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

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.

Comprehensive Molecular Serology of Human Chlamydia trachomatis Infections by Peptide Enzyme-Linked Immunosorbent Assays.

Sensitive species-specific detection of anti-Chlamydia trachomatis antibodies is compromised by cross-reactivity of the C. trachomatis antigens used in standard microimmunofluorescence (MIF) testing and enzyme-linked immunosorbent assays (ELISAs). Previously, we discovered 48 strongly reactive C. trachomatis-specific B cell epitope peptides from 21 immunodominant proteins. Here we comprehensively evaluated the 11 top-ranked C. trachomatis-specific peptide antigens from 8 proteins for use in C. trachomatis serology. Sera from 125 women with nucleic acid amplification test (NAAT)-confirmed active C. trachomatis infection and from 49 healthy women with a low risk of C. trachomatis infection were used as anti-C. trachomatis antibody-positive and -negative sera. Results obtained for detection of IgG1, IgG3, and IgA1 antibodies against the 11 C. trachomatis peptide antigens were compared to results from 4 commercial anti-C. trachomatis IgG ELISAs. Using composite reference standards (CRS) of all assays for anti-C. trachomatis antibody status, commercial ELISAs detected antibodies in antibody-positive women with sensitivities of 51.5% to 64.8%. In contrast, a combination of the results of all 11 peptides detected IgG (IgG1 and IgG3) antibodies with 91.8% sensitivity, and a labor-saving combination of the 5 optimal peptides still detected antibodies in antibody-positive women with 86.5% sensitivity (all at 98% specificity). The superior performance of the combined peptide ELISAs was confirmed by area under the receiver operating characteristic curve (ROC-AUC), likelihood ratio, and predictive value analyses. The higher sensitivity of the peptide assays results from using multiple B cell epitopes of several C. trachomatis immunodominant proteins, including OmpA, compared to exclusively using the OmpA antigens used in commercial ELISAs. Thus, ELISAs with combined use of synthetic peptide antigens for C. trachomatis antibody detection have the advantage of simultaneous high sensitivity and high specificity.IMPORTANCE For detection of anti-Chlamydia trachomatis antibodies by serological assays, use of classical whole-organism chlamydial antigens results in high cross-reactivity. These antigens bind mainly antibodies against the major outer membrane protein (OmpA) and bind antibodies against other immunodominant non-OmpA proteins to a lesser extent, resulting in poor assay sensitivity. The specificity of C. trachomatis serology is also compromised by the high prevalence of cross-reactive anti-C. pneumoniae antibodies in human populations. We previously identified 48 highly specific C. trachomatis B cell epitope peptide antigens of 21 immunodominant proteins. This study validated peptide antigen-based novel ELISAs that provide highly specific and sensitive detection of anti-C. trachomatis antibodies. Compared to four commercial ELISAs that achieved only poor sensitivities (51.5% to 64.8%), the combined signals of 5 to 11 peptides provided high sensitivity (86.5% to 91.8%) at the same 98% specificity. Thus, by using multiple peptide antigens of immunodominant proteins, we created simple ELISAs with specificity and sensitivity superior to standard C. trachomatis serodiagnosis.

Discovery of Blood Transcriptional Endotypes in Women with Pelvic Inflammatory Disease.

Sexually transmitted infections with Chlamydia trachomatis and/or Neisseria gonorrhoeae and rates of pelvic inflammatory disease (PID) in women continue to rise, with reinfection being common because of poor adaptive immunity. Diagnosis remains imprecise, and pathogenesis data are derived primarily from monoinfection of mice with C. trachomatis or N. gonorrhoeae By comparing blood mRNA responses of women with C. trachomatis- and/or N. gonorrhoeae-induced PID and histologic endometritis with those from women with C. trachomatis and/or N. gonorrhoeae infection limited to their cervix and asymptomatic uninfected women determined via microarray, we discovered important pathogenic mechanisms in PID and response differences that provide a pathway to biomarker discovery. Women with N. gonorrhoeae- and/or C. trachomatis-induced PID exhibit overexpression of myeloid cell genes and suppression of protein synthesis, mitochondrial oxidative phosphorylation, and T cell-specific genes. Coinfected women exhibited the greatest activation of cell death pathways and suppression of responses essential for adaptive immunity. Women solely infected with C. trachomatis expressed elevated levels of type I and type II IFN genes, and enhanced type I IFN-induced chemokines in cervical secretions were associated with ascension of C. trachomatis to the endometrium. Blood microarrays reveal discrete pathobiological endotypes in women with PID that are driven by pathogen invasion of the upper genital tract.

A Chlamydia-Specific TCR-Transgenic Mouse Demonstrates Th1 Polyfunctionality with Enhanced Effector Function.

