ ID NO: 11 shows a nucleic acid sequence encoding the IncC C. psittaci protein, with deduced primary amino acid sequence also shown.

SEQ ID NO: 12 shows the amino acid sequence of the IncC C. psittaci protein.

SEQ ID NO: 13 shows a nucleic acid sequence encoding the IncA C. trachomatis protein, with deduced primary amino acid sequence also shown.

SEQ ID NO: 14 shows the amino acid sequence of the IncA C. trachomatis protein.

SEQ ID NO: 15 shows a nucleic acid sequence encoding the IncB C. trachomatis protein, with deduced primary amino acid sequence also shown.

SEQ ID NO: 16 shows the amino acid sequence of the IncB C. trachomatis protein.

SEQ ID NO: 17 shows a nucleic acid sequence encoding the IncC C. trachomatis protein, with deduced primary amino acid sequence also shown.

SEQ ID NO:18 shows the amino acid sequence of the IncC C. trachomatis protein.

SEQ ID NO: 19 shows the upstream oligonucleotide used to amplify the C. psittaci incC

ORF.

SEQ ID NO:20 shows the downstream oligonucleotide used to amplify the C. psittaci incC ORF.

SEQ ID NO:21 shows the upstream oligonucleotide used to amplify the C. psittaci incB

ORF.

SEQ ID NO:22 shows the downstream oligonucleotide used to amplify the C. psinaci incB ORF.

SEQ ID NO:23 shows the upstream oligonucleotide used to amplify the C. psittaci incA

ORF.

SEQ ID NO:24 shows the downstream oligonucleotide used to amplify the C. psittaci incA ORF.

V. DESCRIPTION OF THE INVENTION A. DEFINITIONS Particular terms and phrases used herein have the meanings set forth below.

WO 99/53948 PCT/US99/08744 -6- "EB" refers to the Elementary Body, an environmentally refractile and largely metabolically dormant form of Chlamydia that is infectious and is presented as a small dense body enclosed by a bacterial cell wall.

"RB" refers to the Reticulate Body, a metabolically active form of Chlamydia that is not infectious, and exists only within a host cell, being very fragile, often branched, and appearing larger and less dense that the EB.

"Infection-specific" refers to a protein that shows enhanced expression in the RB form of Chlamydia compared to the EB form. Infection-specific proteins are not necessarily absent from the EB form, but they are significantly more common in the RB form than in the EB form.

"infection-specific antibody" is an antibody that binds specifically to an infection-specific protein.

"Biological sample" refers to any sample of biological origin including, but not limited to a blood sample, a plasma sample, a mucosal smear or a tissue sample.

"Isolated" An isolated nucleic acid has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, other chromosomal and extrachromosomal DNA and RNA. The term "isolated" thus encompasses nucleic acids purified by standard nucleic acid purification methods. The term also embraces nucleic acids prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

"Probes" and "primers." Nucleic acid probes and primers may readily be prepared based on the nucleic acid sequences provided by this invention. A "probe" comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.

"Primers" are short nucleic acids, typically DNA oligonucleotides 15 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.

Probes and primers as used in the present invention typically comprise at least nucleotides of the nucleic acid sequences that are shown to encode infection-specific proteins. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 30 or 40 consecutive nucleotides of the disclosed nucleic acid sequences.

Methods for preparing and using probes and primers are well known in the art and are described in, for example Sambrook et al. (1989); Ausubel et al., (1987); and Innis et al., (1990).

PCR primer pairs can be derived from a known sequence, for example, by using computer WO 99/53948 PCT/US99/08744 -7programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).

"Conservative amino acid substitutions" are those substitutions that, least interfere with the properties of the original protein, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions.

Original Residue Conservative Substitution Ala Ser Arg Lys Asn gin, his Asp Glu Cys Ser Gin Asn Glu Asp Gly Pro His asn, gin Ile leu, val Leu ile, val Lys arg, gin, glu Met leu, ile Phe met, leu, tyr Ser Thr Thr Ser Trp Tyr Tyr trp, phe Val ile, leu Conservative substitutions generally maintain the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain.

The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; a cysteine or proline is substituted for (or by) any other residue; a residue having an electropositive side chain, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, glutamyl or aspartyl; or a residue having a bulky side chain, phenylalanine, is substituted for (or by) one not having a side chain, glycine.

"Sequence identity" The similarity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Variants of naturally occurring infectionspecific peptides useful in the present invention are typically characterized by possession of at least sequence identity counted over the full length alignment with the amino acid sequence of a WO 99/53948 PCT/US99/08744 -8naturally occurring infection-specific peptide when aligned using BLAST 2.0.1 (Altschul et al., 1997). For comparisons of amino acid sequences of greater than about 30 amino acids, the BLAST 2 analysis is employed using the blastp program set to default perameters (open gap 11, extension gap 1 penalty, gap x dropoff 50, expect 10, word size 3, filter on), and using the default BLOSUM62 matrix (gap existence cost 11, per residue gap cost 1, lambda ratio 0.85). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix (gap existence cost 9, per residue gap cost 1, lambda ratio 0.87). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least sequence identity. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at http//www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast help.html.

Similarly, when comparing nucleotides, blastn may be used with default settings (rewards for match 1, penalty for mismatch open gap 5, extension gap 2 penalty, gap x dropoff 50, expect 10, word size 11, filter on), with the default BLOSUM62 matrix (as above). Variants of naturally occurring infection-specific nucleic acid sequences useful in the present invention are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the nucleic acid sequence of a naturally occurring infection-specific ORF when aligned using BLAST 2.0.1. Useful nucleic acids may show even greater percentage identity, and may, for example, possess at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity naturally occurring infection-specific ORF.

"Operably linked" A first nucleic acid sequence is "operably" linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

"Recombinant" A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, by genetic engineering techniques.

"Stringent Conditions" Stringent conditions, in the context of nucleic acid hybridization, are sequence-dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5 degrees to 20 degrees lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (1989), pages 9.49-9.55. Typical high- stringency hybridization conditions (using radiolabeled probes to hybridize to nucleic acids immobilized on a nitrocellulose filter) may include, for example, wash conditions of 0.1 X SSC, 0.5% SDS at a wash temperature of 68°C.

When referring to a probe or primer, the term "specific for (a target sequence)" indicates that the probe or primer hybridizes under high-stringency conditions substantially only to the target sequence in a given sample comprising the target sequence. "Purified" A purified peptide is a peptide that has been extracted from the cellular environment and separated from substantially all other cellular peptides. As used herein, the term peptide includes peptides, polypeptides and proteins. In certain embodiments, a purified peptide is a preparation in which the subject peptide comprises 50% or more of the protein content of the preparation. For certain uses, such as vaccine preparations, even greater purity may be preferable. "Immunostimulatory peptide" as used herein refers to a peptide that is capable of stimulating a humoral or antibody-mediated immune response when inoculated into an animal.

"Vaccine" A vaccine is a composition containing at least one immunostimulatory peptide 9 which may be inoculated into an animal with the intention of producing a protective immunological reaction against a certain antigen. The antigen to be protected against may be, for instance, an infectio-specific antigen of Chlamydia. B. ISOLATION OF INFECTION SPECIFIC CHLAMYDIA

POLPEPTIDES

AND IDENTIFICATION OF GENES ENCODING

THESE

POLYPEPTIDES

S1. ISOLATION OF IncA, lncB AND lncC Bacterial strains. Chlamydia psittaci strain GPIC or C. irachomatis LGV-434, scr.

L2) was cultivated in HeLa 229 cells using standard methods (Caldwell et al., 1981). Purified Chlamydiae were obtained using Renografin R. Squibb Sons, Inc., Princeton, density gradient centrifugation. Escherichia coli DH5 a(Bethesda Research Laboratories, Inc., Gaithersburg, Md.) was used as the host strain for transformations with recombinant DNA. E.

coli XLl-Blue MRF' (Stratagene, La Jolla, Calif.) was used as the host strain for infection with lambda ZAPII phage vector. E. coli SOLR (Stratagene) was used as the host strain for infection with in vivo excised filamentous lambda ZAPII.

WO 99/53948 PCT/US99/08744 Antisera. MBP (Maltose Binding Protein)-Inc fusion proteins were used as antigens for the production of mono-specific antibody reagents in Hartley strain guinea-pigs. The protein was diluted to 100 pg/ml' sterile saline and mixed with the Ribi Trivalent Adjuvant (Ribi Immunochem.). The antigen/adjuvant emulsion was administered to anaesthetized guinea-pigs using a procedure provided by the manufacturer. Sera were collected 14 days after secondary and tertiary immunizations. Control antisera were produced by immunizing guinea-pigs with adjuvant alone, or with adjuvant plus purified maltose-binding protein.

Convalescent guinea-pig antisera, antisera against live EBs, and antisera against formalinfixed EBs were produced using standard methods (Rockey and Rosquist, 1994 and Rockey et al., 1995).

C. psittaci library construction and screening. For the incB and incC genes, C. psittaci strain GPIC DNA was extracted using a genomic DNA extraction kit (Qiagen) with one modification; dithiothreitol (5mM) was added to the suspension buffer to assist EB lysis. DNA was partially digested with Tsp509I and ligated to EcoRI digested X-ZAPII phage arms (Stratagene). The ligation was packaged in vitro with Gigapack extracts according to the manufacturer's instructions (Stratagene). Recombinant phage were plated on E. coli XL-1 Blue at densities of approximately 10" PFU/150-mm (diameter) plate. Following a nine hour incubation to allow development of the plaques, the plates were sequentially overlaid with nitrocellulose disks and the resulting lifts were processed for immunoblotting with convalescent antisera and antisera to fixed EBs. Of approximately 8,000 plaques, 18 had reactivity with the convalescent sera but not sera generated against EBs. One of these was subcloned into pBluescript phagmid by in vitro excision in the E. coli SOLR strain (Stratagene) and designated pBS200-7.

For the incA gene, genomic DNA from C. psittaci strain GPIC was partially digested with Sau3A, size-selected (2-8 kb) by electrophoresis through low-melting-temperature agarose, and blunt-ended with T4 DNA polymerase. This DNA was ligated to an EcoRl/Notl adapter (Life Technologies), kinased, and ligated to EcoRI-digested Lambda ZAP II vector (Stratagene Cloning Systems). Recombinants were packaged (Lambda Gigapack Gold, Stratagene) and used to infect E. coli XLl-Blue (Stratagene). Plaques were allowed to develop for 4 h at 37°C. Nitrocellulose filters laden with 10 mM IPTG (US Biochemical Corp.) were placed onto the plaques and incubated for an additional 4 h at 37°C. These filters were removed and placed into a blocking solution consisting of PBS (150 mM NaCl, 10 mM NaPO4, pH7.2) plus 0.1% Tween-20 (TPBS) and 2% BSA-TPBS. Filters were incubated for 1 h, rinsed twice in TPBS, and incubated overnight in convalescent-guinea-pig sera diluted 1:100 in BSA-TPBS. After three washes in TPBS, the filters were incubated for 1 h in 'l 5 1-staphylococcal protein A (New England Nuclear) diluted to approx. 124 nCiml in BSA-TPBS. Filters were again washed three times in TPBS and positive plaques were detected by exposure of the dried filters to autoradiography film overnight at room temperature. Positive clones were picked and plaque-purified. pBluescript-SK- plasmids WO 99/53948 PCT[US99/08744 -11containing the chlamydial genes of interest were recovered from the purified bacteriophage using ExAssist filamentous bacteriophages (Stratagene).

Identification of antigens recognized by convalescent antisera. Recombinant plaques were identified that showed reactivity with convalescent (anti-RB) antisera, but not with anti-EB serum. The purified recombinant phage were converted into pBluescriptlI SK plasmid by in vivo excision and recircularization and these recombinant DNAs were used to transform E. coli. SDS- PAGE and immunoblot analysis of lysates of these recombinant E. coli showed that each expressed one or more proteins that reacted with convalescent antisera but not with the EB serum.

DNA Cloning and fusion protein production. The plasmid pJC2 contains a 5.0 kb EcoRI GPIC genomic fragment cloned into the pZEro2.1 vector (Invitrogen). To construct pJC2, the incC ORF sequence was 32 P-radiolabeled using random priming (Gibco-BRL) and used to probe EcoRl cut GPIC genomic DNA fragments separated by agarose gel electrophoresis.

Fragments in the size range of the positive signal were excised from the gel and purified by Gene- Clean (Biol01l). The gel-purified fragments were used in a ligation along with EcoRI-digested pZEro2.1. Kanamycin resistant colonies were screened by colony hybridization with radiolabeled incC.

