Title: A Fluorometric High-Throughput Assay for Measuring Chlamydial Neutralizing

23 Chlamydia trachomatis is an obligate intracellular mucosotropic pathogen that causes human 24 infections of global importance. C. trachomatis causes trachoma, the leading cause of 25 preventable blindness worldwide and is the most common cause of bacterial sexually transmitted 26 disease. Although oculogenital infections are treatable with antibiotics, a vaccine is needed to 27 control C. trachomatis infection. Ideally, a vaccine would provide coverage against most, if not 28 all, naturally occurring antigenically distinct serovariants. The development of a subunit vaccine 29 to prevent oculogenital disease could be advanced by identifying chlamydial antigens that elicit 30 pan-neutralizing antibodies, particularly among infected human populations of known risk 31 factors. Currently there is no objective high-throughput in vitro assay to screen human sera for 32 neutralization to aid in identification of these antigens. This study describes an objective, high33 throughput in vitro assay that measures C. trachomatis neutralizing antibodies. Antibody34 mediated neutralization of chlamydial infection was performed in a 96-well microtiter format 35 and neutralization was quantified by immunostaining fixed cells followed by automated 36 fluorometric analysis. This study shows that fluorometric analysis of C. trachomatis infection 37 directly correlates to labor intensive manual inclusion counts. Furthermore, this study shows that 38 fluorometry can be used to identify C. trachomatis serovar and serocomplex-specific 39 neutralization. This objective, high-throughput analysis of serum neutralization is amenable to 40 epidemiological studies of human chlamydial infection, human clinical vaccine trials, and pre41 clinical animal model experiments of Chlamydia infection. 42