Among this panel, we also identified 3 distinct major modes of binding. Within this panel, we also included 5 of 25 GII.4-reactive mAbs from a previous report: NORO-202A,−232A.2,−279A,−310A, and −320 15 mAbs from our previous studies of human GI.1-reactive B cell responses did not exhibit cross-reactivity 16 and are not included in this report. In this study, 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. Numerous studies have assessed the presence of a human polyclonal immune response to HuNoV 10, 11, 12, 13, 14. The intricacies of human antibody-mediated HuNoV cross-reactivity and neutralization remain to be fully elucidated. Genetic diversity in structural proteins also 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. Between genogroups, the genomic nucleotide sequence of structural proteins can differ by more than 50% 9. The HuNoVs can be further subdivided into 49 genotypes 8. The amino acid sequence of the major structural protein is also used to classify noroviruses into 10 different genogroups 8. Recombinant expression of the VP1 sequence in insect cells results in the spontaneous formation and release of virus-like particles that are antigenically and morphologically similar to HuNoV virions 6, 7. VP1 can be divided further into a highly conserved shell (S) domain and a more variable protruding (P) domain 5. The norovirus genome is organized into 3 open reading frames with the first encoding non-structural proteins, the second encoding the major structural protein (VP1), and the third encoding the minor structural protein (VP2) 4. Noroviruses, comprising a genus within the Caliciviridae family, are non-enveloped single-stranded positive-sense RNA viruses. Progress has been made towards the development of a HuNoV vaccine, with several vaccine candidates currently in clinical trials, but it is unclear whether or not a successful vaccine would need to be reformulated regularly due to the periodic emergence of novel pandemic HuNoV variants. Thus, there is a substantial global disease burden caused by HuNoVs and a need for sensitive, accurate diagnostics and efficacious therapeutics and vaccines. Globally, the economic burden resulting from both direct health system costs and societal costs is estimated to be over $60 billion per year 3. In the United States, the Center for Disease Control estimates that there are approximately 19 to 21 million annual cases of acute gastroenteritis caused by HuNoVs 2. In 2010, there were 1.8 billion cases of diarrheal disease worldwide, and about 18% of these were due to human norovirus (HuNoVs) 1. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features. 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. 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. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. 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. 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. 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). The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus.
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