MBP fusions of the five ORFs present in pJC2 were produced using the pMAL-C2 vector (New England Biolabs). The reading frame of incC, with the exception of the first four codons, was amplified using Pwo polymerase (Boehringer Mannheim) and pBS200-7 as the template. The upstream and downstream oligonucleotides for this amplification were 5'-AGAACCGATTTAACTCCAGGCG-3' (SEQ ID NO: 19) and 5'-GCGCGGATCCTTAATGTCCGGTAGGCCTAG-3' (SEQ ID NO: 20), respectively.

The vector was digested with XmnI and BamHI, and the amplication product was digested with BamHI. Ligation of these products resulted in an in-frame fusion between the malE gene in the vector and the incC reading frame from pBS200-7. The stop codon for this construction is provided by the insert. Following ligation, the products were transformed into E.coli strain HD51. The resulting fusion protein (MBP/IncC) was overexpressed and purified by maltose affinity chromatography using an amylose resin supplied by New England Biolabs.

The same approach was used for production of the MBP/IncB fusion protein. The sequence encoding the N-terminal 101 amino acids of the IncB ORF was PCR amplified using the oligonucleotides 5'-ATGTCAACAACACCAGCATCTTC-3' (SEQ ID NO: 21) and 5'-GCGCGGATCCTTAATTAGTGCCTTCTGGATTAGG-3' (SEQ ID NO: 22).

The purified MBP/IncB and MBP/IncC fusion proteins were used as antigen for the production of monospecific antibody in Hartley strain guinea-pigs by standard methods (Rockey et al., 1995). Inserts in each construct were confirmed by DNA sequencing.

For IncA, a maltose-binding protein/IncA fusion protein was produced using the pMAL- C2 vector system from New England Biolabs. The reading frame of incA shown in Fig. 1, with the exception of the initiator ATG, the incA ORF was amplified using Vent DNA polymerase (New England Biolabs) and plasmid pGP17 as template. The upstream and downstream oligonucleotides for this amplification were 5'-CGCAGTACTGTATCCACAGACAAC-3- (SEQ ID NO: 23) and 5'-GTCGGATCCGAGAAACTCTCCATGCC-3' (SEQ ID NO: 24), respectively. The vector.was digested with Xmnl and BamHl, and the amplification product was digested with Scal and BamHl. Ligation of these products resulted in an in-frame fusion between the malE gene in the vector and the incA reading frame from pGP17. The stop codon for this construction is provided by the insert. Following ligation, the products were transformed into E. coli strain a. The resulting fusion protein (MBP/IncA) was overexpressed and purified by maltose affinity chromatography using amylose resin (New England Biolabs).

MBP/IncA was used as antigen for the production of mono-specific antibody reagents in Hartley strain guinea-pigs.

DNA sequencing and sequence analysis. The pBS200-7 and pJC2 genomic clones as well as the MBP fusions were sequenced with the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Perkin Elmer/Applied Biosystems Division). Several internal primers were designed to sequence further into the cloned inserts. Sequence assembly was performed using AssemblyLIGN software and sequence analysis was performed with MacVector software (International Biotechnologies Incorporated). Hydrophilicity profiles were determined using the Kyte-Doolittle scale (Kyte and Doolittle, 1982) with a window of 7. Deduced amino acid sequences were compared with the database using the BLAST program (on default settings) available from the National Center for Biotechnology Information on the world wide web. The entire nucleotide sequence of the pJC2 insert was deposited in the GenBank/EMBL Nucleotide Sequence Data Library, under accession number AF017105. For incA, nucleotide sequencing was conducted using the Sequences system (US Biochemical) with the M13 forward and reverse primers, and internal primers synthesized on an Milligen/Biosearch Cyclone Plus DNA synthesizer. Computer analyses were conducted using the MaGVector Sequence Analysis Software (International Biotechnologies Incorporated).

HYdrophilicity profiles were determined using the Kyte-Doolittle scale (Kyte and Doolittle, 1982) with a window of 7. Secondary-structure predictions were generated using a combination of the Chou-Fasman and Robson-Garnier methods (Robson and Suzuki, 1976; Chou and Fasman, 1978).

Deduced amino acid sequences were compared with those in the EMBL and GenBank databases using the BLASTP program available from the National Center for Biotechnology Information.

Electrophoresis and immunoblotting. Polyacrylamide gel electrophoresis (PAGE) was conducted using standard methods (Rockey and Rosquist, 1994). Immunoblotting was performed using standard methods (Rockey et al., 1995).

Immunofluorescence studies. Chlamydiae grown in HeLa cells on sterile glass coverslips were fixed for microscopy one of two ways. Cells were either incubated in methanol for 5 minutes, or in the combination fixative periodate-lysine-paraformaldephyde (PLP) for three hours at room temperature followed by permeabilization with 0.05% saponin (Brown and Farquhar, 1989). Immunostaining of the fixed coverslips was performed according to standard methods (Rockey et al., 1995) and visualized under a Nikon Microphot FXA microscope using the 63x objective and oil immersion.

RT-PCR analysis. RNA for RT-PCR analysis was extracted from approximately 2 x C. psittaci-infected cells. A Qiagen column was used for extraction and purification according to the manufacturer's instructions (Qiagen). RQ1 RNase DNase (Promega) was used to ensure removal of contaminating genomic DNA. cDNA was prepared by incubating 1.5 pg of RNA, 2.5 uM of the reverse oligonucleotide primer, and AMV reverse transcriptase (Promega) for I hour at 42*C in sodium pyrophosphate buffer, according to the manufacturer's instructions. PCR reactions were carried out using 1 pl of the cDNA reaction, 1.25 1M of each oligonucleotide primer, and Pwo polymerase (Boehringer Mannheim). Each RT-PCR reaction was accompanied by a positive control reaction that utilized the same primer set and 10 ng of C. psittaci genomic DNA, and a negative control reaction in which 1 pl of the same RNA preparation was used as template in the PCR reaction. A control primer set located within the incC gene was also used as an RT-PCR control. Identification of incA, incB and incC genes of C. trachomatis. The nucleotide sequence information obtained for the incA, incB and incC of C. psittaci (above) was used, with standard methods, to identify the inc gene orthologues of C. trachomatis. Probes were made that corresponded to the 3' and 5' ends of the C. psittaci inc open reading frames. Standard PCR amplification (as above) was used, with the C. trachomatis genome as a template, to amplify the corresponding C. trachomatis nucleotide sequence. The amplified DNA was then sequenced, using standard methods..

2. ISOLATION OF p242, TroA AND TroB Bacterial strains. C. trachomatis LGV-434, serotype L2, was cultivated in HeLa 229 cells using standard methods (Caldwell et al., 1981). Purified chlamydiae were obtained using Renografin R. Squibb Sons, Inc., Princton, density gradient centrifugation (Hackstadt et al., 1992). Escherichia coli DH5 (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) was used as the host strain for transformations with recombinant DNA. E. coli XLl-Blue

MRF'

(Stratagene, La Jolla, Calif.) was used as the host strain for infection with lambda ZAPII phage WO 99/53948 PCT/US99/08744 -14vector. E. coli SOLR (Stratagene) was used as the host strain for infection with in vivo excised filamentous lambda ZAPII.

Antisera. Two Cynomolgus monkeys (Macaca fasicularis) were anaesthetized and infected urethrally with C. trachomatis EBs. Each monkey was infected twice and allowed to recover between infections. Symptoms of infection were monitored over time. Antisera from infected monkeys were tested for reactivity to Chlamydia by ELISA (Su et al., 1990).

Sera were collected every two weeks and anti-chlamydial titers were determined. These animals showed mild clinical signs of disease which cleared spontaneously. A second challenge was then administered. Sera were collected from these animals and used to probe a C. trachomatis expression library as discussed below. As a control, Guinea Pigs were immunized with killed C.

trachomatis of the EB form. Sera from these animals were obtained and also used to probe the C.

trachomatis expression library.

C. trachomatis library construction and immunoscreening. A C. trachomatis genomic library was constructed with the lambda ZAPII vector as described above for C. psittaci.

Approximately 15,000 plaques were plated, transferred to nitrocellulose filters (Schleicher and Schuell, Keene, in duplicate, and probed with the monkey convalescent antiserum and with Guinea Pig serum against killed EBs (Bannantine et al., 1998). Plaques that reacted only with the monkey convalescent antisera were selected for further study.

Identification of antigens recognized by convalescent antisera. Four positive recombinant plaques were identified that showed reactivity with convalescent antisera but not with anti-EB serum. The purified recombinant phage were converted into pBluescriptll SK plasmid by in vivo excision and recircularization and these recombinant DNAs (pCtl, pCt2, pCt3 and pCt4) were used to transform E. coli. SDS-PAGE and immunoblot analysis of lysates of these recombinant E. coli showed that each expressed one or more proteins that reacted with convalescent (anti-RB) antisera but not with the anti-EB antiserum. Two of the recombinants clones, pCt2 and pCt3, expressed an identical 19.9 kDa protein (p242). The pCt4 recombinant expressed two different proteins of approximately 32 kDa each that are strongly recognized by convalescent antisera (TroA and TroB).

C. SEQUENCE ANALYSIS Sequence analysis of pCtl, 2, and 3 revealed overlapping inserts with only one open reading frame (ORF) common in all three. This ORF encodes an approximately 19.9 kDa protein (p242) that shows no similarity to other known proteins. The nucleotide sequence encoding C.

trachomatis p242, and the amino acid sequence of the protein are shown in SEQ ID NOS:1 and 2, respectively.

The insert in pCt4 contains two complete ORFs which code for two proteins, each of approximately 32kDa (TroA and TroB) that show some homology with proteins from Treponema WO 99/53948 PCT/US99/08744 pallidum. The nucleotide sequences encoding the 32 kDa proteins (TroA and TroB) and the amino acid sequences of these proteins are shown in SEQ ID NOS: 3, 4, 5, and 6.

D. EMBODIMENTS OF THE INVENTION The present invention includes the nucleotide and amino acid sequences for certain infection-specific proteins from Chlamydia. These proteins are p242, TroA, and TroB from C.

trachomatis, and the IncB, and IncC proteins from C. psittaci. The scope of the invention includes fragments of these proteins that may be used in a vaccine preparation or that may be used in a method of detecting Chlamydia antibodies. Such fragments may be, for example, 5, 10, 20, 25, or 30 contiguous amino acids in length, or may even encompass the entire protein.

The present invention also encompasses the use of infection-specific proteins of Chlamydia, and the use of nucleotides encoding such proteins. Infection-specific proteins include the IncA, IncB and IncC proteins of C. psittaci, the IncA, IncB and IncC proteins of C.

trachomatis, and the TroA, TroB, and p242 proteins of C. trachomatis. The inventors have shown that these proteins are infection-specific by using immunological techniques such as immunofluorescence microscopy and immunoblotting.

The present invention includes a vaccine against chlamydial infections comprising infection-specific proteins or fragments of these proteins or proteins that are homologous or show substantial sequence similarity to these proteins. In one embodiment, one or more purified infection-specific proteins may be mixed with a pharmaceutically acceptable excipient to produce a vaccine that stimulates a protective immunological response in an animal. In one embodiment the vaccine may be administered intra-muscularly or sub-cutaneously or intravenously. In another embodiment, the vaccine may be administered by inoculation into or onto the mucous membranes of the subject animal. For example, the vaccine may be administered urethrally or genitally as a liquid or in the form of a pessary. In another embodiment, it may be administered to the mucosa of the lungs as a spray or vapor suspension.

Since at least three amino acids are required to produce an antigenic epitope, the vaccine should comprise at least three consecutive amino acids, preferably at least five consecutive amino acids, and may comprise at least 10, 15, 25, 30, 40, or 45 consecutive amino acids of the infection-specific proteins as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

The vaccine of the invention may be used to inoculate potential animal targets of any of the chlamydial diseases including those caused by C. psittaci, C. trachomatis, C. pneumoniae or C. pecorum. Indeed the vaccine of the invention may be used to inoculate animals against any disease that shows immunological cross-protection as a result of exposure to infection-specific Chlamydia antigen.

Vaccines of the present invention can.include effective amounts of immunological adjuvants known to enhance an immune response alum). The protein or polypeptide is present in the vaccine in an amount sufficient to induce a protective immune response whether WO 99/53948 PCT/US99/08744 -16through humoral or cell mediated pathways or through both. Such a response protects the immunized animal against chlamydial infections specifically by raising an immune response against the Reticulate Body form of Chlamydia. Protective antibodies may be elicited by a series of two or three doses of the antigenic vaccine given about two weeks apart.

