Structures were solved by molecular replacement in (41). changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are known cofactors for contamination. KEYWORDS: antigenic drift, complex structure, neutralizing antibodies, noroviruses ABSTRACT Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The persistence of GII.4 is believed to be associated with the recognition of numerous histo-blood group antigen (HBGA) types and antigenic drift. We found that one of the earliest known GII.4 isolates (in 1974) and a more recent epidemic GII.4 variant (in 2012) had varied norovirus-specific monoclonal antibody (MAb) reactivities but similar HBGA binding profiles. To better understand the binding conversation of one MAb (10E9) that experienced varied reactivity with these GII.4 variants, we decided the X-ray crystal structure of the NSW-2012 GII.4 P domain name 10E9 Fab complex. We showed that this 10E9 Fab interacted with conserved and variable residues, which could be associated with antigenic drift. Interestingly, the 10E9 Fab binding pocket partially overlapped the HBGA pocket and experienced direct competition for conserved HBGA binding residues (i.e., Arg345 and Tyr444). Indeed, the 10E9 MAb blocked norovirus virus-like particles (VLPs) from binding to several sources of HBGAs. Moreover, the 10E9 antibody completely abolished computer virus replication in the human norovirus intestinal enteroid cell culture system. Our new findings provide the first direct evidence that competition for MGCD-265 (Glesatinib) GII.4 HBGA MGCD-265 (Glesatinib) binding residues and steric obstruction could lead to norovirus neutralization. On the other hand, the 10E9 MAb acknowledged residues flanking the HBGA pocket, which are often substituted as the computer virus evolves. This mechanism of antigenic drift likely influences herd Mouse monoclonal to IgM Isotype Control.This can be used as a mouse IgM isotype control in flow cytometry and other applications immunity and impedes the possibility of acquiring broadly reactive HBGA-blocking antibodies. IMPORTANCE The emergence of new epidemic GII.4 norovirus variants is thought to be associated with changes MGCD-265 (Glesatinib) in antigenicity and HBGA binding capacity. Here, we show that HBGA binding profiles remain unchanged between the 1974 and 2012 GII.4 variants, whereas these variants showed various levels of reactivity against a panel of GII.4 MAbs. We recognized a MAb that bound at the HBGA pocket, blocked norovirus VLPs from binding to HBGAs, and neutralized norovirus virions in the cell culture system. Raised against a GII.4 2006 strain, this MAb was unreactive to a GII.4 1974 isolate but was able to neutralize the newer 2012 strain, which has important implications for vaccine design. Altogether, these new findings suggest that the amino acid variations surrounding the HBGA pocket lead to temporal changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are known cofactors for contamination. KEYWORDS: antigenic drift, complex structure, neutralizing antibodies, noroviruses INTRODUCTION Human noroviruses are a major cause of outbreaks of acute gastroenteritis worldwide. The human norovirus genome contains three open reading frames (ORFs), where ORF1 encodes nonstructural proteins, ORF2 encodes capsid protein, and ORF3 encodes a minor capsid protein (1). Human noroviruses are grouped into several genogroups (i.e., GI, GII, and GIV), which are subsequently subgrouped into numerous genotypes. Norovirus capsid genes frequently evolve into genetic and antigenic variants, which have sometimes resulted in epidemics and even pandemics (2). The GII genotype 4 (GII.4) variants have dominated over the past two decades and have approximately 5% capsid amino acid divergence from previous epidemic GII.4 variants. The capsid of each variant likely accumulates advantageous mutations from your preceding variant. Antigenic drift has therefore been proposed as a major driving pressure in the emergence of novel GII.4 variants (3, 4). Expression of the human norovirus ORF2 in insect cells results in the formation of virus-like particles (VLPs) that are antigenically and morphologically much like native virions. The X-ray crystal structure of norovirus VLPs from your prototype strain (GI.1) identified two domains, termed shell (S) and protruding (P) domains (5). The S domain surrounds the viral RNA, whereas the P domain, which can be further subdivided into P1 and P2 subdomains, contains determinants for cell attachment and antigenicity. Human noroviruses bind to histo-blood group antigens (HBGAs), and this interaction is important for contamination (6,C11). The X-ray crystal structures of the P domains from epidemic GII.4 variants in complex with a panel of HBGAs showed that a regular set of conserved residues (i.e., Asp374, Arg345, Thr344, Tyr444, and Gly443) usually holds both the ABH- and Lewis-fucose of HBGAs (12,C15). Interestingly, the HBGA binding profiles of the antigenically unique GII.4 variants have likely remained unchanged over the past few decades (13, 16). Indeed, the region.