Engineering Antigen Binding and Orientation on Alum Adjuvants for Enhanced Humoral Responses and Immunofocusing

A major challenge in vaccine development, especially against rapidly evolving viruses, is the ability to focus the immune response toward evolutionarily conserved antigenic regions to confer broad protection. For example, while many broadly neutralizing antibodies against influenza have been found to target the highly conserved stem region of hemagglutinin (HA-stem), the immune response to seasonal influenza vaccines is predominantly directed to the immunodominant but variable head region (HA-head), leading to narrow-spectrum efficacy. Here, I will first introduce an approach to controlling antigen orientation based on the site-specific insertion of short stretches of aspartate residues (oligoD) that facilitates antigen-binding to alum adjuvants. I will demonstrate the generalizability of this approach to antigens from the Ebola virus, SARS-CoV-2, and influenza and observe enhanced antibody responses following immunization in all cases. Next, I use this approach to reorient HA in an “upside down” configuration, which I envision increases HA-stem exposure, therefore also improving its immunogenicity compared to HA-head. When applied to HA of H2N2 A/Japan/305/1957, the reoriented H2 HA (reoH2HA) on alum induced a stem-directed antibody response that cross-reacted with both group 1 and 2 influenza A HAs. These results demonstrate the possibility and benefits of antigen reorientation by engeering the interaction between antigens and adjuvants, which represents a generalizable immunofocusing approach readily applicable for designing epitope-focused vaccine candidates.