The present invention also teaches a method of making a vaccine against chlamydial infections. The method of making the vaccine comprises providing a pure (or substantially pure) infection-specific chlamydial peptide or portion thereof, and mixing the peptide with a pharmacologically acceptable excipient or adjuvant. Adjuvants may include commonly used compounds such as alum. Additionally, the vaccines may be formulated using a peptide according to the present invention together with a pharmaceutically acceptable excipient such as water, saline, dextrose and glycerol. The vaccines may also include auxiliary substances such as emulsifying agents and pH buffers. Doses of the vaccine administered will vary depending on the antigenicity of the particular peptide or peptide combination employed in the vaccine and characteristics of the animal or human patient to be vaccinated.

The infection-specific vaccine of the invention is directed towards not only C. psittaci, but against all forms of Chlamydia including C. pneumoniae, C. trachomatis and C. pecorum, and the vaccine may comprise not just peptides derived from C. psittaci, but also orthologous peptides and fragments of such orthologous peptides from other species of Chlamydia and peptides that are substantially similar to such peptides.

The present invention also teaches a method of vaccination comprising administering a vaccine formulated as described above to an animal either intravenously, intramuscularly, subcutaneously, by inhalation of a vapor or mist, or by inoculation in the form of a liquid, spray, ointment, pessary or pill into or onto the mucous membranes of the mouth, nose, lungs or urogenital tract or colon.

The methods of the invention may be practiced equally with human or non-human animal subjects.

The present invention also teaches a method of detecting Chlamydia infection-specific proteins produced by the Reticulate Body form of the organism. In this embodiment, antibodies raised to the infection-specific proteins are used in an immunological assay such as an Enzyme Linked Immunosorbant Assay or Biotin-Avidin assay or a radioimmunoassay or any other assay wherein specific antibodies are used to recognize a specific protein. Such assays mnay be used to detect both the quantity of proteins present and also the specificity of binding of such proteins. In such an assay, antibodies have attached to them, usually at the Fc portion, a detectable label, such as an enzyme, fluorescent marker, a radioactive marker or a Biotin-Avidin system marker that allows detection. A biological sample is provided from an animal that has been putatively exposed to Chlamydia. Such a sample may be, for example, whole blood, serum, tissue, saliva or a mucosal secretion. The sample is then contacted with the labeled antibody and specific binding, if any, is detected. Other methods of using infection-specific antibodies to detect infection-specific WO 99/53948 PCT/US99/08744 -17antigens that are present in cells or tissues include immunofluorescense, indirectimmunofluorescense and immunohistochemistry. In immunofluorescense, a fluorescent dye is bound directly to the antibody. In indirect-immunofluorescence, the dye is bound to an antiimmunoglobulin. Specific binding occurs between antigen and bound antibody is detected by virtue of flourescent emissions from the dye moiety. This technique would be particularly useful, for instance, for detection of Chlamydia antigen present on a urogenital mucosal smear.

Other techniques, such as competitive inhibition assays may also be used to assay for antigen, and one of ordinary skill in the art will readily appreciate that the precise methods disclosed may be modified or varied without departing from the subject or spirit of the invention taught herein.

The present invention also teaches a method of detection of Chlamydia infection-specific antibodies made against the Reticulate Body. In this embodiment a sample is provided from an animal putatively exposed to Chlamydia to determine whether the sample contains infectionspecific antibodies. Such a sample may be, for example, whole blood, serum, tissue, saliva or a mucosal secretion. This sample is contacted with infection-specific antigens such that the amount and specificity of binding of the antibody may be measured by its binding to a specific antigen.

Many techniques are commonly known in the art for the detection and quantification of antigen.

Most commonly, the purified antigen will be bound to a substrate, the antibody of the sample will bind via its Fab portion to this antigen, the substrate will then be washed and a second, labeled antibody will then be added which will bind to the Fc portion of the antibody that is the subject of the assay. The second, labeled antibody will be species specific, if the serum is from a human, the second, labeled antibody will be anti-human-IgG antibody. The specimen will then be washed and the amount of the second, labeled antibody that has been bound will be detected and quantified by standard methods.

The present invention also teaches a method of treating a Chlamydial infection by directing a therapeutic agent against a specific target, such as: an infection-specific protein of Chlamydia, (ii) a gene that encodes an infection-specific protein of Chlamydia and (iii) an RNA transcript that encodes an infection-specific protein of Chlamydia, wherein said therapeutic agent interacts with said target to affect a reduction in pathology.

For example, the present invention teaches a method of treating chlamydial infection wherein antisense technology is used to prevent the expression of infection-specific genes, thereby preventing the pathologies associated these proteins and preventing reproduction of the RB phase of Chlamydia. In this embodiment, RNA molecules complementary to transcripts of infection specific genes are introduced into the host cells that contain Chlamydia, and by binding to the mRNA transcripts of the infection-specific genes, prevent translation and therefore expression of the infection-specific proteins that are associated with pathogenesis.

The invention may be practiced to produce a vaccine against any species of Chlamydia, including C. psittaci, C. pecorum, C. trachomatis and C. pneumoniae.

WO 99/53948 PCT/US99/08744 -18- The following examples illustrate various embodiments of the invention.

EXAMPLE 1: Homologous Sequences The DNA and protein sequences discussed herein are shown in SEQ ID NOS: 1-18.

These sequences refer to infection-specific proteins and to the DNA sequences that encode these proteins. Although these sequences are from C. psittaci and C. trachomatis, it would be equally possible to substitute in the present invention, the orthologs of these sequences from other Chlamydia species such as C. pecorum and C. pneumoniae.

Such orthologous sequences may be obtained from the appropriate organisms by isolation of the genome of the organism, digestion with restriction enzymes, separation of restriction fragments by electrophoresis and purification of these fragments and selection of fragments of appropriate size. Identity of the fragments can be confirmed by dot-blot and by standard DNA sequencing techniques. The orthologous sequences in different Chlamydia species may also be found by selection of appropriate PCR primers (selected from appropriate regions flanking the Chlamydia gene of interest), and the use of these primers in a PCR reaction, using the genome of the particular species of Chlamydia of interest as a template, to amplify the ortholog of interest.

Such PCR primers would be selected from the flanking regions to allow specific amplification of the target gene. The fragments so obtained could then be run on a gel to check size and sequenced and compared against the known sequences to determine sequence identity.

The degree of sequence identity between the infection-specific genes of C. psittaci or C.

trachomatis and their orthologs from C. pecorum and C. pneumoniae, may be determined by comparing sequences using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) as described herein.

Orthologues of interest infection-specific proteins are characterized by possession of at least 50% or greater sequence identity counted over the full length alignment with one of the disclosed amino acid sequences of the C. psittaci or C. trachomatis infection-specific proteins using gapped blastp set to default parameters (described herein).

EXAMPLE 2: Heterologous Expression of Infection-Specific Antigens Methods for expressing large amounts of protein from a cloned gene introduced into Escherichia coli coli) may be utilized for the purification of the Chlamydia peptides. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are well known and are described in Sambrook et al. (1989). Such fusion proteins may be made in large amounts, are relatively simple to purify, and can be used to produce antibodies. Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome binding site upstream of the cloned gene. If low levels of protein are produced, additional steps WO 99/53948 PCT/US99/08744 -19may be taken to increase protein production; if high levels of protein are produced, purification is relatively easy.

Often, proteins expressed at high levels are found in insoluble inclusion bodies. Methods for extracting proteins from these aggregates are described in chapter 17 of Sambrook et al.

(1989). Vector systems suitable for the expression of lacZ fusion genes include the pUC series of vectors (Ruther et al. (1983)), pEX1-3 (Stanley and Luzio (1984)) and pMR100 (Gray et al.

(1982)). Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg (1981)), pKK177-3 (Amann and Brosius (1985)) and pET-3 (Studiar and Moffatt (1986)).

Fusion proteins may be isolated from protein gels, lyophilized, ground into a powder and used as antigen preparations.

Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, may also be used for protein expression, as is well known in the art. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other prokaryotic and eukaryotic cells and cell lines may be appropriate for a variety of purposes, to provide higher expression, post-translational modification, desirable glycosylation patterns, or other features.

Additionally, peptides, particularly shorter peptides, may be chemically synthesized, avoiding the need for purification from cells or culture media. It is known that peptides as short as 3 amino acids can act as an antigenic determinant and stimulate an immune response. Such peptides may be administered as vaccines in ISCOMs (Immune Stimulatory Complexes) as described by Janeway Travers, Immunobiology: The Immune System In Health and Disease, 13.21 (Garland Publishing, Inc. New York, 1997). Accordingly, one aspect of the present invention includes small peptides encoded by the nucleic acid molecules disclosed herein. Such peptides include at least 5, and may be at least 10, 15, 20, 25, or 30 or more contiguous amino acids of the polypeptide sequences described herein.

EXAMPLE 3: Production of Antibodies Specific for Infection-Specific Antigens Antibody against infection-specific antigen is encompassed by the present invention, particularly for the detection of Chlamydia infection-specific antigen. Such antibody may be produced by inoculation of an animal such as a guinea-pig or a monkey with infection-specific antigen produced as described above. Such antigen may be a polypeptide as disclosed herein, such as a complete or partial polypeptide from C. psittaci, C. trachomatis, C. pneumoniae or C.

pecorum. As discussed above, any molecule that can elicit a specific, protective immune response WO 99/53948 PCT/US99/08744 may be used as a vaccine, but since a minimum of three amino acids are required to do this, a vaccine should comprise at least three amino acids.

The peptide for use in the vaccine of the invention may be naturally derived or may be synthetic such as those synthesized on a commercially available peptide synthesizer. The peptide may also comprise a complete or partial peptide derived from the C. pneumoniae or C. pecorum infection-specific orthologs of the C. trachomatis or C. psittaci proteins as set out herein.

In one method of production, a polyclonal antibody is produced by providing a purified peptide which is diluted to 100 micrograms per milliliter in sterile saline and mixed with RiBi Trivalent Adjuvant (RiBi Immunochem Inc). The antigen/adjuvant emulsion is then administered to an anaesthetized guinea pig using a procedure as provided by the manufacturer. Serum is collected 14 days after secondary and tertiary immunizations.

Monoclonal antibody to epitopes of the Chlamydia peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected purified protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin, Hypoxanthene, Aminopterin and Thymidine (HAT) medium. The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.

Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (1988).

An alternative approach to raising antibodies against the Chlamydia peptides is to use synthetic peptides synthesized on a commercially available peptide synthesizer based upon the amino acid sequence of the peptides predicted from nucleotide sequence data.

In another embodiment of the present invention, monoclonal antibodies that recognize a specific Chlamydia peptide are produced. Optimally, monoclonal antibodies will be specific to each peptide, such antibodies recognize and bind one Chlamydia peptide and do not substantially recognize or bind to other proteins, including those found in uninfected human cells.

The determination that an antibody specifically detects a particular Chlamydia peptide is made by any one of a number of standard immunoassay methods; for instance, the western blotting technique (Sambrook et al., 1989). To determine that a given antibody preparation (for instance from a guinea pig) specifically detects one Chlamydia peptide by western blotting, total cellular protein is extracted from a sample of blood from an unexposed subject and from a sample of blood from an exposed subject. As a positive control, total cellular protein is also extracted from WO 99/53948 PCT/US99/08744 -21- Chlamydia cells grown in vitro. These protein preparations are then electrophoreged on a sodium dodecyl sulfate-polyacrylamide gel. Thereafter, the proteins are transferred to a membrane (for example, nitrocellulose) by western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-guinea pig antibody conjugated to an enzyme such as alkaline phosphatase; application of the substrate 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immuno-localized alkaline phosphatase. Antibodies which specifically detect the Chlamydia protein will, by this technique, be shown to bind to the Chlamydia-extracted sample at a particular protein band (which will be localized at a given position on the gel determined by its molecular weight) and to the proteins extracted from the blood of the exposed subject. No significant binding will be detected to proteins from the unexposed subject.

EXAMPLE 4: Use of Infection-Specific Sequences and their Corresponding Peptides and Antibodies in Diagnostic Assays Another aspect of the present invention is a method for detecting the presence of anti- Chlamydia antibodies that react with infection-specific Chlamydia proteins, Chlamydia peptides and Chlamydia nucleic acid sequences in biological samples. These methods include detection of antigen and antibody by ELISA and similar techniques, the detection of proteins in a tissue sample by immunofluorescence and related techniques and the detection of specific DNA sequences by specific hybridization and amplification.

