Seth J. Zost

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014-2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016-2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016-2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016-2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.