A novel whole-bacterial enzyme linked-immunosorbant assay to quantify Chlamydia trachomatis specific antibodies reveals distinct differences between systemic and genital compartments

Abstract

Chlamydia trachomatis (CT) is the leading sexually transmitted bacterial infection. The continued global burden of CT infection strongly predicates the need for a vaccine to supplement current chlamydial control programs. The correlates of protection against CT are currently unknown, but they must be carefully defined to guide vaccine design. The localized nature of chlamydial infection in columnar epithelial cells of the genital tract necessitates investigation of immunity at the site of infection. The purpose of this study was to develop a sensitive whole bacterial enzyme-linked immunosorbent assay (ELISA) to quantify and compare CT-specific IgG and IgA in sera and genital secretions from CT-infected women. To achieve this, elementary bodies (EBs) from two of the most common genital serovars (D and E) were attached to poly-L-lysine-coated microtiter plates with glutaraldehyde. EB attachment and integrity were verified by the presence of outer membrane antigens and the absence of bacterial cytoplasmic antigens. EB-specific IgG and IgA standards were developed by pooling sera with high titers of CT-specific antibodies from infected women. Serum, endocervical and vaginal secretions, and endocervical cytobrush specimens from CT-infected women were used to quantify CT-specific IgG and IgA which were then normalized to total IgG and IgA, respectively. Analyses of paired serum and genital samples revealed significantly higher proportions of EB-specific antibodies in genital secretions compared to sera. Cervical and vaginal secretions and cytobrush specimens had similar proportions of EB-specific antibodies, suggesting any one of these genital sampling techniques could be used to quantify CT-specific antibodies when appropriate normalization methodologies are implemented. Overall, these results illustrate the need to investigate genital tract CT antibody responses, and our assay provides a useful quantitative tool to assess natural immunity in defined clinical groups and CT vaccine trials.

Anti‐chlamydia IgG and IgA are insufficient to prevent endometrial chlamydia infection in women, and increased anti‐chlamydia IgG is associated with enhanced risk for incident infection

“…We determined anti‐EB IgG and IgA antibody levels in serum and cervical secretions to explore both systemic and mucosal responses to multiple outer membrane components of the infectious developmental form of chlamydia . We first determined the strength of relationships among the anti‐EB antibodies measured at enrollment.…”

“…An important limitation is the lack of information regarding specificity of serum‐ and secretion‐derived anti‐chlamydial antibodies. EB antibody ELISA detects antigens exposed on the surface of EBs, such as MOMP, but not cytoplasmic or inclusion membrane chlamydia antigens . The latter may have a protective role; for example, IgG specific for IncA, a chlamydia inclusion membrane protein can be transported intracellularly by FcRn, resulting in recruitment of autophagic proteins and reduced infection .…”

“…Cervical secretions were eluted from sponges as previously described . IgG and IgA assays were performed on cervical secretions, and serum samples collected from the first 151 participants enrolled into the TRAC cohort.…”

“…IgG and IgA assays were performed on cervical secretions, and serum samples collected from the first 151 participants enrolled into the TRAC cohort. Concentrations of anti‐EB IgG and IgA were measured using a whole‐EB ELISA that detects chlamydial outer membrane and periplasmic proteins, but not intracellular proteins . Concentrations of total IgG and IgA in serum and secretions were measured using a previously described ELISA which readily detects secretory IgA in mucosal secretions .…”

“…Concentrations of anti‐EB IgG or IgA antibody in each sample were determined from standard curves that consisted of pooled human serum from CT‐infected subjects, then divided by the concentration of total IgG or IgA to obtain the specific activity. Non‐specific antibody values were quantitated using two IgG and nine IgA myeloma antibodies as well as anti‐HIV‐1 gp120 and gp41 human IgG1 mAbs derived from transformed human B cells or hybridomas as previously described . The cutoff for detection of a specific anti‐EB antibody response was defined by a value that was >the mean + 3 standard deviations (SD) of the non‐specific antibody values, which implies a value >99% of the non‐specific antibody values.…”