One aspect of the invention is an ELISA that detects anti-Chlamydia antibodies in a medical specimen. An immunostimulatory infection-specific Chlamydia peptide of the present invention is employed as an antigen and is preferably bound to a solid matrix such as a crosslinked dextran such as SEPHADEX (Pharmacia, Piscataway, NJ), agarose, polystyrene, or the wells of a microtiter plate. The polypeptide is admixed with the specimen, such as blood, and the admixture is incubated for a sufficient time to allow antibodies present in the sample to immunoreact with the polypeptide. The presence of the positive immunoreaction is then determined using an ELISA assay, usually involving the use of an enzyme linked to an anti-immunoglobulin that catalyzes the conversion of a chromogenic substrate.

In one embodiment, the solid support to which the polypeptide is attached is the wall of a microtiter assay plate. After attachment of the polypeptide, any nonspecific binding sites on the microtiter well walls are blocked with a protein such as bovine serum albumin. Excess bovine serum albumin is removed by rinsing and the medical specimen is admixed with the polypeptide in the microtiter wells. After a sufficient incubation time, the microtiter wells are rinsed to remove excess sample and then a solution of a second antibody, capable of detecting human antibodies is added to the wells. This second antibody is typically linked to an enzyme such as peroxidase, WO 99/53948 PCT/US99/08744 -22alkaline phosphatase or glucose oxidase. For example, the second antibody may be a peroxidaselabeled goat anti-human antibody. After further incubation, excess amounts of the second antibody are removed by rinsing and a solution containing a substrate for the enzyme label (such as hydrogen peroxide for the peroxidase enzyme) and a color-forming dye precursor, such as o-phenylenediamine is added. The combination of Chlamydia peptide (bound to the wall of the well), the human anti-Chlamydia antibodies (from the specimen), the enzyme-conjugated antihuman antibody and the color substrate will produce a color that can be read using an instrument that determines optical density, such as a spectrophotometer. These readings can be compared to a negative control such as a sample known to be free of anti-Chlamydia antibodies. Positive readings indicate the presence of anti-Chlamydia antibodies in the specimen, which in turn indicate a prior exposure of the patient to Chlamydia.

In another embodiment, antibodies that specifically recognize a Chlamydia peptide encoded by the nucleotide sequences disclosed herein are useful in diagnosing the presence of infection-specific Chlamydia antigens in a subject or sample. For example, detection of infectionspecific antigens that are present in cells or tissues may be done by immunofluorescence, indirectimmunofluorescense and immunohistochemistry. In immunofluorescense, a fluorescent dye is bound directly to the antibody. In indirect-immunofluorescence, the dye is bound to an antiimmunoglobulin. Specific binding occurs between antigen and bound antibody is detected by virtue of fluorescent emissions from the dye moiety. This technique may be particularly useful, for instance, for detection of Chlamydia antigen present on a urogenital mucosal smear.

Chlamydia may be present in urogenital mucosa, and a smear on a glass slide may be fixed and bathed in a solution containing an antibody specific to the infection-specific antigen. The slide is then washed to remove the unbound antibody, and a fluorescent anti-immunoglobulin antibody is added. The slide is washed again, and viewed microscopically under an appropriate wavelength of light to detect fluorescence. Fluorescence indicates the presence of Chlamydia antigen.

Alternatively, a urogenital mucosal smear may be taken, the sample cultured with HeLa cells to produce large amounts of the RB form, and immunofluorescence may then be used to detect infection-specific Chlamydia antibodies.

Another aspect of the invention includes the use of nucleic acid primers to detect the presence of Chlamydia nucleic acids that encode infection-specific antigens in body samples and thus to diagnose infection. In other embodiments, these oligonucleotide primers will comprise at least 15 contiguous nucleotides of a DNA sequence as shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, or 17. In other embodiments, such oligonucleotides may comprise at least 20 or at least or more contiguous nucleotides of the aforementioned sequences.

One skilled in the art will appreciate that PCR primers are not required to exactly match the target gene sequence to which they anneal. Therefore, in another embodiment, the oligonucleotides will comprise a sequence of at least 15 nucleotides and preferably at least nucleotides, the oligonucleotide sequence being substantially similar to a DNA sequence set forth WO 99/53948 PCTIUS99/08744 -23in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, and 17. Such oligonucleotides may share at least about 85%, 90% or greater sequence identity.

The detection of specific nucleic acid sequences in a sample by polymerase chain reaction amplification (PCR) is discussed in detail in Innis et al., (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, part 4 in particular. To detect Chlamydia sequences, primers based on the sequences disclosed herein would be synthesized, such that PCR amplification of a sample containing Chlamydia DNA would result in an amplified fragment of a predicted size. If necessary, the presence of this fragment following amplification of the sample nucleic acid could be detected by dot blot analysis. PCR amplification employing primers based on the sequences disclosed herein may also be employed to quantify the amounts of Chlamydia nucleic acid present in a particular sample (see chapters 8 and 9 of Innis et al., (1990)).

Alternatively, probes based on the nucleic acid sequences described herein may be labeled with suitable labels (such a P 32 or biotin) and used in hybridization assays to detect the presence of Chlamydia nucleic acid in provided samples.

Reverse-transcription PCR using these primers may also be utilized to detect the presence of Chlamydia RNA which is indicative of an ongoing infection.

EXAMPLE 5: Production of Chlamydia Vaccines The purified peptides of the present invention may be used directly as immunogens for vaccination. Methods for using purified peptides as vaccines are well known in the art and are described in Yang et al. (1991), Andersen (1994) and Jardim et al. (1990). As is well known in the art, adjuvants such as alum, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may be used in formulations of purified peptides as vaccines. Accordingly, one embodiment of the present invention is a vaccine comprising one or more immunostimulatory C.

trachomatis or C. psittaci peptides encoded by nucleotide sequences as shown in the attached sequence listing, together with a pharmaceutically acceptable adjuvant.

Additionally a vaccine may comprise a defined fraction of the disclosed peptide of C.

trachomatis or C. psittaci or may comprise a peptide wherein the gene coding for the peptide shows substantial similarity to the DNA sequences disclosed herein, such as for orthologous genes of C. pneumoniae or C. pecorum.

Additionally, the vaccines may be formulated using a peptide according to the present invention together with a pharmaceutically acceptable excipient such as water, saline, dextrose and glycerol. The vaccines may also include auxiliary substances such as emulsifying agents and pH buffers.

It will be appreciated by one of skill in the art that vaccines formulated as described above may be administered in a number of ways including subcutaneous, intra-muscular and intra-venous injection. Doses of the vaccine administered will vary depending on the antigenicity of the particular peptide or peptide combination employed in the vaccine, and characteristics of the WO 99/53948 PCT/US99/08744 -24animal or human patient to be vaccinated. While the determination of individual doses will be within the skill of the administering physician, it is anticipated that doses of between 1 microgram and 1 milligram will be employed.

As with many vaccines, the vaccines of the present invention may routinely be administered several times over the course of a number of weeks to ensure that an effective immune response is triggered. Where such multiple doses are administered, they will normally be administered at from two to twelve week intervals, more usually from three to five week intervals.

Periodic boosters at intervals of 1-5 years, usually three years, may be desirable to maintain the desired levels of protective immunity.

Alternatively, multiple immunostimulatory peptides may also be administered by expressing the nucleic acids encoding the peptides in a nonpathogenic microorganism, and using this transformed nonpathogenic microorganism as a vaccine.

Finally, a recent development in the field of vaccines is the direct injection of nucleic acid molecules encoding peptide antigens, as described in Janeway Travers, (1997). Thus, plasmids which include nucleic acid molecules described herein, or which include nucleic acid sequences encoding peptides according to the present invention may be utilized in such DNA vaccination methods.

The vaccine of the invention may be used to inoculate potential animal targets of any of the chlamydial diseases including those caused by C. trachomatis, C. psittaci, C. pneumoniae or C. pecorum. Indeed the vaccine of the invention may be used to inoculate animals against any disease that shows immunological cross-protection as a result of exposure to infection-specific Chlamydia antigen. The protein or polypeptide is present in the vaccine in an amount sufficient to induce a protective immune response whether through humoral or cell mediated pathways or through both. Such a response protects the immunized animal against chlamydial infections specifically by raising an immune response against the Reticulate Body form of Chlamydia.

The above embodiments are set out only by way of example and are not intended to be exclusive, one skilled in the art will understand that the invention may be practiced in various additional ways without departing from the subject of the spirit of the invention.

REFERENCES

Akins, D. et al. (1997) J. Bacteriol. 179: 5076-5086.

Amann and Brosius (1985). Gene 40: 183.

Andersen (1994). Infection Immunity 62: 2536.

Ausubel et al. (1987). Current Protocols in Molecular Biology, ed. Greene Publishing and Wiley Interscience: New York (with periodic updates).

Blanco, D. et al. (1995) J. Bacteriol. 177: 3556-3562.

Bannantine, J. et al. (1997) Abstr. Gen. Mtg. Amer. Soc. Microbiol. D-004. Miami, FL.

Bannantine, J.P. et al., (1998) Infect Immun. 66:6017-6021.

Blanco, D. et al. (1996) J. Bacteriol. 178: 6685-6692.

Brown, W. and Farquhar, M. G. (1989) Meth Cell Biol 31: 553-569.

Caldwell, H. et al. (1981) Infect. Immunol. 31: 1161-1176.

Chou, P. Y. and Fasman, G. D. (1978) Annu Rev Biochem 47: 251-276. Engvall (1980). Enzymol. 70: 419. Gray et al. (1982). Proc. Natl. Acad. Sci. USA 79: 6598. Hackstadt, R. et al. (1992) Infect. Immun. 60: 159-165. Hardham, J. et al. (1977) Gene 197: 47-64. Harlow and Lane (1988). Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

Innis et al. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego. Janeway Travers (1997) Immunobiology: The Immune System in Health and Disease 13.21. Garland Publishing, Inc. New York. Kohler and Milstein (1975) Nature 256: 495. Kyte, J. and Doolittle, R. F. (1982)JMolBiol 157: 105-132. Peeling, R. and Burnham, R. (1996) Emerging Infectious Diseases 2 307-317.

Rockey, D. and Rosquist, J. L. (1994) Infect Immun 62: 106-112.

Rockey, D. et al. (1995) Mol Microbiol 15: 617-626.

Rockey, D. et al. (1996) Infect Immun 64: 4269-4278.

Rockey, D. et al. (1997). Mol Microbiol 24: 217-228.

Robson, B. and Suzuki, E. (1976) JMol Biol 107: 327-356.

Rockey, D. and Rosquist, J. L. (1994). Infect Immun 62: 106-112.

Rothman, J. and F. T. Wieland (1996) Science 272: 227-234.

Z ,uther and Muller-Hill (1983). EMBOJ. 2: 1791.

[R:\LIBVV]02761 .doc:ais Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed.

Cold Spring Harbor Lab. Press: Cold Spring Harbor, NY.

[RA\UBVV]02761 .doc:ais WO 99/53948 PCTIUS99/08744 -26- Shimatake and Rosenberg (1981). Nature (London) 292:128.

Stanley and Luzio (1984). EMBO 1. 3:1429.

Studiar and Moffatt (1986). 1. Mo!. Biol. 189:113.

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Yuan, et al. (1992) Infect Immun 60: 2288-2296.

