RESUMEN
It is still a challenge to develop needle-free mucosal vaccines. Despite progress in the development of the influenza vaccine, it must be reformulated annually because of antigenic changes in circulating influenza viral strains. Due to seasonal drift and shift of circulating strains, the influenza vaccine does not always match the circulating strains, and included adjuvants are not sufficient to induce a protective effect with long-lived memory cells. The adjuvants play a major role in the immune responses to a vaccine. Interestingly, the Bacillus anthracis detoxified anthrax edema toxin, which composes of protective antigen PA and N-fragment of edema factor (EFn), has shown improved effects for humoral and cellular immune responses. Here we describe the design of a universal influenza vaccine construct that consists of three tandem M2e repeats of the influenza antigen plus HA2 and detoxified toxin EFn, which is associated with the PA component, as well as the techniques used to corroborate protection. We present two major parts of description to demonstrate the vaccine strategy, using detoxified anthrax toxin for intranasal delivery of influenza antigen: (1) vaccine candidate design, production, and purification; (2) influenza virus microneutralization assay and cellular responses and lethal challenge with influenza viruses and B. anthracis Sterne spores. In the methods detailed here, we used different versions of the M2e-HA2 proteins.