Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemicand mucosal immune responses in mice

We have generated recombinant influenza A viruses belonging to the H1N1 and H3N2 virus subtypes containing an insertion of the 137 C-terminal amino ac id residues of the human immunodeficiency virus type 1 (HIV-1) Nef protein into the influenza A virus nonstructural-protein (NS1) reading frame. These viral vectors were found to be genetically stable and capable of growing e fficiently in embryonated chicken eggs and tissue culture cells but did not replicate in the murine respiratory tract. Despite the hyperattenuated phe notype of influenza/NS-Nef viruses, a Nef and influenza virus (nucleoprotei n)-specific CD8(+)-T-cell response was detected in spleens and the lymph no des draining the respiratory tract after a single intranasal immunization o f mice. Compared to the primary response, a marked enhancement of the CD8()-T-cell response was detected in the systemic and mucosal compartments, in cluding mouse urogenital tracts, if mice were primed with the HINT subtype vector and subsequently boosted with the H3N2 subtype vector. In addition, Nef-specific serum IgG was detected in mice which were immunized twice with the recombinant H1N1 and then boosted with the recombinant H3N2 subtype vi rus. These findings may contribute to the development of alternative immuni zation strategies utilizing hyperattenuated live recombinant influenza viru s vectors to prevent or control infectious diseases, e.g., HIV-1 infection.