EDITORIAL NOTE-NO.36590/99 This specification contains a sequence listing following the description and in numbered as follows: Sequence listing pages 1 to Claim pages 27 to 29 WO 99/53948 PCTIUS99/08744 SEQUENCE LISTING <110> Oregon State University <120> Methods of use for infection-specific INCA, INCB, and INCC proteins of Chlamydia <130> 52297 <140> <141> <150> 60/082,588 <151> 1998-04-21 <150> 60/082,438 <151> 1998-04-20 <150> 60/086,450 <151> 1998-05-22 <160> 24 <170> PatentIn Ver. <210> <211> <212> <213> <220> <221> <222> 534

DNA

Chlamydia trachomatis

CDS

(534) <400> 1 atg aaa Met Lys 1 aag ttc tta Lys Phe Leu 5 tta ctt agc tta Leu Leu Ser Leu tct ttg tca tct Ser Leu Ser Ser cta cct Leu Pro aca ttt gca Thr Phe Ala cgc tgc cta Arg Cys Leu gct Ala aat tct aca ggc Asn Ser Thr Gly att gga atc gtt Ile Gly Ile Val aat tta cgt Asn Leu Arg gct gaa ttc Ala Glu Phe gaa gag tct get Glu Glu Ser Ala ctt Leu 40 ggg aaa aaa gaa Gly Lys Lys Glu tct Ser gaa aag Glu Lys atg aaa aac caa Met Lys Asn Gln ttc Phe 55 tct aac agc atg Ser Asn Ser Met ggg Gly aag atg gag gaa Lys Met Glu Glu gaa Glu ctg tct tct ate Leu Ser Ser Ile tat Tyr tec aag ctc caa Ser Lys Leu Gln gac gat tac atg Asp Asp Tyr Met 192 240 288 ggt cta tcc gag Gly Leu Ser Glu gca gct gcc gaa Ala Ala Ala Glu tta Leu ggg Gly 1 aga aaa aaa ttc Arg Lys Lys Phe gaa gat Glu Asp cta tct gca gaa tac aac aca gct caa Leu Ser Ala Glu Tyr Asn Thr Ala Gin cag tat tac caa ata tta Gin Tyr Tyr Gln Ile Leu WO 99/53948 100 aac caa agt aat Asn Gin Ser Asn 115 aaa gct tct gaa Lys Ala Ser Glu 130 aac gaa gat att Asn Giu Asp Ile 145 got gtt att aaa Ala Val Ile Lys ago tag Ser <210> 2 <211> 177 <212> PRT <213> Chiamydia PCTIUS99/08744 ttc Phe act Thr gtc Val gt t Val 165 aag Lys gtg Val1 tta Leu 150 ctt Leu cgo Arg cgt Arg 135 tct Ser gat Asp 105 caa Gin caa Gin gat Asp ott Leu at t Ile Gly tcg Ser 155 aaa Lys atg Met ttg Leu 140 goa Ala ata Ile gaa Glu 125 t ca Ser gat Asp att Ile 110 gaa Glu gt o Vali aaa Lys aac Asn gtg Val ott Leu aco Thr atg Met 175 aaa Lys ott Leu gat Asp 160 cga Arg 384 432 480 528 trachornatis <400> 2 Met Lys 1 Thr Phe Arg Cys Glu Lys Giu Leu Gly Leu Leu Ser Asn Gin Lys Ala 130 Asn Giu 145 Ala Val Lys Phe Ala Ala Leu Glu Met Lys Ser Ser Ser Giu Ala Glu 100 Ser Asn 115 Ser Glu Asp Ile Ile Lys Leu 5 Asn Giu Asn Ile Thr Tyr Phe Thr Val Vai 165 Leu Ser Ser Gin T yr 70 Ala As n Lys Vai Leu 150 Leu Leu Thr Al a Phe 55 Ser Ala Thr Arg Arg 135 Ser Asp Ser Leu Met Ser Leu Ser Ser Leu Pro 10 Giy Leu 40 Ser Lys Ala Ala Met 120 Ile Ile Val Thr 25 Gly As n Leu Giu Gin 105 Gin Gin Asp Leu Ile Lys Ser Gin Leu 90 Gly Lys Giu Ser Phe 170 2 Gly Lys Met Asp 75 Arg Gin Ile Giy Ser 155 Lys Ile Giu Gly Asp Lys Tyr Met Leu 140 Ala Ile Val Ser Lys Asp Lys Tyr Giu 125 Ser Asp Ile Asn Aia Met Tyr Phe Gin 110 Giu Val Lys Asn Leu Giu Giu Met Glu I le Val1 Leu Thr Met 175 Arg Phe Giu Giu Asp Leu Lys Leu Asp 160 Arg WO 99/53948 WO 9953948PCT/US99/08744 Ser <210> 3 <211> 846 <212> DNA <213> Chiainydia trachoinatis <220> <221> CDS <222> <400> 3 atg aat cgc atg Met Asn Arg Met 1 tgt gat tgc gtg Cys Asp Cys Val cgc ata act ggg Arg Ile Thr Gly gat cga Asp Arg gtc aaq aat Val Lys Asn gag atg gtg Giu Met Val gtt ctg att gat Val Leu Ile Asp gga Gly gcg att gat cct Ala Ile Asp Pro cat tca tat His Ser Tyr cag ctg att Gin Leu Ile aag ggg gat gaa Lys Gly Asp Giu gac Asp 40 cga atg gct atg Arg Met Aia Met agc Ser ttt tgc Phe Cys aat ggt tta ggt Asn Gly Leu Gly tta Leu gag cat tca gct Glu His Ser Ala tta cgt aaa cat Leu Arg Lys His cta Leu gag ggt aac cca Giu Gly Asn Pro aaa Lys 70 gtc gtt gat tta Val Vai Asp Leu ggt Gly 75 caa cgt ttg ctt Gin Arg Leu Leu aac Asn aaa aac tgt ttt Lys Asn Cys Phe gat Asp ctt ctg agt gaa Leu Leu Ser Giu gaa Giu 90 gga ttc cct gac Gly Phe Pro Asp cca cat Pro His att tgg acg Ile Trp Thr gcg gca tta Ala Ala Leu 115 gat Asp 100 atg aga gta tgg Met Arg Val Trp ggt Giy 105 gct gct gta aaa Ala Ala Val Lys gag atg gct Giu Met Ala 110 ttt caa aag Phe Gin Lys 336 384 att caa caa ttt Ile Gin Gin Phe caa tat gaa gaa Gin Tyr Giu Giu gat Asp 125 aat gcg Asn Ala 130 gat cag atc tta Asp Gin Ile Leu t ca Ser 135 gag atg gag gaa Giu Met Giu Giu ctt Leu 140 gat cgt tgg gca Asp Arg Trp Ala gtg Val 145 cgt tct ctc tct Arg Ser Leu Ser acg Thr 150 att cct gaa aaa Ile Pro Glu Lys aat Asn 155 cgc tat tta gtc Arg Tyr Leu Val ggc cac aat gcg Gly His Asn Ala ttc Phe 165 agt tac ttt act Ser Tyr Phe Thr cgt Arg 170 cgg tat cta tcc Arg Tyr Leu Ser tct gat Ser Asp 175 gcg gag aga Ala Giu Arg tct ggg gaa tgg Ser Giy Giu Trp aga Arg 185 tcg cgt tgc att Ser Arg Cys Ile tct cca gaa Ser Pro Giu 190 576 WO 99/53948 ggg ttg tct Gly Leu Ser 195 gtg gag tat Val Glu Tyr 210 acg tta aat Thr Leu Asn 225 gga caa aag Gly Gln Lys tgt gat aac Cys Asp Asn gaa gaa ttg Glu Glu Leu 275 cct Pro atc Ile caa Gln att Ile tat Tyr 260 gga Gly gag gct Glu Ala tct gca Ser Ala gat get Asp Ala 230 cgt ctc Arg Leu 245 ttt agc Phe Ser ggg act Gly Thr cag Gln aac Asn 215 ttg Leu gct Ala acg Thr gtt Val att Ile 200 gat Asp aga Arg aag Lys ttc Phe ctt Leu 280 agt Ser gta Val aag Lys tct Ser cag Gln 265 gaa Glu atc Ile gaa Glu att Ile cct Pro 250 cac His tag cga Arg gtt Val gtt Val 235 tta Leu aat Asn gat Asp gtc Val 220 tct Ser tat Tyr gtt Val att Ile 205 ttt Phe tgc Cys agc Ser cgc Arg cgt Arc gac

GIL

aac Lys aat Asr 255 att Il PCT/US99/08744 gta 624 Val gat 672 i Asp age 720 Ser 240 gtc 768 SVal aca e Thr 816 846 <210> 4 <211> 281 <212> PRT <213> Chlamydia <400> 4 Met Asn Arg Met 1 Val Lys Asn Ile Glu Met Val Lys Phe Cys Asn Gly Leu Glu Gly Asn Lys Asn Cys Phe Ile Trp Thr Asp 100 Ala Ala Leu Ile 115 Asn Ala Asp Gln 130 Val Arg Ser Leu 145 trachomatis Ile 5 Val Gly Leu Pro Asp Met Gln Ile Ser Cys Asp Leu Ile Asp Glu Gly Leu 55 Lys Val 70 Leu Leu Arg Val Gln Phe Leu Ser 135 Thr Ile 150 Cys Val Asp Gly 25 Asp Arg 40 Glu His Val Asp Ser Glu Trp Gly 105 Pro Gln 120 Glu Met Pro Glu Ser 10 Ala Met Ser Leu Glu 90 Ala Tyr Glu Lys Arg Ile Ala Ala Gly 75 Gly Ala Glu Glu Asn 155 Ile Asp Met Ser Gln Phe Val Glu Leu 140 Arg Thr Gly Pro His Ser Gln Leu Arg Arg Leu Pro Asp Lys Glu 110 Asp Phe 125 Asp Arg Tyr Leu Asp Ser Leu Lys Leu Pro Met Gln Trp Val Arg Tyr Ile His Asn His Ala Lys Ala Thr 160 WO 99/53948 Gly His Asn Ala Giu Arg Gly Leu Ser 195 Val Giu Tyr 210 Thr Leu Asn 225 Gly Gin Lys Cys Asp Asn Glu Glu Leu 275 Ala Val 180 Pro Ile Gin Ile Tyr 260 Giy Phe 165 Ser Giu Ser Asp Arg 245 Phe Giy Ser Gly Al a Ala Ala 230 Leu Ser Thr Tyr Glu Gin Asn 215 Leu Ala Thr Val Phe Trp Ile 200 Asp Arg Lys Phe Leu 280 Thr Arg 185 Ser Val Lys Ser Gin 265 Glu Arg 170 Ser Ile Giu Ile Pro 250 His Arg Arg Arg Val Val1 235 Leu As n T yr Cys Asp Val 220 Ser Tyr Val Leu Ile Ile 205 Phe Cys Ser Arg PCTIUS99/08744 Ser Asp 175 Pro Glu Arg Val Giu Asp Lys Ser 240 Asn Vai 255 Ile Thr <210> <211> 861 <212> DNA <213> Chiamydia trachomatis <220> <221> CDS <222> <400> atg tat gtg ata act att tta gca cgt tcc agc aca atg ttc gca caa Met 1 tta Leu gct Aila tta Leu att Ile gga.

