Antibody recognition of a neutralization epitope on the major outer membrane protein of Chlamydia trachomatis.

Two BALB/c mice were immunized with serovar C Chlamydia trachomatis elementary bodies, and 63 hybridomas producing monoclonal antibodies to C. trachomatis were recovered. Eight hybridomas which were specific for an identical peptide epitope (AGLQND) in serovar C major outer membrane protein variable domain I were identified. Detailed immunochemical study of the antigen-antibody interaction and genetic characterization of the antibody variable-region gene sequences showed that distinct B-cell clonal lineages were elicited by the epitope sequence. Since each antibody had a distinct pattern of fine specificity for recognition of the epitope and displayed different degrees of cross-reactivity with a related serovar (serovar A), we conclude that B-cell recognition of an immunodominant neutralization epitope can be pleiotropic. Differences in B-cell recognition of a neutralization epitope may delay the emergence by mutation of antigenic-drift variants of the C. trachomatis major outer membrane protein.     

Functional and structural mapping of Chlamydia trachomatis species-specific major outer membrane protein epitopes by use of neutralizing monoclonal antibodies.

Three monoclonal antibodies (MAbs), E4, L1-4, and L1-24, to the major outer membrane protein (MOMP) of Chlamydia trachomatis were identified that neutralized in vitro the infectivity of members of the B- and C-related complex as well as the mouse pneumonitis strain. MAbs L1-4, L1-24, and E4 gave a strong signal in an indirect immunofluorescence assay and/or Western immunoblot with all serovars of the lymphogranuloma venereum and trachoma biovars and a weak signal with the mouse biovar. In addition, C. psittaci and C. pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by the technique of pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by by the technique of overlapping peptides, all three MAbs showed reactivity to variable domain (VD) IV of MOMP. While all three MAbs had different recognition patterns, all strongly bound to the peptides TLNPTI and LNPTIA within the species-conserved region of VD IV. MAb E4 also recognized the peptide SATAIF in the subspecies region of VD IV. Peptides corresponding to VD IV of MOMP were synthesized and used in competitive inhibition experiments to determine the functional location of the epitope recognized by these three MAbs. Both the serological and neutralizing activities of MAb E4 were inhibited by the peptides ATAIFDTTTLNPTIAG and FDTTTLNPTIAG; however, none of the peptides made to the VD IV region blocked the neutralizing activity of MAbs L1-4 and L1-24. Therefore, the neutralizable domain of the epitope recognized by MAb E4 is contiguous and may be an important candidate for inclusion in a subunit vaccine.     

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.     

Immunoassay for detecting Chlamydia trachomatis major outer membrane protein.

The development of a solid-phase immunoassay for the detection of the 39,500-dalton major outer membrane protein of the Chlamydia trachomatis lymphogranuloma venereum serotype L2 is described. The test uses immunoadsorbent-purified rabbit anti-L2 major outer membrane protein immunoglobulin G (IgG) passively adsorbed to microtiter plates as a capture antibody. This same IgG antibody was either conjugated to horseradish peroxidase or radioiodinated with 125I and used as a probe to detect major outer membrane protein bound to immobilized IgG. At its greatest sensitivity, the test was capable of detecting 0.5 to 1 ng of purified major outer membrane protein, 5 X 10(3) elementary body inclusion-forming units, and approximately 100 C. trachomatis intracytoplasmic inclusions per assay.     

Chlamydia trachomatis species-specific epitope detected on mouse biovar outer membrane protein.

A common antigenic determinant on the chlamydial major outer membrane protein was detected on each of the three Chlamydia trachomatis biovars (trachoma, lymphogranuloma venereum, and mouse). This determinant was prominently displayed on the surface of chlamydial strains from both the trachoma and lymphogranuloma venereum biovars. However, detection of this determinant on a mouse biovar strain required denaturation by sodium dodecyl sulfate or periodate oxidation. This determinant provides a definable taxonomic link between the three biovars of C. trachomatis.     

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.     

