The GTPase Rab4 interacts with Chlamydia trachomatis inclusion membrane protein CT229

Chlamydiae, which are obligate intracellular bacteria, replicate in a nonlysosomal vacuole, termed an inclusion. Although neither the host nor the chlamydial proteins that mediate the intracellular trafficking of the inclusion have been clearly identified, several enhanced green fluorescent protein (GFP)-tagged Rab GTPases, including Rab4A, are recruited to chlamydial inclusions. GFP-Rab4A associates with inclusions in a species-independent fashion by 2 h postinfection by mechanisms that have not yet been elucidated. To test whether chlamydial inclusion membrane proteins (Incs) recruit Rab4 to the inclusion, we screened a collection of chlamydial Incs for their ability to interact with Rab4A by using a yeast two-hybrid assay. From our analysis, we identified a specific interaction between Rab4A and Chlamydia trachomatis Inc CT229, which is expressed during the initial stages of infection. CT229 interacts with only wild-type Rab4A and the constitutively active GTPase-deficient Rab4AQ67L but not with the dominant-negative GDP-restricted Rab4AS22N mutant. To confirm the interaction between CT229 and Rab4A, we demonstrated that DsRed-CT229 colocalized with GFP-Rab4A in HeLa cells and more importantly wild-type and constitutively active GFP-Rab4A colocalized with CT229 at the inclusion membrane in C. trachomatis serovar L2-infected HeLa cells. Taken together, these data suggest that CT229 interacts with and recruits Rab4A to the inclusion membrane and therefore may play a role in regulating the intracellular trafficking or fusogenicity of the chlamydial inclusion.     

Hypothetical protein Cpn0308 is localized in the Chlamydia pneumoniae inclusion membrane

The hypothetical protein encoded by Chlamydia pneumoniae open reading frame cpn0308 was detected in inclusion membranes of C. pneumoniae-infected cells using antibodies raised with Cpn0308 fusion proteins. The anti-Cpn0308 antibodies did not cross-react with IncA, a known C. pneumoniae inclusion membrane protein, although the anti-Cpn0308 antibody staining overlapped with the anti-IncA antibody labeling. The labeling of the inclusion membrane by the anti-Cpn0308 antibody was specifically blocked by the Cpn0308 but not IncA fusion proteins. The Cpn0308 antigen was detectable 24 h after infection and remained in the inclusion membrane throughout the infection course.     

Localization of Chlamydia trachomatis hypothetical protein CT311 in host cell cytoplasm

The chlamydia-specific hypothetical protein CT311 was detected both inside and outside of the chlamydial inclusions in Chlamydia trachomatis-infected cells. The extra-inclusion CT311 molecules were distributed in the host cell cytoplasm with a pattern similar to that of CPAF, a known Chlamydia-secreted protease. The detection of CT311 was specific since the anti-CT311 antibody labeling was only removed by absorption with CT311 but not CPAF fusion proteins. In addition, both anti-CT311 and anti-CPAF antibodies only detected their corresponding endogenous proteins without cross-reacting with each other or any other antigens in the whole cell lysates of C. trachomatis-infected cells. Although both CT311 and CPAF proteins were first detected 12 h after infection, localization of CT311 into host cell cytosol was delayed until 24 h while CPAF secretion into host cell cytosol was already obvious by 18 h after infection. The host cell cytosolic localization of CT311 was further confirmed in human primary cells. CT311 was predicted to contain an N-terminal secretion signal sequence and the CT311 signal sequence directed secretion of PhoA into bacterial periplasmic region in a heterologous assay system, suggesting that a sec-dependent pathway may play a role in the secretion of CT311 into host cell cytosol. This hypothesis is further supported by the observation that secretion of CT311 in Chlamydia-infected cells was blocked by a C16 compound known to inhibit signal peptidase I. These findings have provided important molecular information for further understanding the C. trachomatis pathogenic mechanisms.     

Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.     

Localization of the hypothetical protein Cpn0585 in the inclusion membrane of Chlamydia pneumoniae-infected cells

Cpn0585, encoded by a hypothetical open reading frame in Chlamydia pneumoniae genome, was detected in the inclusion membrane during C. pneumoniae infection using both polyclonal and monoclonal antibodies raised with Cpn0585 fusion protein. The anti-Cpn0585 antibodies specifically recognized the endogenous Cpn0585 without cross-reacting with IncA (a known inclusion membrane protein of C. pneumoniae) or other control antigens. A homologue of Cpn0585 in the C. caviae species (encoded by the ORF CCA00156) was also localized in the inclusion membrane of the C. caviae-infected cells. The Cpn0585 protein became detectable 24h while CCA00156 as early as 8h after infection. Once expressed, both proteins remained in the inclusion membrane throughout the rest of infection course.     

A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic.

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines.     

