10779/crick.12546593.v1 Kerstin Beer Kerstin Beer Mian Dai Mian Dai Steven Howell Steven Howell Pramila Rijal Pramila Rijal Alain R Townsend Alain R Townsend Yipu Lin Yipu Lin Stephen A Wharton Stephen A Wharton Rodney S Daniels Rodney S Daniels John W McCauley John W McCauley Characterization of neutralizing epitopes in antigenic site B of recently circulating influenza A(H3N2) viruses The Francis Crick Institute 2020 Influenza antigenic drift monoclonal antibodies Animals Antibodies, Monoclonal Antibodies, Neutralizing Antibodies, Viral Antigens, Viral Epitopes Ferrets Glycosylation Hemagglutinins, Viral Humans Influenza A Virus, H3N2 Subtype Influenza, Human Models, Molecular Protein Conformation McCauley FC001030 WIC PRT 06 Biological Sciences 07 Agricultural and Veterinary Sciences 11 Medical and Health Sciences Virology 2020-06-26 09:39:28 Journal contribution https://crick.figshare.com/articles/journal_contribution/Characterization_of_neutralizing_epitopes_in_antigenic_site_B_of_recently_circulating_influenza_A_H3N2_viruses/12546593 Influenza A(H3N2) viruses are associated with outbreaks worldwide and can cause disease with severe complications. The impact can be reduced by vaccination, which induces neutralizing antibodies that mainly target the haemagglutinin glycoprotein (HA). In this study we generated neutralizing mouse monoclonal antibodies (mAbs) against A/Victoria/361/2011 and identified their epitopes by generating and sequencing escape viruses. The epitopes are located in antigenic site B, which is near the receptor-binding site and is immunodominant in humans. Amino acid (aa) substitutions at positions 156, 158, 159, 189, 190 and 193 in antigenic site B led to reduced ability of mAbs to block receptor-binding. The majority of A(H3N2) viruses that have been circulating since 2014 are antigenically distinct from previous A(H3N2) viruses. The neutralization-sensitive epitopes in antigenic site B of currently circulating viruses were examined with these mAbs. We found that clade 3C.2a viruses, possessing an additional potential glycosylation site at HA1 position N158, were poorly recognized by some of the mAbs, but other residues, notably at position 159, also affected antibody binding. Through a mass spectrometric (MS) analysis of HA, the glycosylated sites of HA1 were established and we determined that residue 158 of HA1 was glycosylated and so modified a neutralization-sensitive epitope. Understanding and monitoring individual epitopes is likely to improve vaccine strain selection.