Norovirus-specific monoclonal antibodies that block histo-blood group antigen binding isolated from healthy donors.

Human noroviruses are a leading cause of acute gastroenteritis, yet no licensed vaccine or antiviral is currently available. The extensive genetic diversity of noroviruses and the limited cross-protective immunity between genotypes complicate the development of broadly effective interventions. To systematically characterize norovirus-specific B cell responses from healthy individuals, we applied linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) to peripheral blood mononuclear cells using a genetically diverse panel of norovirus capsid protruding (P) domain antigens. Antigen-specific B cells were identified at single-cell resolution and selected for recombinant monoclonal antibody (mAb) expression. A total of 25 norovirus-specific mAbs were generated and evaluated for binding breadth across multiple genogroup II (GII) genotypes. Binding analyses revealed substantial heterogeneity, with some mAbs displaying narrow, genotype-restricted recognition, whereas others exhibited broad cross-reactivity. Epitope competition assays using a subset of mAbs demonstrated both overlapping and distinct binding sites on the P domain. Functional activity, assessed using a surrogate neutralization assay measuring inhibition of histo-blood group antigen (HBGA) binding, showed that several broadly reactive antibodies inhibited virus-like particle attachment to HBGAs with variable potency across genotypes, whereas others exhibited genotype-restricted inhibition. Together, these results demonstrate that LIBRA-seq enables efficient mapping of human antibody repertoires to norovirus at the population level and reveals that natural exposure generates a diverse B-cell repertoire with varying specificity and functional breadth.IMPORTANCEHuman noroviruses are the leading cause of acute viral gastroenteritis worldwide, yet individuals experience repeated infections throughout life, indicating that protective immunity is incomplete and remains poorly understood. In this study, we applied the high-throughput LIBRA-seq approach to systematically map the norovirus-specific antibody repertoire in healthy human donors, enabling the identification of monoclonal antibodies that recognize and functionally inhibit clinically relevant genotypes. This work provides new insight into the breadth and specificity of naturally acquired humoral immunity by establishing a comprehensive framework for analyzing antigen-specific B cell responses in the general population and generates a well-defined panel of human antibodies that will be useful for future studies of norovirus immunity, vaccine evaluation, and therapeutic development.