Pentameric M2e influenza vaccine candidate generates strong immunity with limited survival benefit

We previously demonstrated that native-like oligomers of Influenza A H1N1 M2e, expressed in Expi293F cells, elicit robust immune responses. Building on this foundation, we engineered a recombinant antigen comprising five tandem repeats of the M2e epitope (M2e-5x) fused to the tGCN4 domain, which promotes oligomerization and stability. BALB/c mice were immunized intramuscularly with M2e-5x antigen formulated in AddaVax^TM, in a prime-boost regimen, which elicited strong humoral and cellular responses. Despite this, vaccinated animals did not survive following high-dose lethal challenge with Influenza A/PR/8/34 (H1N1) and X-31 (H3N2) viruses. However, notable reductions in lung viral titers and less severe histopathological damage were observed compared to unvaccinated controls, suggesting partial viral clearance. Further evaluation of <i>in vivo</i> protective efficacy by combining M2e-5x (35 µg) with a low dose of soluble trimeric HA (5 µg) antigen showed complete protection in challenged mice. The overall results emphasized the importance of multi-antigen formulations for protective and robust influenza immunity. These findings highlight the importance of incorporating conserved and key viral epitopes into vaccine formulations to enhance broad and robust protection, providing valuable guidance for future influenza vaccine development.