Evidence that the major outer membrane protein of Chlamydia trachomatis is glycosylated.

The major outer membrane protein (MOMP) of Chlamydia trachomatis was determined to be a glycoprotein on the basis of susceptibility to glycosidase digestion and the presence of carbohydrate by staining and radiolabeling. The MOMP of the serovar L2 organisms was isolated by electroelution from the protein band excised from the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incubation of MOMP with N-glycosidase F, an endoglycosidase that cleaves the N-glycan, and periodate resulted in two new molecular weight species. While MOMP treated with N-glycosidase F showed a lower-molecular-weight mobility, the periodate-treated MOMP increased in molecular weight. Both treatments abolished the ability of the MOMP to bind to HeLa cell components. In the immunoblot, the reactivity to the monoclonal antibody specific against the C. trachomatis species was preserved. The endoglycosidase specific to O-linked glycan, endo-alpha-N-acetylgalactosaminidase, had no visible effect on the isolated MOMP. Carbohydrate was detected in the MOMP by p-phenylenediamine staining of the protein band in the gel following SDS-PAGE. Autoradiograms of proteins of chlamydial organisms metabolically labeled with [3H]galactose or [3H]glucosamine and separated by SDS-PAGE revealed the MOMP band. The isolated MOMP was shown to bind specifically to concanavalin A, wheat germ agglutinin, and Dolichos biflorus agglutinin in the lectin binding assay. No binding was observed with Ulex europaeus agglutinin I, soybean agglutinin, or Ricinus communis agglutinin.     

Immunoaccessible peptide sequences of the major outer membrane protein from Chlamydia trachomatis serovar C.

The antigenicity of the major outer membrane protein of Chlamydia trachomatis serovar C was assessed by using overlapping hexapeptide homologs of serovar C major outer membrane protein and rabbit antisera in a peptide enzyme-linked immunosorbent assay. Five immunogenic sites were found distributed within variable sequences of the protein: four were immunodominant and three were surface exposed on native elementary bodies of serovar C. None was surface exposed on serovars H, I, and J.     

Characterization of fifty putative inclusion membrane proteins encoded in the Chlamydia trachomatis genome

Although the Chlamydia trachomatis genome is predicted to encode 50 inclusion membrane proteins, only 18 have been experimentally localized in the inclusion membrane of C. trachomatis-infected cells. Using fusion proteins and anti-fusion protein antibodies, we have systematically evaluated all 50 putative inclusion membrane proteins for their localization in the infected cells, distribution patterns, and effects on subsequent chlamydial infection when expressed ectopically, as well as their immunogenicity during chlamydial infection in humans. Twenty-two of the 50 proteins were localized in the inclusion membrane, and 7 were detected inside the inclusions, while the location of the remaining 21 was not defined. Four (CT225, CT228, CT358, and CT440) of the 22 inclusion membrane-localized proteins were visualized in the inclusion membrane of Chlamydia-infected cells for the first time in the current study. The seven intra-inclusion-localized proteins were confirmed to be chlamydial organism proteins in a Western blot assay. Further characterization of the 50 proteins revealed that neither colocalization with host cell endoplasmic reticulum nor inhibition of subsequent chlamydial infection by ectopically expressed proteins correlated with the inclusion membrane localization. Interestingly, antibodies from women with C. trachomatis urogenital infection preferentially recognized proteins localized in the inclusion membrane, and the immunodominant regions were further mapped to the region predicted to be on the cytoplasmic side of the inclusion membrane. These observations suggest that most of the inclusion membrane-localized proteins are both expressed and immunogenic during C. trachomatis infection in humans and that the cytoplasmic exposure may enhance the immunogenicity.     

Neutralization of Chlamydia trachomatis infectivity with antibodies to the major outer membrane protein.

Rabbit immunoglobulin G (IgG) antibodies raised against the major outer membrane protein of the Chlamydia trachomatis lymphogranuloma venereum strain 434 neutralized the infectivity of the parasite for HeLa 229 cells. The mechanism by which anti-major outer membrane protein IgG prevented C. trachomatis from establishing infection was studied by using intrinsically 14C-radiolabeled elementary bodies. Neutralized elementary bodies were filterable through a polycarbonate filter (pore diameter, 600 nm), demonstrating that reduction in infectivity was not due to the aggregation of elementary bodies by cross-linking IgG. Antibody-neutralized elementary bodies attached to and penetrated HeLa cells at rats nearly identical to those for infectious organisms exposed to nonneutralizing control IgG. These results suggest that antibody interferes with the infectious process of the parasite after its internalization. Anti-major outer membrane protein Fab fragments could not be substituted for neutralizing IgG antibodies. The requirement for intact IgG implies that cross-linking of antibodies to the major outer membrane protein on the surfaces of the organisms may be instrumental in neutralization.     

