Inhibitory effects of antibody crosslinking on influenza virus assembly and release.

Abstract

Influenza viruses bud from the plasma membrane of infected cells, where the viral surface proteins hemagglutinin (HA), neuraminidase (NA) and the ion channel M2 are vulnerable to antibodies in the extracellular environment. Although binding of multivalent antibodies is likely to perturb the dynamics and organization of the viral surface proteins, how this affects the assembly and release of new virions remains unclear. This is due in part to limitations of available techniques: while antibody neutralization of virus entry is routinely measured using hemagglutination inhibition and microneutralization assays, there are few approaches to specifically evaluate antibody inhibition of virus assembly and release that are quantitative and mechanistic. Motivated by this need, we developed fluorescence imaging-based assays to quantify virus shedding under antibody challenge and to dissect the underlying inhibition mechanisms. We find that HA-, NA-, and M2-specific monoclonal antibodies targeting a range of antigenic sites significantly decrease the number of viruses released during a single replication cycle. These antibodies inhibit virus release at concentrations ∼1-10-fold higher than their inhibitory concentrations for viral entry, reinforcing the idea that antibody function is multifaceted. Using fluorescence recovery after photobleaching, we observe that HA stem-binding IgG antibodies CR9114 and FI6v3 disrupt the diffusion of cell surface HAs by crosslinking adjacent HAs in cis, thus restricting their contributions to viral assembly. In contrast, head-binding antibodies S139 and C05 are unable to alter the HA diffusion rate but potently inhibit the release of fully formed virions by crosslinking HAs in trans, leading to the sequestration of virions at the cell surface. Collectively, these results suggest that antibody inhibition of virus assembly and release is a general feature of IgG antibodies, and that antibody crosslinking either in cis or in trans can contribute to the inhibition potency.