Xun Cheng

The effect of orientation within a chimeric peptide on the immunogenicity of Chlamydia trachomatis epitopes

Peptides representing the Chlamydia trachomatis major outer membrane protein variable domains (VD) 1 and 4 of serovars C and E, respectively, have been shown to elicit a neutralizing antibody response in mice. To assess whether the position within a chimeric peptide influences the immunogenicity of the epitopes, two constructs, VD 1-4 and VD 4-1, were made in which the position of the VD relative to the amino and carboxy terminals were rotated. C57BL/10 mice were immunized with 100 micrograms of peptide in complete Freunds adjuvant (FA) on day 0, followed by an immunization with peptide (100 micrograms) in complete FA on day 14. By day 21 the immunodominant epitope in both chimeras as measured by ELISA was the one located at the carboxy terminus. A pepscan of the VD 1-4 antisera revealed a main peak in VD 4 which had been previously identified by neutralizing MAbs. The VD 4-1 antisera gave a peak in the VD 1 region which did not correspond to regions previously mapped with neutralizing MAbs. The VD 1-4 antisera but not the VD 4-1 antisera was able to neutralize in vitro serovar E. In summary, the position of these chlamydial epitopes within a chimeric peptide greatly influenced the resulting immune response.

Analysis of the humoral response elicited in mice by a chimeric peptide representing variable segments I and IV of the major outer membrane protein of Chlamydia trachomatis.

A synthetic chimeric peptide representing the variable segments I (VS I) and IV of the major outer membrane protein (MOMP) of Chlamydia trachomatis, serovars C and E respectively, was studied to determine its ability to elicit a neutralizing humoral response in mice. Antibody responses varied to the peptide in the five inbred strains of mice, A/J(H-2a), DBA/1(H-2q), C57BL/10(H-2b), CBA/J(H-2k), Balb/c(H-2d), that were immunized. There was a spectrum of antibody responses which ranged from high ELISA and IFA titres by the C57BL/10 mice to little or no response by Balb/c mice. Antisera from C57BL/10 mice recognized all 15 serovars of C. trachomatis in a dot blot assay. A pepscan of the antisera from C57BL/10 mice showed strong reactivity to both neutralizing epitopes VAGLQNDPT in VS I of serovar C and the species-conserved peptide, TLNPTIA, in the VS IV. This antiserum neutralized, in vitro, the infectivity of serovars representing the B complex (B, D and E), C complex (C and J), B-related (F) and C-related (L3) complexes. In an attempt to elicit a stronger response to the peptide in the weakly reactive Balb/c and the DBA/1 strains, the peptide was conjugated to the carrier, keyhole limpet haemocyanin (KLH). All mice immunized with the KLH-peptide produced high-titred antisera that recognized neutralizing epitopes in VS I and VS IV and strongly neutralized the infectivity of both serovars C and E.

Mapping of a surface-exposed B-cell epitope to the variable sequent 3 of the major outer-membrane protein of Chlamydia trachomatis

A B-cell epitope, AEFPLDIT, was located to the variable sequent 3 of the major outer-membrane protein (MOMP) using the monoclonal antibody L3-1, raised to the Chlamydia trachomatis serovar L3 MOMP. By Western blot and inclusion immunofluorescence assay the monoclonal antibody recognized all the C complex and C-related complex serovars of C. trachomatis, except serovar C. Dot-blot and ELISA data using native elementary bodies indicated that the epitope was surface exposed. The monoclonal antibody, at concentrations of 10 and 100 micrograms per 10(7) chlamydial inclusion-forming units, was able to neutralize the infectivity of chlamydia in an in vivo assay but did not neutralize chlamydia in vitro or in a mouse toxicity assay. A peptide corresponding to the variable sequent 3 has previously been shown to also elicit a T-cell response; thus, careful consideration should be given to inclusion of this region of the major outer-membrane protein in a subunit vaccine.