This work was funded in part by the Spanish Government, Ministerio de Econom?a y Competitividad de España (MINECO), project: AGL2013-48923-C2-2-R, and by the collaborative infrastructure project funded by the European Comission (EC) under Horizon 2020, project Transvac2-730964-INFRAIA-2016-1. IRTA is supported by CERCA Programme/ Generalitat de Catalunya. M.S.O. is supported by MINECO (scholarship n BES-2014-068506). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Swine influenza virus (SIVs) infections cause a significant economic impact to the pork industry. Moreover, pigs may act as mixing vessel favoring genome reassortment of diverse influenza viruses. Such an example is the pandemic H1N1 (pH1N1) virus that appeared in 2009, harboring a combination of gene segments from avian, pig and human lineages, which rapidly reached pandemic proportions. In order to confront and prevent these possible emergences as well as antigenic drift phenomena, vaccination remains of vital importance. The present work aimed to evaluate a new DNA influenza vaccine based on distinct conserved HA-peptides fused with flagellin and applied together with Diluvac Forte as adjuvant using a needle-free device (IntraDermal Application of Liquids, IDAL®). Two experimental pig studies were performed to test DNA-vaccine efficacy against SIVs in pigs. In the first experiment, SIV-seronegative pigs were vaccinated with VC4-flagellin DNA and intranasally challenged with a pH1N1. In the second study, VC4-flagellin DNA vaccine was employed in SIV-seropositive animals and challenged intranasally with an H3N2 SIV-isolate. Both experiments demonstrated a reduction in the viral shedding after challenge, suggesting vaccine efficacy against both the H1 and H3 influenza virus subtypes. In addition, the results proved that maternally derived antibodies (MDA) did not constitute an obstacle to the vaccine approach used. Moreover, elevated titers in antibodies both against H1 and H3 proteins in serum and in bronchoalveolar lavage fluids (BALFs) was detected in the vaccinated animals along with a markedly increased mucosal IgA response. Additionally, vaccinated animals developed stronger neutralizing antibodies in BALFs and higher inhibiting hemagglutination titers in sera against both the pH1N1 and H3N2 influenza viruses compared to unvaccinated, challenged-pigs. It is proposed that the described DNA-vaccine formulation could potentially be used as a multivalent vaccine against SIV infections.