Fluorometric high-throughput assay for measuring chlamydial neutralizing antibody

Abstract

Chlamydia trachomatis is an obligate intracellular mucosotropic pathogen that causes human infections of global importance. C. trachomatis causes trachoma, the leading cause of preventable blindness worldwide, and is the most common cause of bacterial sexually transmitted disease. Although oculogenital infections are treatable with antibiotics, a vaccine is needed to control C. trachomatis infection. Ideally, a vaccine would provide coverage against most, if not all, naturally occurring antigenically distinct serovariants. The development of a subunit vaccine to prevent oculogenital disease could be advanced by identifying chlamydial antigens that elicit pan-neutralizing antibodies, particularly among infected human populations of known risk factors. There is currently no objective high-throughput in vitro assay to screen human sera for neutralization to aid in identification of these antigens. This report describes an objective, high-throughput in vitro assay that measures C. trachomatis-neutralizing antibodies. Antibody-mediated neutralization of chlamydial infection was performed in a 96-well microtiter format, and neutralization was quantified by immunostaining fixed cells followed by automated fluorometric analysis. This report shows that fluorometric analysis of C. trachomatis infection directly correlates to labor-intensive manual inclusion counts. Furthermore, this report shows that fluorometry can be used to identify C. trachomatis serovar- and serocomplex-specific neutralization. This objective, high-throughput analysis of serum neutralization is amenable to epidemiological studies of human chlamydial infection, human clinical vaccine trials, and preclinical animal model experiments of Chlamydia infection.

Fig 1

Flow chart showing neutralization of C. trachomatis infection of HaK cells in vitro. EBs were incubated with antibody followed by inoculation of HaK monolayers and infection by centrifugation. Cells were washed, fed with MDMEM-10 supplemented with cycloheximide, and incubated for 32 h under standard cell culture conditions. Cells were washed and fixed, and chlamydial particles were immunostained for automated fluorescence analysis or manual inclusion counts.

Fig 2

Automated fluorometric analysis of C. trachomatis infection of HaK monolayers in a microtiter format. HaK monolayers were infected with 2-fold serial dilutions of C. trachomatis EBs representing the B complex (L2, D, E), the C complex (A, C, I), and the Intermediate complex (F, G, K). Monolayers were fixed at 32 h postinfection and stained for fluorometric analysis using the anti-HSP60 MAb and rhodamine-conjugated secondary antibody. Fluorescence was evaluated using 8 biological replicates for each multiplicity of infection (MOI). Averages of results and standard deviations are shown. Data shown are representative of the results of 2 independent experiments.

Fig 3

Correlation between manual inclusion counts and fluorometric analysis of C. trachomatis infection. Nine serovars representing B, C, and Intermediate complexes are shown. Fluorometric data are expressed in fluorescence units, and inclusion counts are given in numbers of inclusions (inclusions/well). Coefficient of determination (R²), Pearson's product-moment correlation coefficient (R), and P values are provided for each serovar.

Fig 4

Neutralization of C. trachomatis infection by serovar-specific anti-MOMP MAbs. (A) Western analysis was conducted with known neutralizing MAbs to serovar L2 MOMP (L2-I45) and serovar A MOMP (A-20) with BCA-standardized protein lysates from B complex (L2, D), C complex (A, C), and Intermediate complex (G, K) serovars. (B) Neutralization assays were also conducted using the anti-MOMP MAbs across a wide range of antibody concentrations (μg/ml). Fluorescence was evaluated from 3 biological replicates at each antibody concentration. Averages of results and standard deviations are shown. Data are representative of the results of 3 independent experiments.

Fig 5

Neutralization of C. trachomatis infection by anti-L2 polyclonal rabbit serum. (A) Western analysis was conducted to determine serovar specificity and protein reactivity of the L2 polyclonal serum. BCA-standardized protein lysates from B complex (L2, D), C complex (A, C), and Intermediate complex (G, K) serovars were evaluated. (B) Neutralization assays were conducted using the L2-antiserum across a wide range of antibody dilutions. Fluorescence was evaluated from 3 biological replicates at each serum dilution. Average results and standard deviations are shown. Data are representative of the results of 3 independent experiments.