Determination of neutralizing epitopes in variable domains I and IV of the major outer-membrane protein from Chlamydia trachomatis serovar K

Abstract

Chlamydia trachomatis is a leading cause of sexually transmitted diseases and a number of strategies have been developed to produce vaccines to prevent its transmission. The purpose of this study was to map the neutralizing epitopes of C. trachomatis major outer-membrane protein (MOMP) serovar K by using anti-MOMP antibodies and synthetic peptides. Seven anti-MOMP monoclonal antibodies and three polyclonal antisera were produced and characterized. Their fine specificity was defined by direct binding assay on 15 peptides of 10 amino acid residues, overlapping by five residues, corresponding to the four variable domains (VDI-VDIV : residues 64-85, 139-160, 224-237 and 287-319) of MOMP serovar K. Our data confirmed that a neutralizing epitope is found in VDIV, defined by peptides K12 and K13. This epitope is 296rTLNPTIAG304, which has never been reported as a neutralizing epitope of serovar K. Another neutralizing epitope, defined by peptide K2, has been identified in VDI. This epitope is in the same position as 71VAGLEK76, a peptide with neutralizing activity found in serovar A, but they are not identical because antibodies against peptide K2 do not bind to this epitope. No neutralizing epitope was found in the two other variable domains (VDII and 111). In summary, two neutralizing sites, one in variable domain I and one in variable domain IV, were identified in serovar K.

Defining Species-Specific Immunodominant B Cell Epitopes for Molecular Serology of Chlamydia Species

“…Thus, the probability of high-affinity binding is proportional to the length of a peptide antigen. These recent data suggest that previous studies failed to achieve high sensitivity (23-25, 43, 44), which is most likely due to weak antibody binding to the short peptides used (32)(33)(34)(35)(36) and to steric hindrance of antibody binding to these peptides (45).…”

“…Based on these epitope mapping studies, synthetic OmpA peptides were tested for Chlamydia species-specific serology (23-25, 37, 38). However, these studies used peptides as short as 6 to 10 amino acids in length and did not use spacers between solid support and peptide to minimize steric hindrance of antibody binding (32)(33)(34)(35)(36). Recent computational studies of antigen-antibody complex 3D structures showed that 15-to 25-amino-acid (aa) residues of an epitope are structurally involved in antibody binding (39)(40)(41)(42).…”

“…Chlamydia genus-, species-, subspecies-, and serotype-specific B cell epitopes have been mapped before to the four variable domains of the outer membrane protein A (OmpA) by use of monoclonal antibodies (26,27), recombinant protein fragments (28)(29)(30)(31), and synthetic peptides (32)(33)(34)(35)(36). Based on these epitope mapping studies, synthetic OmpA peptides were tested for Chlamydia species-specific serology (23-25, 37, 38).…”

Prediction of the membrane‐spanning β‐strands of the major outer membrane protein of Chlamydia

“…Already in the original paper by Schirmer and Cowan (1993), it was shown that the Hs' algorithm can produce false positives. Immunological studies and immunoelectronmicroscopy, for example, have shown that mice inoculated with Chlamydia produce antibodies against all four VDs of the MOMP (Kuo and Chi 1987;Baehr et al 1988;Baghian et al 1990;Andersen 1991;Peterson et al 1991;Pal et al 1993Pal et al , 1996Pal et al , 1997aPal et al ,b, 2000Qu et al 1993;Villeneuve et al 1994). Like with Neisseria gonorrhoeae only the long loops L2, L3, L5, and L6 of C. trachomatis and C. psittaci have VDs probably reflecting the fact that they are more immunoaccessible (van der Ley et al 1991).…”

Mutagenesis and Functional Reconstitution of Chlamydial Major Outer Membrane Proteins: VS4 Domains Are Not Required for Pore Formation but Modify Channel Function

“…Bilayers were formed by drawing a dispersion of diphytanoyl phosphatidylcholine (Avanti) in n-decane (Sigma) across a 0.3-mm hole in a polystyrene partition separating two solution-filled chambers (35,36,38). The contents of both chambers were stirred (using stirbars) as required, and all the bilayers used had a final capacitance of 200 to 300 pF and a conductance of 5 to 10 pS.…”