Broadly cross-reactive human antibodies that inhibit genogroup I and II noroviruses

Gabriela Alvarado,Mary K Estes,Banumathi Sankaran,Liya Hu,Wilhelm Salmen,James E Crowe,Khalil Ettayebi,B V Venkataram Prasad

doi:10.1038/s41467-021-24649-w

Abstract

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.

Highlights

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus
To isolate cross-reactive human norovirus (HuNoV) human monoclonal antibodies (mAbs), we used EBV and additional B cell stimuli to transform memory B cells in PBMCs obtained from patients who were overall healthy but with a previous history of acute gastroenteritis, as previously described[16]
A week later, transformed PBMCs supernatants were tested by ELISA to screen for the expression of mAbs that bound to more than one representative strain HuNoV Virus-like particles (VLPs)

Summary

Introduction

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. The Fab form of NORO-320 neutralizes GII.[4] infection more potently than the mAb, does not block HBGA binding. Dynamic light scattering analysis of GII.[4] virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Genetic diversity in structural proteins causes changes in antigenic properties, so it is imperative that we understand the HuNoV-mediated human immunological response to infection and the antigenic variation among circulating strains of HuNoV to develop an effective vaccine. We identified and characterized a panel of broadly binding and neutralizing human IgM, IgG, and IgA antibodies from subjects who were infected previously with GII.[4] Sydney 2012 HuNoV

Objectives

Methods

Results

Conclusion