Failure to detect Chlamydia trachomatis in cell culture by using a monoclonal antibody directed against the major outer membrane protein.

Two commercially available monoclonal antibodies for cell culture confirmation of Chlamydia trachomatis were compared in two prospective studies and one large retrospective study. In total, more than 33,000 genital specimens were cultured in parallel and stained with both antibodies, one of which was directed against the major outer membrane protein (MOMP) and one of which was directed against the lipopolysaccharide (LPS). We found the anti-LPS-based assay to be more sensitive and as specific as the anti-MOMP-based assay for C. trachomatis cell culture confirmation of genital specimens.     

Mimicry of a neutralizing epitope of the major outer membrane protein of Chlamydia trachomatis by anti-idiotypic antibodies.

The major outer membrane protein (MOMP) is a primary target antigen for the development of chlamydial vaccine. This protein is composed of four variable domains (I to IV) flanked by constant regions. Some of the variable domains contain antigenic determinants that elicit a neutralizing antibody response. Murine monoclonal antibodies (MAbs) against three nonoverlapping epitopes of MOMP were developed. One of these, called DP10, bound to all serovars, as shown by immunoblot analysis, and neutralized chlamydial infectivity for hamster kidney (HaK) cells in a complement-independent in vitro assay. Furthermore, analysis of the fine specificity of this MAb showed that it recognized a synthetic peptide contained within variable domain IV of the MOMP. Anti-idiotypic antibodies (aId) directed against this anti-MOMP MAb were produced in rabbits. These aId specifically bound to the relevant idiotype (DP10) and inhibited the binding of anti-MOMP MAb (DP10) to MOMP preparations in a dose-dependent fashion. The specificity of our aId for the binding site of anti-MOMP MAb is further suggested by the binding inhibition of affinity-purified aId to DP10 by the synthetic peptide defined by the idiotype. In addition, these aId also reacted with anti-MOMP antisera from rats and mice, suggesting an idiotypic cross-reactivity between these species. Finally, immunization of naive mice with aId induced an antibody response directed against the peptide defined by our anti-MOMP MAb and with neutralizing activity. Taken together, these data suggest that aId mimic a neutralization site on MOMP and could serve as a surrogate antigen to induce protective immunity against Chlamydia trachomatis.     

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.     

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.     

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.     

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.     

Characterization of the humoral response induced by a peptide corresponding to variable domain IV of the major outer membrane protein of Chlamydia trachomatis serovar E.

A 30-amino-acid peptide corresponding to variable domain IV (VD IV) of the major outer membrane protein of Chlamydia trachomatis serovar E was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize mice. The resulting antisera (anti-KLH-VD IV sera) recognized all 15 serovars of C. trachomatis when assayed by indirect immunofluorescence and Western blotting. Probing of overlapping hexameric peptides representing VD IV with mouse anti-KLH-VD IV sera revealed that two main regions of the peptide were recognized by the antisera, the N terminus of the peptide, which contains B-complex-specific epitopes, and the middle region of the peptide, which contains a species-conserved domain. When used in an in vitro neutralization assay, these antisera were able to neutralize mainly serovars in the B complex. These data provide evidence that a linear peptide corresponding to VD IV can induce in vitro protection from C. trachomatis infectivity that is subspecies specific.     

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.     

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.     

Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis.

During studies in which we serotyped large numbers of Chlamydia trachomatis clinical isolates by using monoclonal antibodies, three novel serological variants (D-, D*, and I-) were identified. To determine the molecular basis for the altered monoclonal antibody reactions of these strains and other previously identified variants (Da, Ia, and L2a), we determined the nucleotide sequences of the variable domains in their major outer membrane protein genes. Da, D-, and D* variants differed by a single nucleotide and also an amino acid in the carboxy terminus of variable domain IV (VDIV) from the D serovar. The L2a variant also differed from L2 by a single amino acid but in VDII. Ia variants differed in VDI, III, and IV and I- variants differed in all four VDs from the I serovar. These studies demonstrate the potential for using major outer membrane protein VD sequencing as a highly sensitive typing method and further identify immunologically reactive major outer membrane protein epitopes.     