Protective monoclonal antibodies to Chlamydia trachomatis serovar- and serogroup-specific major outer membrane protein determinants.

Monoclonal antibodies exhibiting Chlamydia trachomatis serovar specificity (serovar A, B-Ba, or C) and serogroup specificity (B, intermediate, or C serogroup) were produced and characterized. These antibodies reacted with the major outer membrane protein, recognized epitopes located at the chlamydial cell surface, and passively neutralized chlamydial toxicity for mice. The antibodies should be useful reagents for defining the molecular structure of these protective epitopes, a necessary step toward the development of a subunit or recombinant C. trachomatis vaccine.     

Immunologic characterization of a cloned fragment containing the species-specific epitope from the major outer membrane protein of Chlamydia trachomatis.

A 183-bp fragment encoding variable domain IV (VD IV) of Chlamydia trachomatis serovar B major outer membrane protein (MOMP) (amino acids 273 to 333) and containing the species-specific epitope was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-VD IV). The fusion protein was affinity purified under nondenaturing conditions and used to immunize rabbits. Antisera were characterized by microimmunofluorescence, immunoblot, dot blot, peptide enzyme-linked immunosorbent, and in vitro neutralization assays. Antisera recognized MOMP from all 12 tested serovars of C. trachomatis but not from Chlamydia psittaci. In a dot blot assay, antisera bound to elementary bodies of serovars B, D, E, L2, and K in a strong fashion and to elementary bodies of serovars F, G, A, and H in a weak fashion but not to elementary bodies of serovars C, J, and I. High-resolution peptide mapping with synthetic overlapping serovar B MOMP peptides in a solid-phase enzyme-linked immunosorbent assay showed that immunization with GST-VD IV produced a serologic response that closely mimicked the response produced with purified serovar B elementary bodies. Antipeptide antibodies with strong binding to species- and subspecies-specific epitopes were elicited. Antisera were able to neutralize only those C. trachomatis serovars that bound antibodies in the dot blot assay. These results suggest that antigenic fragments from VD IV containing the species-specific epitope may be useful in the construction of a chlamydial vaccine for some but not all C. trachomatis serovars.     

The major outer membrane protein of a single Chlamydia trachomatis serovar can possess more than one serovar-specific epitope.

The major outer membrane proteins (MOMPs) of human Chlamydia trachomatis serovars exhibit four regions of variable amino acid sequences (VS1 to VS4) harboring serovar-specific B-cell epitopes. Antibody responses to these epitopes may contribute to acquired protection against human chlamydial infection. MOMP B-cell epitopes defined by 22 different serovar-specific or bispecific murine monoclonal antibodies were localized with synthetic peptides representing the four VS regions of seven genital serovars (D, Da, E, F, G, H, and K). Serovar F possessed two distinct serovar-specific epitopes, located in VS2 and VS4, while serovar K possessed three distinct serovar-specific epitopes, located in VS1, VS2, and VS4. Serovar D- and serovar Da-specific epitopes were located in VS1. Regardless of whether the serovar was from the B (serovars D, Da, and E), C (serovars H and K), or F-G (serovars F and G) serogroup, all serovar-specific epitopes were found in three discrete subgroups of MOMPs. These subregions comprised all central portion of VS1, residues 70 to 77; the amino-terminal half of VS2, residues 139 to 149; and the carboxyl-terminal third of VS4, residues 305 to 315. Monoclonal antibodies to each of these subregions neutralized infectivity in standard HaK cell culture assays. These findings are relevant to the development of an MOMP or MOMP subunit vaccine.     

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.     

Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera.

We have identified two families of novel Chlamydia trachomatis isolates with amino acid changes within the major outer membrane protein (MOMP) variable domains: one family of Da, D*, and D- and one family of Ia and I-. In order to determine whether these MOMP variants can escape antibody neutralization of infectivity, we tested both the D and I prototype strains and the variants in a complement-independent in vitro neutralization assay. We found that variants can indeed escape neutralization by both monoclonal antibodies and polyclonal human immune sera that neutralize the prototype strain.     