Chlamydia is responsible for millions of new infections annually, and current efforts focus on understanding cellular immunity for targeted vaccine development. The Chlamydia-specific CD4 T cell response is characterized by the production of IFN-γ, and polyfunctional Th1 responses are associated with enhanced protection. A major limitation in studying these responses is the paucity of tools available for detection, quantification, and characterization of polyfunctional Ag-specific T cells. We addressed this problem by developing a TCR-transgenic (Tg) mouse with CD4 T cells that respond to a common Ag in Chlamydia muridarum and Chlamydia trachomatis Using an adoptive-transfer approach, we show that naive Tg CD4 T cells become activated, proliferate, migrate to the infected tissue, and acquire a polyfunctional Th1 phenotype in infected mice. Polyfunctional Tg Th1 effectors demonstrated enhanced IFN-γ production compared with polyclonal cells, protected immune-deficient mice against lethality, mediated bacterial clearance, and orchestrated an anamnestic response. Adoptive transfer of Chlamydia-specific CD4 TCR-Tg T cells with polyfunctional capacity offers a powerful approach for analysis of protective effector and memory responses against chlamydial infection and demonstrates that an effective monoclonal CD4 T cell response may successfully guide subunit vaccination strategies.

Identification of Chlamydia trachomatis Antigens Recognized by T Cells From Highly Exposed Women Who Limit or Resist Genital Tract Infection.

BACKGROUND: Natural infection induces partial immunity to Chlamydia trachomatis Identification of chlamydial antigens that induce interferon γ (IFN-) secretion by T cells from immune women could advance vaccine development. METHODS: IFN-γ production by CD4+ and CD8+ peripheral blood T cells from 58 high-risk women was measured after coculture with antigen-presenting cells preincubated with recombinant Escherichia coli expressing a panel of 275 chlamydial antigens. Quantile median regression analysis was used to compare frequencies of IFN-γ responses in women with only cervical infection to those in women with endometrial infection and frequencies in women who remained uninfected for over 1 year to those in women who developed incident infection. Statistical methods were then used to identify proteins associated with protection. RESULTS: A higher median frequency of CD8+ T-cell responses was detected in women with lower genital tract chlamydial infection, compared with those with upper genital tract chlamydial infection (13.8% vs 9.5%; P =04), but the CD4+ T-cell response frequencies were not different. Women who remained uninfected displayed a greater frequency of positive CD4+ T-cell responses (29% vs 18%; P < .0001), compared with women who had incident infection, while the frequencies of CD8+ T-cell responses did not differ. A subset of proteins involved in central metabolism, type III secretion, and protein synthesis were associated with protection. CONCLUSIONS: Investigations in naturally exposed women reveal protective responses and identify chlamydial vaccine candidate antigens.

Analysis of Factors Driving Incident and Ascending Infection and the Role of Serum Antibody in Chlamydia trachomatis Genital Tract Infection.

BACKGROUND: Chlamydia trachomatis genital tract infection is a major cause of female reproductive morbidity. Risk factors for ascending infection are unknown, and the role for antibody in protection is not well established. METHODS: We recruited 225 women from urban outpatient clinics and followed them for a median of 12 months. We performed a cross-sectional analysis of serum anti-chlamydial immunoglobulin G (IgG), behavioral factors, and microbiological factors associated with endometrial infection at enrollment, and a longitudinal analysis of factors associated with incident infection. RESULTS: Oral contraceptives (adjusted relative risk [RR], 2.02 [95% confidence interval {CI}, 1.38-2.97]) and gonorrhea (adjusted RR, 1.66 [95% CI, 1.07-2.60]) were associated with endometrial infection. Gonorrhea (adjusted hazard ratio [HR], 3.09 [95% CI, 1.41-6.78]), cervical infection at enrollment (adjusted HR, 2.33 [95% CI, 1.07-5.11]), and exposure to uncircumcised partners (adjusted HR, 2.65 [95% CI, 1.21-5.82]) or infected partners (adjusted HR, 4.99 [95% CI, 2.66-9.39]) significantly increased the risk of incident infection. Seropositivity was associated with a reduced cervical burden (P < .05) but no differences in rates of ascending infection (adjusted RR, 1.24 [95% CI, .71-2.19]) or incident infection (adjusted HR, 0.94 [95% CI, .52-1.69]). CONCLUSIONS: Serum anti-chlamydial IgG is not associated with a lowered rate of ascending or repeat infection. Identification of factors associated with ascending infection and increased risk of incident infection provide guidance for targeted screening of women at increased risk for sequelae.

Comparable Genital Tract Infection, Pathology, and Immunity in Rhesus Macaques Inoculated with Wild-Type or Plasmid-Deficient Chlamydia trachomatis Serovar D.

Rhesus macaques were studied to directly address the potential for plasmid-deficient Chlamydia trachomatis to serve as a live attenuated vaccine in the genital tract. Five repeated cervical inoculations of rhesus macaques with wild-type serovar D strain D/UW-3/Cx or a plasmid-deficient derivative of this strain, CTD153, resulted in infections with similar kinetics and induced comparable levels of protective immunity. After all animals received five challenges with D/UW-3/Cx, levels of inflammation observed grossly and histologically were similar between the groups. Animals in both groups developed evidence of oviduct dilatation; however, reduced oviduct dilatation was observed for "controllers," i.e., animals without detectable chlamydial DNA in the fimbriae at weeks 5 and 12. Grouping animals into "ascenders" and "controllers" revealed that elevated early T cell responses were associated with protection, whereas higher antibody responses were associated with ascension. Protected animals shared common major histocompatibility complex (MHC) alleles. Overall, genetic differences of individual animals, rather than the presence or absence of the chlamydial plasmid in the primary infecting strain, appeared to play a role in determining the outcome of infection.