Combining innovative molecular adjuvanting approaches with novel adenoviral vector delivery to generate a universal influenza vaccine

SUMMARY: Influenza A viruses (IAVs) are important human pathogens, which cause seasonal epidemics and sporadic pandemics. The ongoing threat posed by emerging zoonotic influenza viruses, for which humans are immunologically naïve, represents a major global concern. Current influenza vaccines elicit narrow, strain- specific immunity, are overly reliant on egg-based manufacturing, have a prolonged production process, and fail to elicit robust cellular and humoral immune responses to multiple IAV antigens (Ags) simultaneously. The variable effectiveness of seasonal influenza vaccines, has highlighted the importance of investing in the early pre-clinical evaluation of innovative vaccines which could elicit immune responses with increased breadth against emerging viruses/variants. Efforts to develop a universal influenza virus vaccine, capable of providing broad and long-lived protection against seasonal and pandemic subtypes, are focused on inducing immune responses directed towards highly conserved epitopes on influenza virus Ags such as the stalk of the major surface glycoprotein hemagglutinin (HA), the neuraminidase (NA) or the internal nucleoprotein (NP). In this R01, we will develop an innovative, optimized, universal influenza vaccine platform to overcome issues associated with current vaccines. Preliminary data generated through R21 funding enabled the identification of lead headless HAs for group 1 IAVs, and demonstrated that we can successfully encode at least two Ags in a single expression cassette. We will now build upon these data and engineer bi- or tri-cistronic Ag cassettes encoding our lead headless HAs in combination with NA and/or NP. We will augment and broaden immune recognition of the immunosubdominant HA stalk, or long overlooked NA, by using employing a fusion- Ag based molecular adjuvanting approach called "exosome-display", which facilitates targeting of Ags to host- derived extracellular vesicles including exosomes in vivo. Exosomes play important roles in the regulation of immunity, and we have demonstrated that exosome-display can dramatically increase the immunogenicity of a model Ag encoded by two distinct adenoviral (Ad) vector platforms. Optimized, "adjuvanted" Ag expression cassettes will be engineered into Ad vectored vaccines with low seroprevalence in humans, allowing in vivo tethering of Ag to host-derived exosomes. This could potentiate immune responses by increasing recognition of the encoded Ag by the immune system. Finally, we will comprehensively evaluate and phenotype the immune profile of these universal vaccines in single-shot regimens, and test efficacy in lethal challenge with heterologous and heterosubtypic IAVs. Ad vectors have risen to prominence during the coronavirus pandemic, due to their ease of manufacturing, cheap cost, the possibility for thermostabilization with minimal losses to immunogenicity under cold-chain free conditions, making them ideal candidates for equitable global distribution. Therefore, the Ad-based universal influenza vaccines described in this R01 would be suited to stockpiling for pandemic preparedness, and could provide "universal" protection following a single shot.