Structure-guided Neuraminidase Immunogen Design for Next-generation Influenza Vaccines

Abstract

Strain-specific immune responses to influenza infection and vaccination drive recurring cycles of viral escape and the need for continual vaccine reformulation. The annual burden of influenza disease is high, and the threat of new pandemics due to genetic reassortment is constant. Thus, substantial research effort is devoted to creating a universal influenza vaccine—one that elicits robust immunity to broadly conserved features of influenza proteins and thereby offers protection against diverse virus strains simultaneously. While current research largely focuses on strengthening immune resilience against the virus receptor-binding protein, hemagglutinin (HA), there is growing interest in also generating robust protection against neuraminidase (NA), the protein critical for viral egress and subsequent spread. NA evolution occurs more slowly and discordantly from HA, and immunity against NA is independently protective against influenza infection. There are relatively few examples of NA-based vaccines that have been experimentally tested in vivo and even fewer that incorporate strategies to optimize the resulting humoral immune response. This thesis applies rational immunogen design approaches to NA to understand and characterize the elicited humoral immunity. Guided by NA structure and sequence conservation, we engineered sites of glycosylation throughout the variable surface of the NA protein to shield epitopes of variability from circulating B cells and focus the elicited humoral immunity toward the conserved catalytic site (CS). Through this work, we showed the effectiveness of hyperglycosylation as an immune-focusing strategy, contributed to our understanding of the NA antigenic landscape and its sites of immunodominance, established a genetic reference for NA-reactive B cells in the C57BL/6 murine model, and characterized a subtype-conserved and partially protective epitope on the NA underside. Using a distinct but complementary immunogen design approach, we engineered neuraminidase-on-a-string (NoaS) nanoparticles (NPs) to display tandemly-linked NA dimers. By varying the sequence diversity between the two NA strains in the dimers, we investigated how antigenic distance between the NA strains influenced the breadth and cross-reactivity of antibody responses. We found that incorporating an antigenically distinct second strain enhanced the breadth and enzymatic inhibition potential of the immune sera. However, strain-specific responses also increased as a function of the antigenic distance between the two NA strains. Collectively, these data guide iterative designs of NA immunogens. More broadly, they inform the development of next-generation influenza vaccines that include NA components to enhance the breadth of vaccine-induced immunity.