(P77) Development and validation of a high-throughput serum bactericidal assay to measure functional antibody responses to Neisseria gonorrhoeae

Background: In addition to the specificity of Neisseria gonorrhoeae (Ng) for the human host, identification of correlates of protection for gonococcal vaccines has been hampered by the lack of standardized assays to measure functional anti-Ng antibody responses. To begin to address this problem, we sought to develop and validate a standardized high-throughput human complement serum bactericidal assay (hSBA) that could be used to compare data among different laboratories.

Methods: Ng strain MS11 was grown to mid-exponential phase in liquid GC medium containing CMP-NANA to simulate in vivo lipooligosaccharide sialylation. Bacteria were then incubated in a 96-well plate with 10% IgG/IgM-depleted human complement serum and a panel of ~40 test serum samples obtained from rabbits immunized with cross-reactive meningococcal vaccines. Following incubation, an agar overlay consisting of a modified GCB agar medium was added to each well, and plates were incubated overnight at 37°C. Titers were calculated as the reciprocal dilution at which ≥50% killing was observed relative to the mean of control wells containing bacteria and complement alone. Precision and linearity were evaluated over the proposed analytical range by calculation of assay variability and regression analyses, respectively. Additional studies included testing of serum sensitive gonococcal and unencapsulated meningococcal strains, as well as mouse, baboon, and human sera.



Results: The high-throughput anti-Ng hSBA was validated over the reciprocal titer range of [4, 64]. The experimental layout allowed for simultaneous testing of up to five different test sera per plate, with five different dilutions tested per serum. Supplemental studies with serum-sensitive strains F62, M45193, and M38546, as well as test sera obtained from multiple mammalian species, demonstrated robust assay performance.



Conclusions: Standardization of hSBA conditions permits testing of a variety of strains and sera without the necessity for protocol alterations or the cumbersome practice of pre-screening complement for endogenous killing activity. As divergent test strains, buffers, and complement concentrations are currently used in hSBAs for gonococcal vaccine development, widespread adoption of the high-throughput assay may enable direct comparisons of bactericidal responses across different laboratories and evaluation of the importance of elicitation of bactericidal antibodies in preventing Ng infection.