Protein Engineering Strategies to Modulate B Cell Responses

Abstract

The majority of antibody responses raised against influenza hemagglutinin (HA) preferentially target highly variable epitopes, and thus do not confer lasting protection. Antibodies against conserved epitopes such as the receptor binding site (RBS), head interface, and stem region have the potential to be broadly protective yet are poorly elicited through traditional vaccination strategies. To focus immune responses to the RBS, we have designed an immunogen called a resurfaced HA trimeric chimera (rsHAtCh) that is enriched for the H1 RBS epitope relative to all other epitopes. Additionally, we have designed two versatile platforms for the generation of heterotrimeric immunogens. We have characterized immune responses against our RBS-enriched immunogen in two separate partially-humanized mouse models. We have shown that immunization with rsHAtCh in huVH1- 2 knock-in mice focuses both serum and B cell responses to the H1 RBS and elicits a genetically stereotyped response making primarily germline-encoded contacts. We have also shown that rsHAtCh immunization in huJH6 knock-in mice preferentially elicits a genetically diverse RBS- directed response both in primary and secondary germinal centers. Representative structures of RBS-directed antibodies show multiple examples of receptor-like contacts previously characterized in human RBS-directed broadly neutralizing antibodies. Next-generation influenza vaccines seek to preferentially alter patterns of dominance in favor of conserved subdominant epitopes. Our data, which show that an epitope-enriched immunogen can focus to the subdominant hemagglutinin receptor binding site, support the use of epitope enrichment as an immunofocusing strategy.