Discovery of Human-Specific Immunodominant Chlamydia trachomatis B Cell Epitopes

Abstract

Current serological assays for species-specific detection of anti-Chlamydia species antibodies suffer from well-known shortcomings in specificity and ease of use. Due to the high prevalences of both anti-C. trachomatis and anti-C. pneumoniae antibodies in human populations, species-specific serology is unreliable. Therefore, novel specific and simple assays for chlamydial serology are urgently needed. Conventional antigens are problematic due to extensive cross-reactivity within Chlamydia spp. Using accurate B cell epitope prediction and a robust peptide ELISA methodology developed in our laboratory, we identified immunodominant C. trachomatis B cell epitopes by screening performed with sera from C. trachomatis-infected women. We discovered 38 novel human host-dependent antigens from 20 immunodominant C. trachomatis proteins, in addition to confirming 10 host-independent mouse serum peptide antigens that had been identified previously. This extended set of highly specific C. trachomatis peptide antigens can be used in simple ELISA or multiplexed microarray formats and will provide high specificity and sensitivity to human C. trachomatis serodiagnosis.

Mixed Chlamydia trachomatis Peptide Antigens Provide a Specific and Sensitive Single-Well Colorimetric Enzyme-Linked Immunosorbent Assay for Detection of Human Anti-C. trachomatis Antibodies

“… a Only the actual C. trachomatis serovar D strain D/UW-3/Cx peptide antigen sequences are shown, without N-terminal biotin and serine-glycine-serine-glycine spacer that is attached to each peptide ( 16 ). b Reactivity scores are log 2 weighted averages of seven reactivities of total and subpooled sera as described previously ( 20 ). c Reactivity with only human C. trachomatis -specific immune sera (yes) or also with mouse anti- C. trachomatis hyperimmune mouse sera (no) is shown ( 16 , 20 ).…”

“… b Reactivity scores are log 2 weighted averages of seven reactivities of total and subpooled sera as described previously ( 20 ). c Reactivity with only human C. trachomatis -specific immune sera (yes) or also with mouse anti- C. trachomatis hyperimmune mouse sera (no) is shown ( 16 , 20 ). d Average percent amino acid sequence identity with 22 C. trachomatis strains representing all major C. trachomatis clades or 5 C. pneumoniae strains representing all major C. pneumoniae clades is shown ( 20 ).…”

“…An important finding was that 16- to 30-amino-acid peptide antigens combined with an N-terminal hydrophilic serine-glycine-serine-glycine spacer were required for optimal signal strength ( 16 – 18 ). Further testing of predicted peptide antigens with sera from C. trachomatis - infected women revealed additional human host-specific C. trachomatis B-cell epitopes that were recognized only by the natural human host ( 20 ) but did not react with sera from mice that were hyperimmunized to C. trachomatis ( 16 ). Subsequently, we comprehensively evaluated the utility of 11 top-ranked peptide antigens from 8 C. trachomatis proteins for use in human C. trachomatis serology ( 21 ).…”

“…Importantly, the high sensitivity of the peptide assays was achieved mainly by the use of multiple B-cell epitopes of several C. trachomatis immunodominant proteins, including OmpA ( 21 ), compared to exclusive OmpA antigens used in commercial ELISAs. Given the stochasticity of antibody responses to individual B-cell epitopes ( 16 , 20 , 21 ), only the combined use of multiple peptide antigens can reliably measure host antibodies produced in response to C. trachomatis infection ( 21 ), similar to the quantitative results obtained with complex antigens. However, this requires single serum testing of individual peptide antigens in 11 separate microtiter wells ( 21 ).…”

“…In the present study, we established a simple colorimetric ELISA using an antigen mixture of 12 strongly reactive C. trachomatis -specific peptide antigens ( 20 ), requiring per serum only single-well signal detection of anti- C. trachomatis antibodies. For test evaluation, we used sera from 125 women with NAAT-confirmed active C. trachomatis infection with high prevalence of anti- C. trachomatis antibodies ( 20 , 21 ) and 87 negative-control sera. Importantly, this simple colorimetric ELISA outperformed all four commercial ELISAs for detection of anti- C. trachomatis IgG antibodies, with the advantage of higher sensitivity and specificity.…”