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.     

In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein.

A murine monoclonal antibody, which binds to an epitope on the major outer membrane protein of Chlamydia trachomatis and with species specificity in the micro-immunofluorescent assay, effectively neutralized in vitro two antigenically distinct serovars of C. trachomatis. Optimal concentrations of both organism and antibody were required to produce maximal neutralization of the organism. Neutralization was less effective and more variable at lower dilutions of antibody than at higher dilutions, suggesting a prozone phenomenon. A radiolabeled attachment assay demonstrated that attachment of elementary bodies was unaffected by earlier treatment with antibody and that neutralization occurred at a step after attachment. The epitope to which this antibody is directed, on the major outer membrane protein of C. trachomatis, may have an important role in determining infectivity of the organism.     

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.     

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.     

Characterization of a neutralizing monoclonal antibody directed at variable domain I of the major outer membrane protein of Chlamydia trachomatis C-complex serovars.

A monoclonal antibody (MAb), C10, that neutralized in vitro the infectivity of serovars C, I, J, and L3 (members of the C and C-related complexes) of Chlamydia trachomatis was identified. Of the 15 major serovars and the mouse pneumonitis strain of C. trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae, which were used as nontreated and heat-treated (56 degrees C, 30 min) antigens in a dot blot assay, only serovars C, I, J, and L3 were recognized with both the native and treated antigens. Western blot (immunoblot) results showed that MAb C10 recognized the major outer membrane protein of these four serovars. Overlapping hexameric peptides corresponding to variable domains (VDs) I, II, III, and IV of the major outer membrane protein of C. trachomatis serovar C were synthesized, and peptide screening showed that MAb C10 mapped to the VD I amino acid sequence VAGLQNDPT. Results of an in vitro neutralization assay correlated with those of the indirect immunofluorescence assay, Western blot, and dot blot assay in that only serovars C, I, J, and L3 were neutralized by MAb C10. In vitro competitive neutralization experiments, using a peptide representing VD I of serovar C to compete with C. trachomatis serovar C for MAb C10 binding, revealed that both serological and neutralizing activities of MAb C10 were inhibited by the VD I peptide. In an in vivo toxicity/infectivity assay using serovar L3 pretreated with MAb C10, there was 100% survival of mice infected with a lethal dose at 48 h. In contrast, the control group, consisting of mice injected with the same dose of L3 pretreated with a MAb that does not recognize L3, had no survivors during a 48-h observation period. In summary, since the surface-exposed contiguous epitope recognized by MAb C10 binds neutralizing antibodies that are subspecies specific for the C and C-related complexes, it should be considered for inclusion in the development of a chlamydial vaccine.     

Typing of Chlamydia trachomatis by restriction endonuclease analysis of the amplified major outer membrane protein gene.

A procedure was developed for characterization of Chlamydia trachomatis strains by using restriction endonuclease analysis of amplified genes of the major outer membrane protein (MOMP). Reference strains of the 15 serovars (A through K and L1 through L3) and clinical isolates were tested. The nucleotide sequences of the MOMP genes of each of the 15 serovars were arbitrarily constructed by using the sequences of the four variable domains known for each serovar and the constant domains of serovar L1. Computer analysis of these sequences indicated that two restriction digestions performed in parallel, one with AluI and the other with IIpaII, followed by HinfI and EcoRI, would allow the theoretical differentiation of 13 serovars. Serovars Ba and L1 presented the same theoretical restriction profile. Our typing method consisted of polymerase chain reaction amplification of a fragment of about 1,200 bp of the MOMP gene, followed by restriction endonuclease digestion with the aforementioned enzymes. From the 15 serovars, we obtained 14 different patterns; 13 profiles were serovar specific, while serovars B and Ba presented the same pattern. Application of this typing method to C. trachomatis strains isolated from clinical material gave the same results as the immunotyping method for 14 of 17 strains. Furthermore, restriction endonuclease analysis detected differences within a serovar. This method seems to be promising for epidemiological studies.     

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.