In vitro neutralization of Chlamydia trachomatis by monovalent Fab antibody specific to the major outer membrane protein.

Monovalent Fab antibodies to serovar- and subspecies-specific epitopes of the major outer membrane protein (MOMP) of Chlamydia trachomatis neutralized infectivity for hamster kidney cells by preventing chlamydial attachment. These findings exclude the aggregation of chlamydiae as a mechanism of anti-MOMP neutralization and provide additional evidence in support of the MOMP as a chlamydial adhesin.     

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.     

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.     

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.     

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.     

Characterization of the murine antibody response to peptides representing the variable domains of the major outer membrane protein of Chlamydia pneumoniae.

In an attempt to gain more knowledge about the immunogenicity of the variable domains (VDs) of the major outer membrane protein (MOMP) of Chlamydia pneumoniae, peptides representing these areas were used to immunize BALB/c and C57BL/6 mice. Antisera to the peptides and to peptides conjugated to keyhole limpet hemocyanin (KLH) were characterized by their ability to recognize the immunizing peptide and elementary bodies (EBs) of C. pneumoniae by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot). In addition, antiserum was analyzed for its molecular specificity by a pepscan as well as its in vitro neutralizing ability. In general, results obtained with antisera to the peptides paralleled the results obtained with the antisera to the KLH-conjugated peptides except that the titers or strength of reaction in the assays was less. Antisera to the VDs in both strains of mice gave ELISA titers to the homologous VD peptide ranging from 1,000 to >64,000. The strength of reactivity with the reduced MOMP as judged by Western blot, in most cases, paralleled the ELISA titer to the peptide. However, only antisera raised in both strains of mice to the VD1 and VD4 peptides reacted strongly with the EBs, suggesting surface exposure of these VDs. In addition, antisera to VD3 from C57BL/6 mice gave strong reactivity to EBs. By pepscan analysis antisera from both strains of mice reacted with several VD1 and VD3 octameric peptides, with weaker reactivity being seen with the octameric peptides in the other two VDs. This was in contrast to antisera raised to EBs of C. pneumoniae TW-183, which identified two immunogenic regions, one in VD1 and the other mapped to VD4. While antisera raised to EBs strongly neutralized the infectivity of C. pneumoniae, none of the peptide antisera was able to neutralize. In addition, peptides to the VDs were not able to block the neutralizing ability of the antisera to EBs of C. pneumoniae. Therefore, these results suggest that the VDs of the MOMP of C. pneumoniae are surface exposed but do not elicit neutralizing antibodies when linear peptides representing them are used as the immunogen.     

Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars.

The amino acid sequences of major outer membrane proteins (MOMPs) from Chlamydia trachomatis serovars A, B, C, L1, and L2 are predominantly conserved but have four variable domains (VDs) in which major neutralizing and serotyping antigenic determinants are located. Because these MOMP VDs are primarily responsible for antigenic differences between serovars and are associated with important immunological and biological properties, we undertook studies focused on defining these sequences within the MOMPs of all 15 C. trachomatis serovars. We used oligonucleotide primer extension sequencing of MOMP mRNA to determine the nucleotide and deduced amino acid sequences of the four MOMP VDs of the 15 C. trachomatis serovars. Comparative amino acid sequence homologies of all four domains separated the serovars into three groups: group 1, serovars B, Ba, D, E, L1, and L2; group 2, serovars G and F; and group 3, serovars A, C, H, I, J, K, and L3. Hydrophilicity and charge values for each domain were determined. The MOMP VDs of given serovars with the greatest total hydrophilicity and charge values were found to be the location of antigenic determinants recognized by MOMP-specific monoclonal antibodies. These findings should be useful for predicting MOMP antigenic determinants and testing the antigenic properties of these VDs by using synthetic peptides corresponding to each MOMP VD. The potential usefulness of the VD sequence information is discussed in relation to the development of defined synthetic peptides and oligonucleotides that may be used to develop new serological and diagnostic assays for C. trachomatis infections.     

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.     

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.