Optimization and Miniaturization of a Human Intestinal Organoid Neutralization Assay for Evaluating Human Norovirus...

Human noroviruses (HuNoVs) are the principal etiological agents of acute viral gastroenteritis worldwide. While conventional 96-well human intestinal organoid (HIO) neutralization assays provide a physiologically relevant assessment of neutralizing antibody (nAb) responses, sample volume requirements (50µL for 96 wells vs. 12.5µL serum for 384 wells per titration) and 96-well plate capacity limit the application for large-scale clinical and vaccine studies. Here, we describe the development of a miniaturized 384-well HIO neutralization assay for nAb responses to HuNoV. Sera from a phase 2b trial of a bivalent GI.1 and consensus GII.4 virus-like particle norovirus vaccine candidate demonstrated high concordance in nAb responses to the predominant GII.4 Sydney variant between conventional 96-well and miniaturized 384-well assay formats (r= 0.95). Further, a strong correlation between nAb and histo-blood group antigen (HBGA)-blocking antibodies enables serum-sparing (25µL per titration, 4-fold reduction) norovirus immune profiling, supporting scalable deployment in large clinical trials. The 384-well plate neutralization assay maintains assay sensitivity, and concordance with the 96-well format increases scalability by allowing more samples per plate and reducing serum requirements.