Gly Ser cag Gin tgg Trp tgt Cys ggg Gi y ctg Leu Val aag Lys tc Ser 35 cac His cag Pro att Ile Ile a at Asn 20 gta Val att .Ile aat Asn cgt Arg ga Al a Thr 5 tgg Trp gag Giu act Thr gga Gly gct Al a cat His Ile gag Giu gat Asp ttt Phe gat Ala 70 t ct Ser cat His Leu Ala gga ctg Gly Leu ctt tgt Leu Cys 40 tct atg Ser Leu 55 ggt aaa Gly Lys tct ggc Ser Gly aga ata Arg Ile Arg tta Phe 25 gtt Val act Pro agt Ser caa Gin gc Al a Ser 10 ttg Leu aat Asn gca Al a act Thr agc Ser 90 tat Tyr Ser aat As n tat T yr ggg Gly ttg Leu 75 ttg Leu atg Met Thr aga Arg gat Asp gca Ala 60 att Leu ttc Phe act Pro Met gat Asp a His 45 atg Met aag Lys ttt Phe caa Gin Phe aat Asn 30 ta Ser gat Ala gat Al a ggt Gly aga Arg Al a g ca Ala gac Asp gat Ala tat Ser cag Gin gag Al a Gin att Ile gt a Val1 att Ile tt a Leu aga Arg agt Ser 48 96 144 192 240 288 336 ttt tac aag Phe Ser Lys WO 99/53948 gtg gat tgg Val Asp Trp 115 PCT/US99/08744 gat ttc cca atg Asp Phe Pro Met act Thr 120 gtt ctt gat ctc Val Leu Asp Leu gtg Val 125 ttg atg ggg Leu Met Giy tgt tac Cys Tyr 130 ggc tat aaa gga Giy Tyr Lys Gly ata Ile 135 tgg aat cgt att Trp Asn Arg Ile t cc Ser 140 act gat gat cgt Thr Asp Asp Arg cag Gin 145 gag gct atg cgt Giu Aia Met Arg att Ile 150 tta gag cgg gtt Leu Giu Arg Val ttg gaa gat ttt Leu Giu Ala Phe 432 480 528 aat cgt caa ata Asn Arg Gin Ile ggt Gly 165 aag ctc tct gga Lys Leu Ser Giy caa caa cag aga Gln Gin Gin Arg gct ttt Ala Phe 175 tta gcg cgg Leu Ala Arg ctg ttc tct Leu Phe Ser 195 tca Ser 180 tta atg caa aaa Leu Met Gin Lys gca Aila 185 gat ttg tat ctc Asp Leu Tyr Leu atg gat gag Met Asp Glu 190 gta gat gtt Val Asp Vai 576 624 gag atc gat atg Aia Ile Asp Met tct tat cag atg Ser Tyr Gin Met gtt Val 205 ttg caa Leu Gin 210 gag ctt aaa agc Giu Leu Lys Ser ggg aag act att Giy Lys Thr Ile gt g Vai 220 gtc att cat cat Val Ile His His gat Asp 225 ttg agt aat gta Leu Ser Asn Val cgg Arg 230 aag att ttt gat Lys Leu Phe Asp gtg att tta tta Val Ile Leu Leu aag cat ctt gtg Lys His Leu Vai tga dys 245 tat gga agc gta Ser Giy Ser Vai gaa Giu 250 gaa tga ttg act Giu Cys Leu Thr aaa gaa Lys Giu 255 gac att ttt Aia Ile Phe tca aat tgt Ser Asn Cys 275 cag Gin 260 gat tat ggg tgt Aia Tyr Giy Cys gac Asp 265 ttg aga ttt tgg Leu Ser Phe Trp att aca cac Ile Thr His 270 816 861 ata gag gca agt Leu Giu Ala Ser aca Thr 280 aag gat agt gat Lys Asp Arg Ala aga tgc tga Arg Cys 285 <210> 6 <211> 286 <212> PRT <213> Chiamydia trachoinatis <400> 6 Met Ser Val Ile 1 Leu Gin Lys Asn Ala Trp Ser Val Thr Ile Leu Ala Arg 5 Ser Thr Met Phe Ala Gin Trp Giu Gly Leu Phe 25 Leu Asn Arg Asp Asn Ala Ile Ser Asp Val Giu Asp Leu Cys Vai Asn Tyr Asp 40 His WO 99/53948 PCT/US99/08744 Ile Thr Phe Ser Leu Pro Ala Gly Ala Met Ala Ala Ile 55 Leu Ile Gly Phe Val Cys Gin 145 Asn Leu Leu Leu Asp 225 Lys Al a Ser Cys Giy Leu Ser Asp Tyr 130 Giu Arg Ala Phe Gin 210 Leu His Ile Asn Asn Arg Al a 100 Asp Tyr Met Ile Ser 180 Al a Leu Asn Val Gin 260 Ala 70 Ser His Pro Gly Ile 150 Lys Met Asp Ser Arg 230 Ser Tyr Gly Ser Arg Met Ile 135 Leu Leu Gin Met Giu 215 Lys Gly Gly Lys Gly Ile Thr 120 Trp Glu Ser Lys Ala 200 Gly Leu Ser Cys Ser Gin Ala 105 Val Asn Arg Gly Al a 185 Ser Lys Phe Vai Asp 265 Thr Ser Tyr Leu Arg Val1 Gly 170 Asp Tyr Thr Asp Giu 250 Leu Leu 75 Leu Met Asp Ile Gly 155 Gin Leu Gin Ile His 235 Glu Ser Leu Phe Pro Leu Ser 140 Leu Gin Tyr Met Val 220 Val Cys Phe Lys Phe Gin Val 125 Thr Glu Gin Leu Val 205 Vai Ile Leu Trp Al a Gly Arg 110 Leu Asp Ala Arg Met 190 Val Ile Leu Thr Ile 270 Ser Gin Al a Met Asp Phe Al a 175 Asp Asp His Leu Lys 255 Thr Leu Arg Ser Gly Arg Al a 160 Phe Glu Val1 His Asn 240 Glu His Leu Glu Ala Ser Thr Lys Asp Arg Ala Arg Cys <210> 7 <211> 1068 <212> DNA <213> Chiamydia psittaci <220> <221> CDS <222> (1068) <400> 7 atg aca gta tcc aca gac aac aca agt cct gta ata tcg aga gcg tcc 48 Met Thr Val Ser Thr Asp Asn Thr Ser Pro Val Ile Ser Arg Ala Ser 1 5 10 7 WO 99/53948 tca cct act Ser Pro Thr ata ccc att Ile Pro Ile PCT/US99/08744 gga gat cat ggt aag gat ttc gac Gly Asp His Gly Lys Asp Phe Asp aac aat aaa att Asn Asn Lys Ile tca ata gaa gct Ser Ile Glu Ala cca Pro 40 act tct tca gct Thr Ser Ser Ala gct Ala gct gta ggg Ala Val Giy gct aaa Ala Lys acg gct atc gag Thr Ala Ile Giu gaa gga aga agc Glu Gly Arg Ser cca Pro cta ctt caa agg Leu Leu Gin Arg att Ile tgc tat ctt gtt Cys Tyr Leu Val aaa Lys att atc gct gcc Ile Ile Ala Ala gcc ctc ttt gtt Ala Leu Phe Val ggt atc gca gcc Gly Ile Ala Ala gtt tgc tta tat Val Cys Leu Tyr ct c Leu ggt agc gtt atc Gly Ser Val Ile tca acg Ser Thr cct tct ctt Pro Ser Leu gtt att acg Val Ile Thr 115 at t Ile 100 ctt atg ctt gcg Leu Met Leu Ala atg ctt gta tcc Met Leu Val Ser ttt gtg atc Phe Vai Ile 110 gtc cgt cac Val Arg His 336 384 gca att cga gat Aia Ile Arg Asp ggc Gly 120 aca ccg tct caa Thr Pro Ser Gin gtg Val 125 atg aaa Met Lys 130 cag caa att cag Gin Gin Ile Gin ttt ggc gaa gaa Phe Gly Giu Glu aac Asn 140 acg cgt tta cat Thr Arg Leu His acc Thr 145 gca gta gaa aat Ala Val Giu Asn aaa gct gtt aac Lys Ala Val Asn gt t Val 155 gag ctc tca gag Giu Leu Ser Giu caa Gin 160 432 480 528 att aac caa ctt Ile Asn Gin Leu aaa Lys 165 caa cta cat act Gin Leu His Thr aga Arg 170 tta tcg gat ttt Leu Ser Asp Phe ggt gat Gly Asp 175 agg ctt gaa Arg Leu Giu caa ctc agt Gin Leu Ser 195 aat acc ggt gat Asn Thr Gly Asp ttt Phe 185 act gca ctt att Thr Ala Leu Ile gcg gat ttc Ala Asp Phe 190 gtt gaa acc Val Giu Thr ctg gaa gag ttt Leu Giu Giu Phe aag Lys 200 tct gtt ggt act Ser Vai Gly Thr aaa Lys 205 atg ctc Met Leu 210 tct cca ttt gag Ser Pro Phe Giu tta gct cag tct.

