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Discovery of Human-Specific Immunodominant Chlamydia trachomatis B Cell Epitopes - PubMed

  • ️Mon Jan 01 2018

Discovery of Human-Specific Immunodominant Chlamydia trachomatis B Cell Epitopes

K Shamsur Rahman et al. mSphere. 2018.

Abstract

Chlamydia species-specific serology is compromised by cross-reactivity of the gold standard microimmunofluorescence (MIF) or commercial enzyme-linked immunosorbent assays (ELISAs). This study was conducted to discover novel C. trachomatis-specific peptide antigens that were recognized only by the antibody response of the natural human host. We evaluated a library of 271 peptide antigens from immunodominant C. trachomatis proteins by reactivity with 125 C. trachomatis antibody-positive sera from women with PCR-confirmed C. trachomatis infection and 17 C. trachomatis antibody-negative sera from low-risk women never diagnosed with C. trachomatis infection. These C. trachomatis peptide antigens had been predicted in silico to contain B cell epitopes but had been nonreactive with mouse hyperimmune sera against C. trachomatis We discovered 38 novel human host-dependent antigens from 20 immunodominant C. trachomatis proteins (PmpD, IncE, IncG, CT529, CT618, CT442, TarP, CT143, CT813, CT795, CT223, PmpC, CT875, CT579, LcrE, IncA, CT226, CT694, Hsp60, and pGP3). Using these human sera, we also confirmed 10 C. trachomatis B cell epitopes from 6 immunodominant C. trachomatis proteins (OmpA, PmpD, IncE, IncG, CT529, and CT618) as host species-independent epitopes that had been previously identified by their reactivity with mouse hyperimmune sera against C. trachomatis ELISA reactivities against these peptides correlated strongly with the C. trachomatis microimmunofluorescence (MIF) text results (Pearson's correlation coefficient [R] = 0.80; P < 10-6). These C. trachomatis peptide antigens do not cross-react with antibodies against other Chlamydia species and are therefore suitable for species-specific detection of antibodies against C. trachomatis This study identified an extended set of peptide antigens for simple C. trachomatis-specific ELISA serology.IMPORTANCE 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.

Keywords: B cell epitopes; Chlamydia pneumoniae; Chlamydia trachomatis; ELISA; cross-reactivity; diagnosis; microimmunofluorescence; peptide antigens; serology; serovar; species-specific.

Copyright © 2018 Rahman et al.

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Figures

FIG 1
FIG 1

Reactivities of 60 peptide antigens from 11 Chlamydia species with Chlamydia species-specific mouse sera. Each peptide was ELISA tested with 11 pools of 9 to 50 hyperimmune mouse sera obtained by 3× intranasal inoculation with live inocula of a single chlamydial species (39). Green cells represent the reactivity of peptide antigens with their corresponding homologous antiserum pools. Red cells indicate peptide antigen cross-reactivity with nonhomologous antisera (ELISA signals > background + 2 standard deviations [SD]). Green and red color intensities indicate signal strength, and white cells indicate nonreactivity. Peptide designations consist of three-letter Chlamydia species acronyms (defined in the headings of columns 3 to 13) followed by strain, source protein, and the amino acid positions of the peptide in the protein. RLU indicates relative light units per second.

FIG 2
FIG 2

Reactivities of the 60 mouse antiserum-reactive chlamydial peptide antigens with C. trachomatis-positive (Ctr-Pos) and -negative (Ctr-Neg) human serum pools. The reactivity of each of the 60 Chlamydia species-specific peptide antigens (Fig. 1) was tested with the human serum pools. The C. trachomatis-positive pool consisted of sera from 125 women with C. trachomatis infection, and the C. trachomatis-negative pool consisted of sera from 17 women never diagnosed with C. trachomatis infection who were EB ELISA negative for anti-C. trachomatis antibodies. Polyclonal anti-human IgG HRP conjugate was used for detection of bound total IgG, and monoclonal antibody conjugates were used to detect bound long-lived IgG1 or short-lived IgG3 isotypes. Peptide reactivities are shown in Log2 RLU signal bars. Different colors are used for the chlamydial species for convenient visualization.

FIG 3
FIG 3

Anti-C. trachomatis MIF titer-dependent IgG reaction intensity of the 60 chlamydial peptide antigens. C. trachomatis-positive sera from 108 women with known anti-C. trachomatis microimmunofluorescence (MIF) test results were combined by MIF titer into 4 subpools of 19 to 35 sera. Reactivities of the 4 subpools, indicated by color intensity, with peptides from all 11 chlamydial species (Fig. 1) are shown in Log2 RLU IgG signal bars on the scale as described for Fig. 2. All 10 C. trachomatis peptides showed a signal above background, and the signals correlated highly significantly with MIF titers (R = 0.80; P < 10−6). Three C. pneumoniae peptides and six peptides from other Chlamydia species showed a signal above background that did not correlate with MIF titers (P = 0.78).

FIG 4
FIG 4

Host-dependent C. trachomatis and C. pneumoniae peptide antigens specifically reactive with human C. trachomatis-positive sera but not with immune mouse sera. A library of 271 C. trachomatis and 153 C. pneumoniae peptide antigens was screened with the C. trachomatis-positive and -negative human serum pools. These peptides had previously been nonreactive in screens performed with hyperimmune monospecies-specific anti-C. trachomatis and anti-C. pneumoniae mouse sera (39). A total of 100 peptides showed reactivity above background, and the top-ranked 38 C. trachomatis and 8 C. pneumoniae peptide antigens derived from immunodominant proteins are shown. Signal intensities are shown in Log2 RLU bars as described for Fig. 2.

FIG 5
FIG 5

Anti-C. trachomatis MIF titer-dependent IgG reactivity of C. trachomatis but not C. pneumoniae peptide antigens. Thirty-eight C. trachomatis and 8 C. pneumoniae peptide human host-dependent peptide antigens (Fig. 4) were probed with C. trachomatis-positive sera pooled by MIF titer (Fig. 3). Signals are shown in Log2 RLU IgG signal bars on the scale as described for Fig. 2. The signals of the C. trachomatis peptides correlated strongly with anti-C. trachomatis MIF titers (R = 0.79; P < 10−6), but those of the C. pneumoniae peptides did not (P > 0.61).

FIG 6
FIG 6

IgG1 and IgG3 antibodies against C. trachomatis and C. pneumoniae peptides in 48 individual sera. Sera from 48 C. trachomatis-positive women that covered the spectrum of C. trachomatis and C. pneumoniae MIF titers (rows 2 and 3), as well as the racial origin of the study subject, were selected. A total of 56 unique C. trachomatis and C. pneumoniae peptide antigens with potential utility for human population surveys were tested with these individual sera. These peptides are divided into 6 sets based on C. trachomatis and C. pneumoniae species, serovar specificity, source proteins, reaction frequency, and the immunoglobulin subclasses detected. The host-independent peptides shown in black font had previously reacted with mouse anti-C. trachomatis sera (39), while the host-dependent peptides shown in red font were not recognized by mouse sera but showed reactivity in this study with human sera. Blue font indicates eight non-serovar D peptides from OmpA variable domains 1 and 2. The column following the peptide designations shows C. trachomatis or C. pneumoniae specificity. Individual sera are numerically identified in the top row and are arranged in order of their average level of IgG1 reactivity with the first set of 11 C. trachomatis peptides. Peptide reactivities with the individual 48 sera are indicated by color intensities (legend at bottom), and absence of reactivity is shown by white cells.

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