A universal influenza vaccine must provide protection against antigenically divergent influenza viruses either through broadly neutralizing antibodies or cross-reactive T cells. Here, intranasal immunizations with recombinant adenoviral vectors (rAd) encoding hemagglutinin (HA) and nucleoprotein (NP) in combination with rAd-Interleukin-(IL)-1β or rAd-IL-18 were evaluated for their efficacy in BALB/c mice. Mucosal delivery of rAd-IL-1β enhanced HA-specific antibody responses including strain-specific neutralizing antibodies. Nevertheless, the beneficial effects on the local T cell responses were much more impressive reflected by increased numbers of CD103CD69 tissue-resident memory T cells (T). This increased immunogenicity translated into superior protection against infections with homologous and heterologous strains including H1N1, pH1N1, H3N2, and H7N7. Inhibition of the egress of circulating T cells out of the lymph nodes during the heterologous infection had no impact on the degree of protection underscoring the unique potential of T for the local containment of mucosal infections. The local co-expression of IL-1β and antigen lead to the activation of critical checkpoints in the formation of T including activation of epithelial cells, expression of chemokines and adhesion molecules, recruitment of lung-derived CD103 DCs, and finally local T imprinting. Given the importance of T-mediated protection at mucosal barriers, this study has major implications for vaccine development.

The Respiratory Syncytial Virus (RSV) and Influenza A Virus (IAV) are both two major causative agents of severe respiratory tract infections in humans leading to hospitalization and thousands of deaths each year. In this study, we evaluated the immunogenicity and efficacy of a combinatory DNA vaccine in comparison to the single component vaccines against both diseases in a mouse model. Intramuscular electroporation with plasmids expressing the hemagglutinin (HA) of IAV and the F protein of RSV induced strong humoral immune responses regardless if they were delivered in combination or alone. In consequence, high neutralizing antibody titers were detected, which conferred protection against a lethal challenge with IAV. Furthermore, the viral load in the lungs after a RSV infection could be dramatically reduced in vaccinated mice. Concurrently, substantial amounts of antigen-specific, polyfunctional CD8+ T-cells were measured after vaccination. Interestingly, the cellular response to the hemagglutinin was significantly reduced in the presence of the RSV-F encoding plasmid, but not vice versa. Although these results indicate a suppressive effect of the RSV-F protein, the protective efficacy of the combinatory vaccine was comparable to the efficacy of both single-component vaccines. In conclusion, the novel combinatory vaccine against RSV and IAV may have great potential to reduce the rate of severe respiratory tract infections in humans without increasing the number of necessary vaccinations.

In respect to the heterogeneity among influenza A virus strains and the shortcomings of current vaccination programs, there is a huge interest in the development of alternative vaccines that provide a broader and more long-lasting protection. Gene-based approaches are considered as promising candidates for such flu vaccines. In our study, innate signalling molecules from the RIG-I and the NALP3 pathways were evaluated as genetic adjuvants in intramuscular DNA immunizations. Plasmids encoding a constitutive active form of RIG-I (cRIG-I), IPS-1, IL-1β, or IL-18 were co-administered with plasmids encoding the hemagglutinin and nucleoprotein derived from H1N1/Puerto Rico/8/1934 via electroporation in BALB/ c mice. Immunogenicity was analysed in detail and efficacy was demonstrated in homologous and heterologous influenza challenge experiments. Although the biological activities of the adjuvants have been confirmed by in vitro reporter assays, their single or combined inclusion in the vaccine did not result in superior vaccine efficacy. With the exception of significantly increased levels of antigen-specific IgG1 after the co-administration of IL-1β, there were only minor alterations concerning the immunogenicity. Since DNA electroporation alone induced substantial inflammation at the injection site, as demonstrated in this study using Mx2-Luc reporter mice, it might override the adjuvants´contribution to the inflammatory microenvironment and thereby minimizes the influence on the immunogenicity. Taken together, the DNA immunization was protective against subsequent challenge infections but could not be further improved by the genetic adjuvants analysed in this study.

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