Leu Ala Gin Ser aaa gag acc ttt Lys Glu Thr Phe tct Ser 225 caa gaa gct gtt Gin Giu Ala Val cag Gin 230 gca atg atg tcc Ala Met Met Ser t ct Ser 235 gta act gag tta Val Thr Glu Leu acc aat ttg aat Thr Asn Leu Asn gca Al a 245 ttg aaa gag ctt Leu Lys Glu Leu at a Ile 250 aca. gag aat aaa Thr Glu Asn Lys acc gta Thr Vai 255 ata gag caa cta aaa gct gat gct caa ctt aga gaa gag caa gtg cgg 81 816 WO 99/53948 Ile Glu Gin ttt tta gaa Phe Leu Glu 275 tcc cat tca Ser His Ser 290 tct aca act Ser Thr Thr 305 gtt cag gat Val Gin Asp gat agc gcg Asp Ser Ala aaa gat agt Lys Asp Ser 355 Lys cgt Arg gcg Al a tta Leu gca Al a 325 ccc Pro Ala Asp aaa caa Lys Gin act cta Thr Leu 295 cat gca His Ala 310 gag tcc Giu Ser caa gat Gin Asp Leu qaa Giu tcc Ser agt Ser gta Val 330 caa Gin Arg gag Glu aca Thr cgt Arg 315 gag Giu t ct Ser Giu gct Ala acc Thr 300 ctt Leu gaa Glu gat Asp Glu tgt Cys 285 ctt Leu atc Ile gct Al a gct Al a Gin 270 tca Ser ct a Leu ggt Gly tca Ser gga Gly 350 PCT/US99/08744 Val Arg aca ttg 864 Thr Leu aag gac 912 Lys Asp gtt atg 960 Val Met 320 caa gat 1008 Gin Asp 335 gag cat 1056 Glu His gzz gaa Val Glu tcc acc Ser Thr gaa aat Giu Asn 345 1068 <210> 8 <211> 355 <212> PRT <213> Chiamydia <400> 8 Met Thr Val Ser 1 Ser Pro Thr Phe Ile Pro Ile Ser Ala Lys Thr Ala Ile Cys Tyr Leu Gly Ile Ala Ala Pro Ser Leu Ile 100 Val Ile Thr Ala 115 Met Lys Gin Gin 130 psittaci Thr Asp Asn 5 Giy Asp His Ile Glu Ala Ile Glu Pro 55 Val Lys Ile 70 Leu Val Cys Leu Met Leu Ile Arg Asp Ile Gin Gin 135 Thr Gly Pro 40 Giu Ile Leu Al a Gly 120 Phe Ser Lys 25 Thr Gly Al a Tyr Ile 105 Thr Gly Pro 10 Asp Ser Arg Ala Leu 90 Met Pro Giu Ile Asp Al a Pro Ala Ser Val Gin Asn 140 Ser Asn Al a Leu Leu Vai Ser Vai 125 Thr Arg Asn Aia Leu Phe Ile Phe 110 Val Arg Ala Lys Val1 Gin Val Ser Val1 Arg Leu Ser Ile Gly Arg Val1 Thr Ile His His WO 99/53948 Thr Ala Val 145 Ile Asn Gin Arg Leu Giu Gin Leu Ser 195 Met Leu Ser 210 Ser Gin Giu 225 Thr Asn Leu Ile Giu Gin Phe Leu Giu 275 Ser His Ser 290 Ser Thr Thr 305 Val Gin Asp Asp Ser Aia Lys Asp Ser 355 Giu Leu Al a 180 Leu Pro Ala Asn Leu 260 Lys Ile Asn Gly Gin 340 Leu 150 Gin Thr Giu Glu Gin 230 Leu Al a Lys Thr His 310 Giu Gin Lys Leu Giy Phe Lys 215 Ala Lys Asp Gin Leu 295 Al a Ser Asp Val Thr Phe 185 Ser Al a Met Leu Gin 265 Leu Giu Giu Thr Asn 345 Glu Ser Leu Thr Leu 220 Val Glu Glu Al a Thr 300 Leu Glu Asp Leu Asp Ile Lys 205 Lys Thr Asn Glu Cys 285 Leu Ile Al a Ala Ser Phe Al a 190 Val Glu Giu Lys Gin 270 Ser Leu Gly Ser Gly 350 PCTIUS99/08744 Giu Gin 160 Gly Asp 175 Asp Phe Glu Thr Thr Phe Leu Arg 240 Thr Val 255 Val Arg Thr Leu Lys Asp Val Met 320 Gin Asp 335 Giu His <210> 9 <211> 597 <212> DNA <213> Chiamydia psittaci <220> <221> CDS <222> <400> 9 atg tca aca aca cca gca. tct tca gca agt cga gao gta tta tta gat Met Ser Thr Thr Pro Ala Ser Ser Ala Ser Arg Asp Val Leu Leu Asp 1 5 10 gac gtt tta ata gct ttt. aat aga aag cta. aat ctc gta qaa caa caa Asp Val Leu Ile Ala Phe Asn Arg Lys Leu Asn Leu Val Giu Gin Gin 25 WO 99/53948 PCT/US99/08744 gcg aaa gaa ctt gaa acg aaa gtc agt ttg gta gac aga aca gct act Ala Lys tta tca Leu Ser aaa gat Lys Asp cta tta Leu Leu cca gaa Pro Glu gct aaa Ala Lys aca gta Thr Val 130 ttt att Phe Ile 145 ccg atc Pro Ile tct att Ser Ile aat ttt Asn Phe Giu ctt Leu gag Glu atc Ile Gly ctt Leu 115 ctt Leu tcc Ser att Ile aac As n caa Gin 195 Leu Giu Thr Lys Val Ser Leu Val Asp Arg Thr Ala Thr acc act Thr Thr gtt gca Val Ala cgc tca Arg Ser act aat Thr Asn 100 aca gcg Thr Ala gtt atc Val Ile cta ctc Leu Leu tcg tgt Ser Cys 165 tct tta Ser Leu 180 ggC Gly gaa Giu 70 ggc Gly tac Tyr tta Leu tgt Cys aac Asn 150 gcc Ala tta Leu aat Asn 55 cta Leu tca Ser ct a Leu gcg Al a att Ile 135 atg Met gca Al a aga Arg aat Asn aaa Lys t ca Ser ata Ile tta Leu 120 gtt Val tac Tyr gt t Val aac Asn gta Val1 gga Giy aga Arg gga Gly 105 aca Thr act Thr aca Thr tca Ser gcc Al a tgt Cys aca Thr 90 tgc Cys at t Ile gt t Val1 gtt Val atg Met 170 acg Thr ttg Leu 75 cct Pro a ca Thr ata Ile t gc Cys ggt Gly 155 atg Met gat Asp t ct Ser 9gg Gly cct Pro gcc Ala ggc Gly 140 gct Ala att Ile gta ctc Vai Leu gca gtt Ala Val ggt gct Gly Ala cct tct Pro Ser 110 ctc att Leu Ile 125 ggt ttc Gly Phe tgt ata Cys Ile cta tgc Leu Cys ctt tta Leu Leu acg gat Thr Asp cct aat Pro Asn ctt tgc Leu Cys gct atc Ala Ile ccc cta Pro Leu tcc tta Ser Leu 160 tca cat Ser His 175 144 192 240 288 336 384 432 480 528 576 agg cct gcg atc tat atg act aac Arg 185 Pro Ala Ile Tyr Met Thr Asn 190 aca gaa tct Thr Glu Ser 597 <210> <211> 198 <212> PRT <213> Chiamydia <400> Met Ser Thr Thr 1 Asp Val Leu Ile Ala Lys Giu Leu Leu Ser Leu Thr psittaci Pro Ala Ser Ser Ala Ser Arg Asp Val Leu Leu Asp 5 10 Ala Phe Asn Arg Lys Leu Asn Leu Val Glu Gin Gin 25 Giu Thr Lys Val Ser Leu Val Asp Arg Thr Ala Thr 40 Thr Gly Asn Asn Vai Ala Thr Asp Val Leu Leu Leu 11 WO 99/53948 PCT/US99/08744 55 Lys Asp Glu Val Ala Glu Leu Lys Gly Cys Leu Ser Ala Val Thr Asp 70 75 Leu Leu Ile Arg Ser Gly Ser Ser Arg Thr Pro Gly Gly Ala Pro Asn 90 Pro Glu Gly Thr Asn Tyr Leu Ile Gly Cys Thr Pro Pro Ser Leu Cys 100 105 110 Ala Lys Leu Thr Ala Leu Ala Leu Thr Ile Ile Ala Leu Ile Ala Ile 115 120 125 Thr Val Leu Val Ile Cys Ile Val Thr Val Cys Gly Gly Phe Pro Leu 130 135 140 Phe Ile Ser Leu Leu Asn Met Tyr Thr Val Gly Ala Cys Ile Ser Leu 145 150 155 160 Pro Ile Ile Ser Cys Ala Ala Val Ser Met Met Ile Leu Cys Ser His 165 170 175 Ser Ile Asn Ser Leu Leu Arg Asn Arg Pro Ala Ile Tyr Met Thr Asn 180 185 190 Asn Phe Gln Thr Glu Ser 195 <210> 11 <211> 561 <212> DNA <213> Chlamydia psittaci <220> <221> CDS <222> <400> 11 atg acc tct gta aga acc gat tta act cca ggc gac acc tca ctc caa 48 Met Thr Ser Val Arg Thr Asp Leu Thr Pro Gly Asp Thr Ser Leu Gin 1 5 10 tct tct tta tta aat ccg agt gat ctc aca aca caa cta tcc aac ctc 96 Ser Ser Leu Leu Asn Pro Ser Asp Leu Thr Thr Gln Leu Ser Asn Leu 25 cag act gtt ctc gca ggg ata caa caa caa cat cct tta aac ggt ggt 144 Gln Thr Val Leu Ala Gly Ile Gln Gln Gln His Pro Leu Asn Gly Gly 40 tgg cct cag cat cat cct act ggc gct gca gat caa aat tat ctc atg 192 Trp Pro Gln His His Pro Thr Gly Ala Ala Asp Gln Asn Tyr Leu Met 55 cgt ctg atg caa tct cat atg gca agt acc gta tca gca gta tct gaa 240 Arg Leu Met Gln Ser His Met Ala Ser Thr Val Ser Ala Val Ser Glu 70 75 tta aga acc gaa gtc act gca atc aag aca aaa ttg cac ggg cta tct 288 12 WO 99/53948 Leu Arg Thr act cca got Thr Pro Ala cta got ata Leu Ala Ile 115 ggc ctt gca Gly Leu Ala 130 gca aac tct Ala Asn Ser 145 tgc tta att Cys Leu Ile tta cot att Leu Pro Ile Glu aat Asn 100 t ct Ser ggc Gi y ata Ile ago Ser gaa Giu 180 Val gtt Val1 tta Leu ata Ile t gg T rp gt g Val 165 acot Thr Thr Ala tgo ago Cys Ser gtt gog Vai Ala ota cot Leu Pro 135 got att Ala Ile 150 ota tgo Leu Cys agg oot Arg Pro Lys oct Pro 105 ato Ile got Al a ago Ser acg Thr Thr 90 atg Met at o Ile gc Al a got Al a ot a Leu 170 oat His Leu ota Leu gtt Vai ato Ile 140 ata Ile oga Arg His gc Al a tta Leu 125 tta Leu gto Val cac His PCT/US99/08744 Leu Ser ttt ott 336 Phe Leu too tta 384 Ser Leu aat aca 432 Asn Thr gtt ato 480 Val Ile 160 aaa 000 528 Lys Pro 175 aco gga Thr Gly 185 <210> 12 <211> 186 <212> PRT <213> Chiamydia <400> 12 Met Thr Ser Vai 1 Ser Ser Leu Leu Gin Thr Vai Leu Trp Pro Gin His Arg Leu Met Gin Leu Arg Thr Glu Thr Pro Ala Asn 100 Leu Ala Ile Ser 115 Gly Leu Ala Gly 130 psittaci Arg Thr Asp Leu 5 Asn Pro Ser Asp Ala Gly Ile Gin 40 His Pro Thr Gly 55 Ser His Met Ala 70 Val Thr Ala Ile Val Cys Ser Gly Leu Val Ala Ile 120 Ile Leu Pro Gin 135 Thr Leu 25 Gin Ala Ser Lys Pro 105 Ile Ala Pro 10 Thr Gin Ala Thr Thr 90 Met Ile Al a Gly Thr His Asp Val 75 Lys Ala Ile Ala Asp Gin Pro Gin Ser Leu Leu Val Ile 140 Thr Leu Leu Asn Ala His Al a Leu 125 Leu Ser Ser Asn Tyr Val Gly Al a 110 Ala Val Leu Asn Gly Leu Ser Leu Phe Ser Asn Gin Leu Gi y Met Glu Ser Leu Leu Thr WO 99/53948 PCT/US99/08744 Ala Asn Ser Ile Trp Ala Ile Val Ser Ala Ser Ile Val Thr Val Ile 145 150 155 160 Cys Leu Ile Ser Val Leu Cys Ile Thr Leu Ile Arg His His Lys Pro 165 170 175 Leu Pro Ile Glu Thr Arg Pro Thr Gly His 180 185 <210> 13 <211> 822 <212> DNA <213> Chlamydia trachomatis <220> <221> CDS <222> <400> 13 atg aca acg cct act cta atc gtg att cct cca tct ccc cct gca cct 48 Met Thr Thr Pro Thr Leu Ile Val Ile Pro Pro Ser Pro Pro Ala Pro 1 5 10 tcc tac tca gcc aat cgc gta cct caa cct tct ttg atg gac aaa att 96 Ser Tyr Ser Ala Asn Arg Val Pro Gln Pro Ser Leu Met Asp Lys Ile 25 aag aaa ata gca gcc att gcc tcc cta att ctt ata ggc aca ata ggc 144 Lys Lys Ile Ala Ala Ile Ala Ser Leu Ile Leu Ile Gly Thr Ile Gly 40 ttt tta gct ctt ttg gga cat ctt gtt ggc ttt ctg atc gct cca caa 192 Phe Leu Ala Leu Leu Gly His Leu Val Gly Phe Leu Ile Ala Pro Gln 55 atc act att gtt ctt ctt gcc cta ttc att acc tca tta gca ggg aat 240 Ile Thr Ile Val Leu Leu Ala Leu Phe Ile Thr Ser Leu Ala Gly Asn 70 75 gct ctt tat cta cag aaa acc gct aat cta cat cta tac cag gat ctg 288 Ala Leu Tyr Leu Gln Lys Thr Ala Asn Leu His Leu Tyr Gln Asp Leu 90 caa aga gaa gtt ggg tct cta aaa gaa att aat ttc atg ctg agc gtt 336 Gln Arg Glu Val Gly Ser Leu Lys Glu Ile Asn Phe Met Leu Ser Val 100 105 110 cta cag aaa gaa ttt ctt cat tta tct aaa gaa ttt gca acg aca tct 384 Leu Gln Lys Glu Phe Leu His Leu Ser Lys Glu Phe Ala Thr Thr Ser 115 120 125 aaa gac ctc tct gct gta tct caa gat ttt tat tct tgt ttg caa gga 432 Lys Asp Leu Ser Ala Val Ser Gln Asp Phe Tyr Ser Cys Leu Gln Gly 130 135 140 ttt aga gat aac tat aaa ggt ttt gaa tct ctt ttg gat gag tat aaa 480 Phe Arg Asp Asn Tyr Lys Gly Phe Glu Ser Leu Leu Asp Glu Tyr Lys 145 150 155 160 aac tct aca gaa gaa atg cgc aaa ctc ttt tcg caa gaa atc ata gca 528 14 WO 99/53948 Asn Ser Thr Giu gat ctt aaa ggc Asp Leu Lys Gly 180 acc cca tta gca Thr Pro Leu Ala 195 tta aca gcg gct Leu Thr Ala Ala 210 gat gaa att gga Asp Glu Ile Gly 225 att gca tta caa Ile Ala Leu Gln gag acg ctc tcc Glu Thr Leu Ser 260 tcc tag Ser <210> 14 <211> 273 <212> PRT <213> Chiamydia <400> 14 Met Thr Thr Pro 1 Ser Tyr Ser Ala Lys Lys Ile Ala Phe Leu Ala Leu Ile Thr Ile Val Ala Leo Tyr Leo Gin Arg Glu Val 100 Leo Gin Lys Giu 115 Glu 165 t ct Ser gaa Glu att Ile caa Gin cga Arg 245 tcc Ser Arg Lys Leo gcc tca tta Ala Ser Leu 185 gtt cgc cga Val Arg Arg 200 gaa tta aaa Gbu Leo Lys 215 tca caa ctt Ser Gin Leu gag agc tca Glu Ser Ser aga aag tct Arg Lys Ser 265 Phe 170 aga Arg tta Leo aca Thr tct Ser gat Asp 250 gca Ala Ser gag Glu gcg Al a att Ile aaa Lys 235 ctg Leo tca Ser Gin gaa Glu cat His cgt Arg 220 act Thr t gt Cys ccc Pro Glu atc Ile aac As n 205 gat Asp ctt Leu tcc Ser tct Ser Ile cga Arg 190 ca g Gin agc Ser acc Thr cag Gin aca Thr 270 PCTJUS99/08744 Ala cta 576 Leo tca 624 LSer cga Arg caa Gin 240 aga Arg agc Ser 672 720 768 816 822 trachomatis Thr Le Ile 5 Asn Arg Val Ala Ile Ala Leo Gly His 55 Leu Leu Ala 70 Gin Lys Thr Gly Ser Leo Phe Leo His Val Pro Ser 40 Leo Leu Ala Lys Leu 120 Pro 10 Pro Ile Gly Ile Leo 90 Ile Lys Pro Ser Leo Phe Thr 75 His Asn Glu Ser Leo Ile Leu Ser Leo Phe Phe Pro Met Gly Ile Leo Tyr Met Ala 125 Al a Lys Ile Pro Gly Asp Ser Thr Pro Ile Gly Gin Asn Leo Val1 Ser WO 99/53948 Lys Asp Leu Ser Ala Val Ser 130 135 Phe Arg Asp Asn Tyr Lys Gly 145 150 Asn Ser Thr Giu Giu Met Arg 165 Asp Leu Lys Giy Ser Val Ala 180 Thr Pro Leu Ala Glu Giu Val 195 Leu Thr Ala Ala Ile Giu Glu 210 215 Asp Giu Ile Gly Gin Leu Ser 225 230 Ile Ala Leu Gin Arg Lys Glu 245 Glu Thr Leu Ser Ser Pro Arg 260 Ser <210> <211> 348 <212> DNA <213> Chiamydia trachomatis <220> <221> CDS <222> (348) <400> atq gtt cat tct gta. tac aat Met Val His Ser Val Tyr Asn 1 5 gtc tct att caa ccc agt cag Val Ser Ile Gin Pro Ser Gin gcg ata atg act ctt ttt gca.