A Chlamydia trachomatis OmcB C-terminal fragment is released into the host cell cytoplasm and is immunogenic in humans

The Chlamydia trachomatis outer membrane complex protein B (OmcB) is an antigen with diagnostic and vaccine relevance. To further characterize OmcB, we generated antibodies against OmcB C-terminal (OmcBc) and N-terminal (OmcBn) fragments. Surprisingly, the anti-OmcBc antibody detected dominant signals in the host cell cytosol, while the anti-OmcBn antibody exclusively labeled intrainclusion signals in C. trachomatis-infected cells permeabilized with saponin. Western blot analyses revealed that OmcB was partially processed into OmcBc and OmcBn fragments. The processed OmcBc was released into host cell cytosol, while the OmcBn and remaining full-length OmcB were retained within the chlamydial inclusions. The organism-associated OmcB epitopes became detectable only after the C. trachomatis-infected cells were permeabilized with strong detergents such as SDS. However, the harsh permeabilization conditions also led to the leakage of the already secreted OmcBc and chlamydia-secreted protease (CPAF) out of the host cells. The OmcBc processing and release occurred in all biovars of C. trachomatis. Moreover, the released OmcBc but not the retained OmcBn was highly immunogenic in C. trachomatis-infected women, which is consistent with the concept that exposure of chlamydial proteins to host cell cytosol is accompanied by increased immunogenicity. These observations have provided important information for further exploring/optimizing OmcB as a target for the development of diagnosis methods and vaccines.     

The Chlamydia trachomatis parasitophorous vacuolar membrane is not passively permeable to low-molecular-weight compounds.

Chlamydia trachomatis is an obligately intracellular bacterial parasite of eucaryotic cells that undergoes a biphasic life cycle within a parasitophorous vacuole (PV) called an inclusion. The parasitophorous vacuolar membrane (PVM) constitutes a barrier between the replicating bacteria and the nutrient-rich environment of the host cytoplasm. To determine whether the chlamydial PVM contains pores that allow passive diffusion of metabolites between the host cytoplasm and the PV, fluorescent tracer molecules were introduced directly into the cytoplasm of infected cells by transfection or microinjection. Fluorescence microscopy and laser scanning confocal microscopy were subsequently employed to determine whether equilibration of the fluorescent tracers between the cytoplasm and the PV occurred. No movement of tracer molecules as small as 520 Da from the cytoplasm to the PV was observed. These data suggest that the chlamydial PV is not passively permeable to small molecules through open channels in the PVM.     

Expression of the major outer membrane protein of Chlamydia trachomatis in Escherichia coli.

The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein.     

Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis.

Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The soluble extract obtained from SDS-treated COMC was adsorbed to a hydroxylapatite column and eluted with a linear sodium phosphate gradient. The 39,500-dalton protein was eluted from the column as a single peak at a phosphate concentration of approximately 0.3 M. The eluted protein was nearly homogeneous by SDS-PAGE and appeared free of contaminating carbohydrate, glycolipid, and nucleic acid. Hyperimmune mouse antiserum prepared against the 39,500-dalton protein from serotype L2 reacted with C. trachomatis serotypes Ba, E, D, K, L1, L2, and L3 by indirect immunofluorescence with EB but failed to react with serotypes A, B, C, F, G, H, I, and J, with the C. trachomatis mouse pneumonitis strain, or with the C. psittaci feline pneumonitis, guinea pig inclusion conjunctivitis, or 6BC strains. Thus, the 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.     

Mucosal and peripheral immune responses to chlamydial heat shock proteins in women infected with Chlamydia trachomatis

Most of the studies on 60-kDa and 10-kDa chlamydial heat shock proteins (HSPs) to date have been carried out with blood lymphocytes or serum antibody responses, which do not provide a clear picture of the actual pathogenesis as they do not differentiate primary infection from recurrent infection. Thus, in the present study induction of the immune response was evaluated by studying lymphoproliferation of both cervical and peripheral lymphocytes to synthetic peptides of cHSP60, cHSP10 and major outer membrane protein (MOMP) antigen. In addition, cervical antibody prevalence to MOMP antigen, cHSP60 and cHSP10 and cytokine levels in cervical washes was also determined. Positive proliferative responses of cervical lymphocytes to cHSP10 peptide were significantly higher (P < 0.05) in women with recurrent infections and that to MOMP antigen were significantly higher in primary infection. On proliferation of PBMCs with the above antigens, no significant difference was observed between primary and recurrent infection. Prevalence of cervical IgG and IgA antibodies to Chlamydia trachomatis was significantly higher (P < 0.05) during primary infection than recurrent infections. In contrast, prevalence of IgG and IgA antibodies to cHSP10 and IgG antibodies to cHSP60 was higher during recurrent infections than primary infections. Interferon (IFN)-gamma levels were significantly higher in cervical washes of women with recurrent infection and correlated strongly with cHSP60 antibody titres. Our data thus suggest that mucosal responses are more appropriate in understanding the pathogenesis of chlamydial infection and IFN-gamma could be involved in the modulation of immune responses towards chlamydial infection directly, by causing acute inflammation, or indirectly through modulation of HSP expression.     

Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides.

The antigenicity of the major outer membrane protein (MOMP) of Chlamydia trachomatis was comprehensively evaluated by using overlapping hexapeptide homologs of serovar B MOMP and polyclonal rabbit antisera in a peptide enzyme-linked immunosorbent assay. Of 367 hexapeptides, 152 showed reactivities with at least one antiserum. Seven hexapeptides located within variable domain (VD) IV (residues 288 to 316) were found to be most reactive in terms of their binding titer and frequency, suggesting that VD IV is the immunodominant region within the MOMP as detected by this assay. Peptide-reactive antibodies could also recognize corresponding epitopes on either viable or acetone-permeabilized organisms. The antigenic specificity and immunoaccessibility of epitopes located in VD IV were resolved by absorbing antisera with chlamydial elementary bodies. Six antigenic sites were found in this region and included a B-type-specific site (S1), four subserogroup-specific sites (S2 and S4 to 6), and one species-specific site (S3), each displaying varying degrees of surface exposures on elementary bodies from different C. trachomatis serovars.     

Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies.

We prepared monoclonal antibodies against prototype strains of the 15 serovars of Chlamydia trachomatis and identified a subset of reagents that reacted with the major outer membrane protein(s) (MOMPs) of one or more serovars. We then determined the specificities of these anti-MOMP monoclonal antibodies by radioimmunoassay and immunoblot assays against the 15 serovars of C. trachomatis and a C. psittaci strain. We identified 14 different anti-MOMP antibody specificities, including serovar-, several orders of subspecies-, and species-specific determinants. In addition, one antibody reacted with all C. trachomatis serovars and a C. psittaci strain, indicating the presence of a genus-specific epitope on MOMP. Many of the cross-reactions of the subspecies-specific antibodies were similar to those previously reported by use of the microimmunofluorescence technique. We also observed a number of cross-reactions that were unexpected but consistent with data derived by the microimmunofluorescence test. All antibodies, except the genus-specific antibodies, reacted with whole elementary bodies in a radioimmunoassay, suggesting surface exposure of the epitopes. These data confirm and extend previous observations that MOMPs among C. trachomatis serovars are antigenically complex and diverse. In addition, these data indicate that the cross-reaction patterns of some monoclonal antibodies directed against MOMP are similar to those detected by the microimmunofluorescence test and are consistent with the hypothesis that such determinants are contained within MOMPs.     

The 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide is a DnaK-like protein.

The gene coding for the 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide has been cloned in Escherichia coli, and the nucleotide sequence has been determined. The cloned DNA fragment contained the coding region as well as the putative promoter. The deduced amino acid sequence of the 1,980-base-pair open reading frame revealed 94% homology with a 75-kilodalton protein from C. trachomatis serovar D and 57% homology with the DnaK proteins of E. coli and of Bacillus megaterium, while amino acid homology with human heat shock protein 70 (hsp70) was 42%. The promoter region was identified by computer search and by primer extension of mRNA synthesized in recombinant E. coli. The promoter region which differed from the putative promoter region in serovar D was shown to be a mixed promoter type in which the -10 region showed a regular TATA box configuration while the -35 region showed high homology with heat shock promoters. This mixed promoter was recognized in E. coli.     

Primary human T-cell responses to the major outer membrane protein of Chlamydia trachomatis.

The major outer membrane protein (MOMP) of Chlamydia trachomatis is the main candidate antigen for a synthetic vaccine against chlamydial infection. Antibodies to surface-exposed epitopes on MOMP neutralize chlamydial infectivity but little is known about T-cell recognition of the molecule. We have measured primary human T-cell responses to recombinant fragments of MOMP as well as to the whole organism and synthetic MOMP peptides. Using antigen-pulsed low density cells (LDC) we were able to stimulate proliferative responses with T cells from most naive individuals. This response was antigen dose dependent and displayed an absolute requirement for dendritic cells in the antigen-presenting cell (APC) population. Several T-cell epitopes were identified in MOMP and one which stimulated T cells from 80% of donors was resolved as a 12 amino acid synthetic peptide. Dual cell surface labelling and cell cycle analysis by FACS revealed that both CD4+ and CD8+ T cells were stimulated in these cultures. The fact that we were able to obtain proliferative responses and interferon-gamma (IFN-gamma) production to MOMP using cells from cord bloods confirmed that these are genuine primary responses. These experiments have identified a region on MOMP, to which T cells from most humans make a primary response, which may be useful in a chlamydial vaccine. The approach is useful for vaccine development in general.