Development and Validation of Sixplexed Opsonophagocytic Killing Assay for Serotype-Specific Functional Pneumococcal Antibody Measurement

<b>Background</b>: Although pneumococcal conjugate vaccines (PCVs) have substantially reduced invasive pneumococcal disease, the emergence of non-vaccine serotypes and antimicrobial-resistant strains has driven the development of higher-valency vaccines. To support functional immune evaluation of these vaccines, we developed and validated a sixplexed opsonophagocytic killing assay (OPA) covering 24 pneumococcal serotypes. <b>Methods</b>: Eight additional serotypes, beyond the 16 included in the conventional fourplex OPA, were generated through stepwise natural mutation under increasing concentrations of ciprofloxacin or doxycycline. Assay conditions were optimized by evaluating multiple effector-to-target (E:T) ratios and baby rabbit complement (BRC) concentrations to minimize non-specific killing (NSK). Validation assessed (1) specificity using inhibition OPA with homologous and heterologous polysaccharides, (2) accuracy by comparison with the single-serotype OPA (SOPA), and (3) precision across five independent experiments using the coefficient of variation (CV). <b>Results</b>: An E:T ratio of 200:1 combined with 10% BRC consistently maintained NSK below 30% across all assay sets. High serotype specificity was demonstrated by near-complete inhibition following homologous polysaccharide adsorption for all serotypes except serotypes 4 and 8, which exhibited very low opsonic indices. Results from the sixplexed OPA showed strong concordance with SOPA, and overall assay precision was acceptable, with CVs generally below 30% when serotypes with very low opsonic activity were excluded. <b>Conclusions</b>: The sixplexed OPA expands functional antibody assessment from 16 to 24 serotypes within four assay sets, providing an efficient and scalable platform for immunogenicity evaluation of current and next-generation high-valency pneumococcal vaccines.