N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies.

Abstract

Vaccination is the most effective way to prevent influenza virus infections. However, the diversity of antigenically distinct isolates is a challenge for vaccine development. In order to overcome the antigenic variability and improve the protective efficacy of influenza vaccines, our research group has pioneered the development of computationally optimized broadly reactive antigens (COBRA) for hemagglutinin (HA). Two candidate COBRA HA vaccines, P1 and X6, elicited antibodies with differential patterns of hemagglutination inhibition (HAI) activity against a panel of H1N1 influenza viruses. In order to better understand how these HA antigens elicit broadly reactive immune responses, epitopes in the Cb, Sa, or Sb antigenic sites of seasonal-like and pandemic-like wild-type or COBRA HA antigens were exchanged with homologous regions in the COBRA HA proteins to determine which regions and residues were responsible for the elicited antibody profile. Mice were vaccinated with virus-like particles (VLPs) expressing one of the 12 modified HA antigens (designated V1 to V12), COBRA HA antigens, or wild-type HA antigens. The elicited antisera was assessed for hemagglutination inhibition activity against a panel of historical seasonal-like and pandemic-like H1N1 influenza viruses. Primarily, the pattern of glycosylation sites and residues in the Sa antigenic region, around the receptor binding site (RBS), served as signatures for the elicitation of broadly reactive antibodies by these HA immunogens. Mice were vaccinated with VLPs expressing HA antigens that lacked a glycosylation site at residue 144 and a deleted lysine at position 147 residue were more effective at protecting against morbidity and mortality following infection with pandemic-like and seasonal-like H1N1 influenza viruses.IMPORTANCE There is a great need to develop broadly reactive or universal vaccines against influenza viruses. Advanced, next-generation hemagglutinin (HA) head-based vaccines that elicit protective antibodies against H1N1 influenza viruses have been developed. This study focused on understanding the specific amino acids around the receptor binding site (RBS) that were important in elicitation of these broadly reactive antibodies. Specific glycan sites and amino acids located at the tip of the HA molecule enhanced the elicitation of these broadly reactive antibodies. A better understanding of the HA structures around the RBS will lead to more effective HA immunogens.