In the last decades, several cases of human infection with the highly pathogenic avian influenza virus (HPAIV) H5N1 have been reported by the World Health Organization http://www.who.int/influenza/human_animal_interface/avian_influenza/en/
). It is a common assumption that the pig may act as mixing vessel to generate new reassortant influenza viruses due to the presence of receptors for both avian and mammalian influenza viruses in the epithelial cells of their respiratory tract 
. A recent example of the latter caused the first pandemia of the 21st century, starting in 2009 as a consequence of the global spread of a swine-origin influenza virus A H1N1 (pH1N1). This was a virus that contained genes from avian, pig and human origin 
. Although the virus was not as pathogenic to humans as expected, severe disease cases associated with pH1N1 have been more recently reported in England (http://www.who.int/influenza/surveillance_monitoring/updates/2010_12_30_GIP_surveillance/en/
). The future evolution of this or any emergent influenza virus (IV) is uncertain. This is a distressing matter particularly because available vaccines and therapies are strictly restricted to phylogenetically closely related circulating viruses. Therefore, finding universal and effective vaccines and therapeutic measures to fight against future IV is a must for public health.
IV hemagglutinin (HA) is a viral surface polypeptide that mediates both, the binding of IV to the host cell surface and the fusion of viral and endosomal membranes 
. HA is formed by subunit 1 (HA1) and subunit 2 (HA2) and both the N- and C- terminal parts of HA1 together with HA2 comprise the stalk of the molecule 
. Vaccines designed to elicit antibodies against the stalk of HA are reported to confer protection against IV infection in mice 
. HA1, although highly variable, encodes specific and highly conserved domains which may be involved in determining the recognition and targeting (RTD) of influenza viruses to their receptor as revealed by the Informational Spectrum Method (ISM) 
. This includes the VIN1 domain, located within the site E in the N-terminus of HA1 
. In contrast with the high variability suffered by the globular part of the HA1 molecule, which is directly responsible for the receptor tropism, the site E remains relatively highly conserved 
. Thus, representing potential targets to develop broad array of protective therapies and vaccines against IV infection.
Due to the already mentioned recent cases
related to H5N1 and H1N1 IV subtypes, and because their potential to cause future outbreaks among the population, we focused our efforts on designing a vaccine capable of confering protection against both viral subtypes. As previously reported, RTD of HA1 from different H1N1 strains and HA1 from the recently emerged in Egypt H5N1 IV encode the same information. However, HA1 from H3N2 and all other H5N1 viruses encode different RT information 
. Thus, aiming to increase the vaccine coverage, one HA1-peptide from the VIN1 domain of H1N1 and three HA1-peptides from two different H5N1 IV strains were designed and selected based on ISM.
In order to test the immunogenicity of our experimental vaccine, we decided to immunize conventional pigs with the combination of the synthesized peptides. Pigs allow the evaluation of the protective efficacy of experimental vaccines against several viral strains, including the recently pandemic H1N1 virus, pH1N1 
. Confirming the rationale behind their use as a pre-clinical animal model, immunization of conventional pigs with the VIN1-peptide cocktail allow us to demonstrate the induction of peptide-specific antibody and T-cell responses in every single animal, independently of their swine leukocyte antigen (SLA)-haplotype. Specific B and T-cell responses were induced against each one of the H1 and H5-peptides used, confirming their immunogenicity in vivo.
Interestingly, the elicited antibodies also recognized several heterologous viruses in vitro
, including the pH1N1, two swine influenza field isolates (SwH1N1 and SwH3N2) and a H5N1 highly pathogenic avian virus. This, together with the fact that the specific T-cell responses induced were also able to recognize the inactivated pH1N1, encouraged us to challenge all pigs with the pH1N1 influenza virus. Albeit preliminary, our results demonstrate that VIN1-vaccination was able to confer a partial protection against intranasal challenge with pH1N1, as demonstrated with the partial and total viral clearance from the lung lavages in two out of four immunized pigs. We believe that our results could contribute to the obtainment of a broader array of protective vaccines against future influenza outbreaks or even pandemics.