Influenza (flu) remains a major global health challenge nearly ninety years after its discovery. A major goal of my dissertation research projects was to develop a novel influenza vaccine platform to improve cross-protection and to better understand innate and adaptive immune correlates. In chapter one, to address the issue of low vaccine effectiveness, I designed a new cross-protective mRNA construct encoding influenza B virus (IBV) NA (NA mRNA) encapsulated into lipid nanoparticles (LNP) and co-administered at a low dose with inactivated IBV split vaccine (B/Florida/4/2006) as a proof-of-concept test. The combination of NA mRNA and inactivated split IBV vaccines was found to induce significantly higher levels of cross-reactive IgG responses, NA and HA inhibition titers, effector and memory cellular responses, as well as cross-lineage protection than singular vaccines. These results suggest that the NA mRNA vaccine enhances the cross-protective efficacy of the combined NA mRNA and split vaccines.

In chapter two, I investigated the underlying innate and adaptive immune responses by which a combination of NA mRNA-LNP and split vaccines enhances IBV-specific immunogenicity. The NA mRNA-LNP could enhance split IBV vaccine efficacy by rapidly eliciting cytokine and chemokine releases, inducing the activation of innate immune cells traveling to draining lymph nodes for antigen presentation within a day after immunization. Furthermore, NA mRNA-LNP co-administered with IBV split vaccine showed a significant enhancement of effector memory T cells and humoral immune responses compared to either split or mRNA-LNP vaccination only. These findings demonstrated that co-administration of NA mRNA-LNP with split vaccines could enhance immunogenicity and cross-protection by effectively activating innate immune cells, thereby eliciting stronger adaptive responses than singular vaccines.

In chapter three, I investigated the molecular biomarkers and signaling pathway involved in the immunogenicity and cross-protection of combination NA mRNA and sFL vaccine. I found that both NA mRNA only and the NA mRNA+sFL could significantly elicit type I IFN productions and cellular responses to type I IFNs signaling pathway. Interestingly, only the NA mRNA+sFL significantly increased expression of Klrk1 and NKG2D‑ligand genes. Consequently, loss of CD94/NKG2A inhibitory checkpoint, vaccinated mice experienced severe weightloss, indicating the role of educated NK cells. By contrast, the combination NA mRNA and sFL vaccines operate dependent on type I IFN–mediated immunity.