Norovirus Escape from Broadly Neutralizing Antibodies Is Limited to Allostery-Like Mechanisms

The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region—one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from—is therefore ideal for vaccine development. ABSTRACT Ideal antiviral vaccines elicit antibodies (Abs) with broad strain recognition that bind to regions that are difficult to mutate for escape. Using 10 murine norovirus (MNV) strains and 5 human norovirus (HuNoV) virus-like particles (VLPs), we identified monoclonal antibody (MAb) 2D3, which broadly neutralized all MNV strains tested. Importantly, escape mutants corresponding to this antibody were very slow to develop and were distal to those raised against our previously studied antibody, A6.2. To understand the atomic details of 2D3 neutralization, we determined the cryo-electron microscopy (cryo-EM) structure of the 2D3/MNV1 complex. Interestingly, 2D3 binds to the top of the P domain, very close to where A6.2 binds, but the only escape mutations identified to date fall well outside the contact regions of both 2D3 and A6.2. To determine how mutations in distal residues could block antibody binding, we used molecular dynamics flexible fitting simulations of the atomic structures placed into the density map to examine the 2D3/MNV1 complex and these mutations. Our findings suggest that the escape mutant, V339I, may stabilize a salt bridge network at the P-domain dimer interface that, in an allostery-like manner, affects the conformational relaxation of the P domain and the efficiency of binding. They further highlight the unusual antigenic surface bound by MAb 2D3, one which elicits cross-reactive antibodies but which the virus is unable to alter to escape neutralization. These results may be leveraged to generate norovirus (NoV) vaccines containing broadly neutralizing antibodies. IMPORTANCE The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region—one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from—is therefore ideal for vaccine development.