High-Throughput Luminescence-Based Serum Bactericidal Assay Optimization and Characterization to Assess Human Sera Functionality Against Multiple Shigella flexneri Serotypes

Shigellosis represents a significant global health concern particularly affecting children under 5 years in low- and middle-income countries (LMICs) and is associated with stunting and antimicrobial resistance. There is a critical need for an effective vaccine offering broad protection against the different <i>Shigella</i> serotypes. A correlate of protection has not yet been established but there is a general consensus about the relevant role of anti-O-Antigen-specific IgG and its functionality evaluated by the Serum Bactericidal Assay (SBA). This study aims to characterize a high-throughput luminescence-based SBA (L-SBA) against seven widespread <i>Shigella</i> serotypes. The assay was previously developed and characterized for <i>S. sonnei</i> and <i>S. flexneri</i> 1b, 2a, and 3a and has now been refined and extended to an additional five serotypes (<i>S. flexneri</i> 4a, 5b, 6, X, and Y). The characterization of the assay with human sera confirmed the repeatability, intermediate precision, and linearity of the assays; both homologous and heterologous specificity were verified as well; finally, limit of detection and quantification were established for all assays. Moreover, different sources of baby rabbit complement showed to have no impact on L-SBA output. The results obtained confirm the possibility of extending the L-SBA to multiple <i>Shigella</i> serotypes, thus enabling analysis of the functional response induced by natural exposure to <i>Shigella</i> in epidemiological studies and the ability of candidate vaccines to elicit cross-functional antibodies able to kill a broad panel of prevalent <i>Shigella</i> serotypes in a complement-mediated fashion.