A Multiplexed Bactericidal Assay for Shigella Lipopolysaccharide Antibodies

Shigella, a major cause of diarrhea for military personnel and civilians, is often associated with Shigella flexneri serotypes 2a, 3a, and 6, and Shigella sonnei. These shigella strains express serologically distinct O-antigen polysaccharides at the distal end of LPS molecules. To control shigella diseases, various shigella antigens are being investigated as vaccine candidates. In the wake of the highly successful pneumococcal conjugate vaccines, multivalent shigella conjugate vaccines (SCVs) are actively being developed. SCVs contain a mixture of several epidemiologically important serotype-specific lipopolysaccharides (LPS) that are conjugated with a carrier protein. SCVs are designed to elicit antibodies to O-antigens that can kill shigella expressing the targeted O-antigen (but not others) in the presence of complement. In developing SCVs, it is necessary to measure the functional capacity of antibodies such as the bactericidal activity induced by the vaccine. Since a serum bactericidal assay (SBA) measures antibody-mediated killing activity in vitro, that killing activity can be used as a surrogate for vaccine efficacy in vaccine studies. However, as explained below, SBAs are difficult assays that must be run once for each serotype, and SCVs have multiple serotypes. Consequently, SCV development requires a sample to be analyzed multiple times with a difficult SBA. Thus, it is highly desirable to have a simple, high-throughput, multiplexed SBA. In this proposal, we recommend two approaches for a two-fold multiplexed SBA: one using two antibiotic-resistant bacterial strains and the other using two fluorescent strains. Specifically, we propose to develop the necessary target strains, assess the two approaches, develop a data acquisition pipeline generating a “killing index” as an output parameter, and begin to evaluate assay performance with a panel of sera and other types of samples.