Double-attenuated influenza virus elicits broad protection against challenge viruses with different serotypes in swine.

Influenza A viruses (IAV) have caused seasonal epidemics and severe pandemics in humans. Novel pandemic strains as in 2009 may emerge from pigs, serving as perpetual virus reservoir. However, reliably effective vaccination has remained a key issue for humans and swine. Here, we generated a novel double-attenuated influenza live vaccine by reverse genetics and subjected immunized mice and pigs to infection with the homologous wild-type, another homosubtypic H1N1, or a heterosubtypic H3N2 virus to address realistic challenge constellations. This attenuated mutant contains an artificial, strictly elastase-dependent hemagglutinin cleavage site and a C-terminally truncated NS1 protein from the IAV A/Bayern/74/2009 (H1N1). Prior to challenge, we immunized mice once and pigs twice intranasally. In vitro, the double-attenuated mutant replicated strictly elastase-dependently. Immunized mice and pigs developed neither clinical symptoms nor detectable virus replication after homologous challenge. In pigs, we observed considerably reduced clinical signs and no nasal virus shedding after homosubtypic and reduced viral loads in respiratory tracts after heterosubtypic infection. Protection against homosubtypic challenge suggests that an optimized backbone strain may require less frequent updates with recent HA and NA genes and still induce robust protection in relevant IAV hosts against drifted viruses.