Development and qualification of a multiplexed immunoassay to assess the immunogenicity of Shigella vaccines.

BACKGROUND

Shigella is a major cause of diarrheal disease in young children worldwide, particularly in low- and middle-income countries (LMICs). The emergence of antibiotic resistance and limited access to healthcare in LMICs underscores the urgent need for an effective Shigella vaccine. Several next-generation Shigella vaccines are under development, many of which target the O-antigen component of lipopolysaccharide (LPS) from multiple serotypes to achieve broad protective coverage. Reliable, multiplexed assays are needed to evaluate vaccine immunogenicity across Shigella serotypes and vaccine platforms.

METHODS

A Luminex-based multiplex immunoassay was developed incorporating purified LPS from five Shigella serotypes (S. sonnei, S. flexneri 1b, 2a, 3a, and 6) along with Shigella IpaB protein and common vaccine carrier proteins. Assay performance was evaluated using pooled post-vaccination serum samples with qualification parameters including linearity, accuracy, precision, and specificity.

RESULTS

Dilutional linearity was confirmed in all antigens across a broad dynamic range (R2 ≥ 0.98), with slopes ranging from 0.91 to 1.16. Within-run and between-run accuracy and intra-assay and inter-assay precision met predefined criteria for all antigens, with variability primarily observed at high dilutions for S. flexneri 1b and 3a. The assigned lower and upper limits of quantitation (LLOQ-ULOQ) spanned up to two orders of magnitude per antigen. The multiplex assay demonstrated specificity for homologous antigens, with some cross-reactivity patterns reflecting known structural similarities among LPS serotypes.

CONCLUSION

This multiplex immunoassay enabled accurate and efficient measurement of vaccine-induced antibody responses across multiple Shigella antigens and carrier proteins. It therefore offers a qualified scalable solution for evaluating multivalent Shigella vaccines in clinical trials, particularly in resource-limited settings.