Ala Ile Met Thr Leu Phe Ala tcc atc gtt aca gtt tgt gga.

Ser Ile Val Thr Val Cys Gly 55 aac acc gta act att ggt gca Asn Thr Val Thr Ile Gly Ala 70 ata gct aca acg tta tta ctt Gin Phe Lys Ser Arg 200 Leu Gin Ser Lys tca Ser att Ile ctc Leu 40 ggg Giy tgc Cys ctt Asp Giu Leu Leu 185 Arg Lys Leu Ser Ser 265 ttg Leu cca Pro 25 aca.

Thr ttt Phe gta Val tgt Phe Ser Phe 170 Arg Leu Thr Ser Asp 250 Al a gct Al a 10 acc Thr gcc Ala cct Pro tcc Ser ctc 16 Tyr Leu 155 Ser Giu Al a Ile Lys 235 Leu Ser Ser 140 Leu Gin Giu His Arg 220 Thr Cys Pro Cys Asp Giu Ile As n 205 Asp Leu Ser Ser Leu Giu Ile Arg 190 Gin Ser Thr Gin Thr 270 ttt Phe gta Val1 at a Ile gct Al a ttc Phe gaa Gir Tyi IiE 17~ PhE G1i Lei Se Il 25! Ly ag Se: 1 at Mei gt Va gc Al.

a c Th: ct.

PCTJUS99/08744 ~Gly Lys 160 SLeu .i Ser .i Arg SGin 240 SArg s Ser cca gaa. ggt Pro Glu Giy agc aaa. aaa Ser Lys Lys att gca. gca Ile Ala Ala ttt ctt ctt Phe Leu Leu ttg ccg gta Leu Pro Val cgt aat atc 9 1 t r c 48 96 144 192 240 288 WO 99/53948 PCTIUS99/08744 Ile Ala Thr Thr Leu Leu Leu Leu Cys Leu Arg Asn Ile Giu Leu Leu 90 gcc aga ccg caa gta ttt acc ctc tcc act caa ttc aqc cca aca aaa 336 Ala Arg Pro Gin Val Phe Thr Leu Ser Thr Gin Phe Ser Pro Thr Lys 100 105 110 cct caa gaa tag 348 Pro Gin Giu 115 <210> 16 <211> 115 <212> PRT <213> Chiarnydia <400> 16 Met Val His Ser 1 Val Ser Ile Gin Ala Ile Met Thr Ser Ile Val Thr Asn Thr Val Thr Ile Ala Thr Thr Ala Arg Pro Gin 100 Pro Gin Giu 115 trachomatis Vai Tyr Asn 5 Pro Ser Gin Leu Phe Ala Val Cys Gly 55 Ile Gly Ala 70 Leu Leu Leu Val Phe Thr Ser Ile Leu 40 Gly Cys Leu Leu Leu Pro Thr Phe Val1 Cys Ser 105 Pro Ser Ile Phe Leu Arg Gin Glu Lys Al a Leu Pro Asn Phe Gly Lys Al a Leu Val Ile Ser Ser Met Val1 Ala Thr Leu Thr <210> 17 <211> 537 <212> DNA <213> Chiamydia trachomatis <220> <221> CDS <222> (537) <400> 17 atg acg tac tct ata too gat ata qca cac aaa tct gat att tct aat Met Thr Tyr Ser Ile Ser Asp Ile Ala His Lys Ser Asp Ile Ser Asn 1 5 10 ccc acg tct ccc gct oca tca aga aaa cga gga tcc ttt ccc cca caa Pro Thr Ser Pro Ala Pro Ser Arg Lys Arg Gly Ser Phe Pro Pro Gin 25 WO 99/53948 tct cct tct gcc Ser Pro Ser Ala PCTIUS99/08744 gga gct aat ttc tct aca tgg 144 Giy Ala Asn Phe Ser Thr Trp gtg ggc tct tta gag Val Giy Ser Leu Giu ggg cca Gly Pro gca atg Ala Met aag aaa Lys Lys ggc ccg Gly Pro gta gct Val Ala ttt gtc Phe Val 130 gtg agc Val Ser 145 ggC Gly caa Gin aaa Lys ca g Gin gtt Val 115 ct t Leu gcc Ala ccc Pro aac As n tta Leu ttt Phe 100 atc Ile gt t Val tcc Ser ttc Phe aac Asn gt t Val1 tta Leu gta Val1 t gt Cys atc Ile ttc Phe ct t Leu 70 cag Gin gog Ala ctt Leu ctg Leu act Thr 150 act Thr 55 ttt Phe t ct Ser gca Ala gct Ala gct Al a 135 aca Thr gt c Val a ca Thr agt Ser t gc Cys tcc Ser 120 ggg Giy ct g Leu cct Pro ttg Leu cag Gin tta Leu 105 tta Leu tca Ser att Ile gtt Val1 caa Gin a ca Thr 90 gtt Vai gga Giy act Thr tgt Cys tat T yr aca Thr 75 cgc Arg gct Al a ctt Leu aat As n tgc Cys 155 cca Pro 60 gag Giu gga Giy gcg Al a ggc Gly gca Ala 140 gtt Val1 caa caa Gin Gin gtt tct Val Ser tct tta Ser Leu aca atc Thr Ile 110 ggt gtt Gly Val 125 att tgg Ile Trp tcc atc Ser Ile ctc gct Leu Ala gct ctc Ala Leu gga ctc Gly Leu ctt gca Leu Ala ctt cct Leu Pro gct att Ala Ile gct tgc Ala Cys 160 192 240 288 336 384 432 480 atc ttc tta gca aaa tgt gat aag Ile Phe Leu Ala Lys 165 gta agc taa Val Ser <210> 18 <211> 178 <212> PRT <213> Chiamydia tra <400> 18 Met Thr Tyr Ser Ile 1 5 Pro Thr Ser Pro Ala Ser Pro Ser Ala Val Gly Pro Gly Pro Phe Ala Met Gin Asn Asn Cys Asp Lys chomat is Ser Asp Pro Ser Gly Ser Phe Thr 55 Leu Phe 70 gga Giy Ala Lys 25 Giu Pro Leu tct gat cct caa act tta tat Ser Asp Pro Gin Thr Leu Tyr Ile Arg Leu 40 Val Thr H-is 10 Arg Gly Val Gin Lys Gly Ala Tyr Thr 75 Ser Ser Asn Pro Glu Asp Phe Phe Gin Val Ile Pro Ser Gin Ser Ser Pro Thr Leu Ala As n Gin Trp Ala Leu WO 99/53948 Lys Lys Lys Leu Val Gln Ser Ser Gln Thr Arg Gly Ser I Gly Pro Gln Phe Leu Ala Ala Cys Leu Val Ala Ala Thr 100 105 Val Ala Val Ile Val Leu Ala Ser Leu Gly Leu Gly Gly 115 120 125 Phe Val Leu Val Cys Leu Ala Gly Ser Thr Asn Ala Ile 130 135 140 Val Ser Ala Ser Ile Thr Thr Leu Ile Cys Cys Val Ser 145 150 155 Ile Phe Leu Ala Lys Cys Asp Lys Gly Ser Asp Pro Gln 165 170 Val Ser <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 19 agaaccgatt taactccagg cg <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> gcgcggatcc ttaatgtccg gtaggcctag <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 21 atgtcaacaa caccagcatc ttc <210> 22 <211> 34 <212> DNA <213> Artificial Sequence PCT/US99/08744 Leu i Ala i Pro Ile I Cys 160 1 Tyr 22 23 WO 99/53948 <220> <223> Description of Artificial Sequence: PCR primer <400> 22 gcgcggatcc ttaattagtg ccttctggat tagg <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 23 cgcagtactg tatccacaga caac <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 24 gtcggatccg agaaactctc catgcc PCT/US99/08744 34 24 26

Claims (27)

1. A vaccine preparation comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

2. The vaccine preparation of claim 1 wherein the at least one peptide comprises at least 10 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:14.

3. The vaccine preparation of claim 1 wherein the at least one peptide comprises at least 15 contiguous amino acid of the amino acid sequences set forth in SEQ ID NO:8 or SEQ ID NO:14.

4. The vaccine preparation of claim 1 wherein the at least one peptide comprises at least 20 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:14. A vaccine preparation comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8, or SEQ ID NO:14.

6. A method of preparing a vaccine comprising combining a pharmaceutically acceptable excipient with at least one purified peptide having an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

7. A method of vaccination, comprising administering a vaccine preparation according to any one of claims 1 to 5 to a mammal.

8. The method of claim 7, wherein the mammal is a human.

9. A composition for inducing an immune response in a subject, comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

10. The composition of claim 9 wherein the sequence of the at least one peptide comprises at least 10 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:14.

11. The composition of claim 9 wherein the sequence of the at least one peptide comprises at least 15 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:14.

12. The composition of claim 9 wherein the sequence of the at least one peptide comprises at least 20 contiguous amino acids of the amino acid sequence set forth in SEQ SID NO:8 or SEQ ID NO:14. [R:\LIBVV]02759.doc:ais

13. A composition for inducing an immune response comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

14. A method of making a composition for inducing an immune response comprising combining a pharmaceutically acceptable excipient with at least one purified peptide having an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14. A method of inducing an immune response in a subject, comprising administering a composition according to any one of claims 9 to 13 to a mammal.

16. The method of claim 15 wherein the mammal is a human.

17. A vaccine preparation comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 when used to vaccinate a mammal.

18. A vaccine preparation comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8, or SEQ ID NO:14 when used to vaccinate a mammal.

19. A composition when used for inducing an immune response in a subject, said composition comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14. A composition when used for inducing an immune response in a subject, said composition comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14.

21. Use of at least one purified peptide comprising at least 5 contiguous amino S acids of amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 for the manufacture of a vaccine preparation for use in vaccinating a mammal.

22. Use of at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 for the manufacture of a vaccine preparation for use in vaccinating a mammal.

23. Use of a composition comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 for the manufacture of a medicament for inducing an immune response in a subject.

24. Use of a composition comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 for the manufacture of a medicament for inducing an immune response in a subject. [R:\LIBVV]02759.doc:ais A vaccine preparation comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 substantially as hereinbefore described with reference to any one of the examples.

26. A vaccine preparation comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8, or SEQ ID NO:14 substantially as hereinbefore described with reference to any one of the examples.

27. A method of preparing a vaccine comprising combining a pharmaceutically acceptable excipient with at least one purified peptide having an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 substantially as hereinbefore described with reference to any one of the examples.

28. A composition for inducing an immune response in a subject, comprising at least one purified peptide comprising at least 5 contiguous amino acids of an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 substantially as hereinbefore described with reference to any one of the examples.

29. A composition for including an immune response comprising at least one purified peptide comprising an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 substantially as hereinbefore described with reference to any one of the examples.

30. A method of making a composition for inducing an immune response comprising combining a pharmaceutically acceptable excipient with at least one purified peptide having an amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:14 substantially as hereinbefore described with reference to any one of the examples.

31. A method of inducing an immune response in a subject, comprising administering a composition according to any one of claims 9 to 13 to a mammal substantially as hereinbefore described with reference to any one of the examples. Dated 30 August, 2002 The State of Oregon, acting by and through The State Board of Higher Education, on behalf of Oregon State University Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\LIBVV]02759.doc:ais