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

Abstract

bUrgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16-to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens.

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction

“…Hyperimmune Sera-Hyperimmune sera were raised in mice as described previously (2). Briefly, 9 -50 mice were challenged three times with high but non-lethal intranasal chlamydial inocula, to mimic antibody production following natural infections.…”

“…Bovine sera used were obtained from animals with PCRconfirmed natural Chlamydia spp. infection (2). Peptide antigens were chemically synthesized with N-terminal biotin, captured onto streptavidin-coated microtiter plates, and incubated with hyperimmune sera.…”

“…This underperformance prompted us to examine current approaches to B-cell epitope prediction by use of extensive data on epitopes and confirmed non-epitope regions of the Chlamydia spp. proteome, accumulated in research on chlamydial molecular serology (2).…”

“…Recent three-dimensional antibody-antigen complex studies (3)(4)(5)(6)(7) show that about 15-22-aa 2 antigen peptide residues are structurally involved in binding of epitopes to ϳ17-aa residues in antibody complementarity-determining regions (CDRs; paratopes). Among these 15-22 structural epitope residues, about 2-5 aa, termed functional residues, contribute most of the total binding energy to antibodies (6).…”

“…(2). After encountering numerous failures of in silico B-cell epitope prediction, we used the first principles established above to analyze the shortcomings of B-cell prediction methodology.…”