Self-Assembling Protein Nanoparticles Presenting Stabilized Hemagglutinin, Neuraminidase, and M2e Antigens for Universal Influenza Vaccine Development - Project Summary Influenza is an acute respiratory illness that infects ~1 billion people annually, causing 3–5 million cases of severe disease and up to 600,000 deaths worldwide. Approximately 75% of infections are caused by influenza A viruses (IAVs) and 25% by influenza B viruses (IBVs). Seasonal vaccines remain the primary tool for prevention, but their effectiveness (10–60%) is limited by mismatches with circulating strains. The ongoing risk of emerging H5 pandemic strains in dairy and poultry industries further underscores the urgent need for a universal influenza vaccine that provides broad, durable protection against both IAV and IBV. Developing such a vaccine requires rational strategies that address the structural and immunological challenges of hemagglutinin (HA), the major viral surface glycoprotein and primary target of neutralizing antibodies (NAbs). Neuraminidase (NA) and the highly conserved extracellular domain of matrix protein 2 (M2e) are also promising targets for eliciting non-NAb– mediated protection. We hypothesize that a multicomponent nanoparticle vaccine platform combining stabilized HA, NA, and M2e antigens will elicit complementary immune mechanisms — NAb responses to HA, NA-inhibitory antibodies to NA, and Fc-mediated functions targeting M2e — thereby providing broad protection against seasonal and pandemic influenza strains. Aim 1 will stabilize, optimize, and evaluate HA trimers containing the N95L/Q442L mutation, alone or in combination with pH-switch (pHS) 1/2 mutations, across representative IAV and IBV strains. We hypothesize that replacing a conserved buried hydrophilic residue (N95 in IAV, Q442 in IBV) with leucine enhances HA trimer stability. Preliminary data show favorable in vitro properties, robust NAb titers, and protection conferred by N95L/Q442L-bearing HA constructs. Our goal is to establish a broadly applicable HA stabilization strategy for universal vaccine design. Aim 2 will present stabilized HA trimers or NA monomers on single-component self-assembling protein nanoparticles (1c-SApNPs) with embedded IAV/IBV M2e epitopes to assess induction of NAb, NA-inhibitory, and effector responses, as well as protective breadth against diverse influenza strains. We have generated data demonstrating the efficacy of 1c-SApNPs displaying M2e (IAV M2ex3, IAV+IBV M2ex6) or stabilized HA trimers compared with their soluble forms. This project will integrate HA, NA, and M2e into a unified design. Aim 3 will investigate how glycan composition influences vaccine immunogenicity and protection. Preliminary data show that oligomannose enrichment via kifunensine treatment enhanced HA- induced NAb responses compared with wild-type or enzymatically trimmed glycans. Importantly, we will examine a novel glycoengineering strategy that incorporates the highly immunogenic core Į(1,3)-fucose into HA and NA glycans to enhance immune recognition. This research aligns with the NIAID Strategic Plan for the Development of a Universal Influenza Vaccine, addressing key priorities such as rational immunogen design, induction of both humoral and cellular responses, and mechanistic evaluation of vaccine breadth and durability. The proposed studies will yield critical insights and a practical platform for the development of universal influenza vaccines.
