Investigation of neuraminidase as a target antigen for a universal influenza virus vaccine

PROJECT SUMMARY Influenza A (IAV) viruses cause significant global morbidity and mortality annually, and unpredictable pandemic viruses emerging at irregular intervals pose a significant threat to public health. Current seasonal influenza virus vaccines elicit strain-specific immunity with variable effectiveness, depending on the degree of matching with circulating virus strains. Importantly, these vaccines do not confer protection against novel pandemic or emergent viruses. As such, a more effective “universal” influenza virus vaccine that provides broader protection against the increasing diversity of strains and subtypes is urgently needed. Such efforts should prioritize the identification and targeting of novel broadly protective epitopes capable of eliciting heterosubtypic and/or pan-group immunity. Moreover, this work should prioritize the investigation of exotic IAV subtypes from emerging zoonotic viruses, as highly pathogenic avian influenza (HPAI) infections are increasingly prevalent, and these viruses are considered a serious global health threat. In addition to IAV hemagglutinin (HA), the viral neuraminidase (NA) represents a worthy, but currently understudied universal influenza virus vaccine target. In this F31 application, I will address these gaps in our knowledge, generating tools to identify broadly protective epitopes on influenza neuraminidases (NA) from exotic, under-investigated subtypes from emerging zoonotic viruses and deepen our understanding of NA-based mechanisms of protection. In Aim 1, we will validate a panel of NAs as structurally, functionally, and antigenically authentic. Lead candidates will be used for the development of unique nanobodies which we will use as tools to aid the identification of novel broadly cross-reactive epitopes. In Aim 2, lead NA candidates will be incorporated into non-replicating adenovirus (Ad) vectors, and we will assess the in vivo immunogenicity of Ad5-NA vaccines to identify innovative heterologous prime:boost regimens best suited for eliciting breadth. Monoclonal antibodies (mAbs) will be isolated from vaccinated mice and evaluated, in parallel with isolated nanobodies, in vitro for breadth and functionality, and in vivo for efficacy in IAV challenge studies. Finally, we will use structure-based approaches to identify the binding footprints of promising lead mAbs/nanobodies. Efficacy data will be linked with data from structural (i.e. epitope mapping) and in vitro analyses to identify broadly protective, cross-reactive epitopes. By completing the aims of the proposed research, we will improve our understanding of protective epitopes on under-studied NA subtypes from emerging zoonotic viruses and generate unique tools and reagents for pandemic preparedness. Together, these efforts to improve our understanding of NA-based immunity at an epitope level will contribute to the next-generation design of more broadly protective universal influenza vaccines for pandemic preparedness.