Improved hatchability and efficient protection after in ovo vaccination with live-attenuated H7N2 and H9N2 avian influenza viruses

Mass in ovo vaccination with live attenuated viruses is widely used in the poultry industry to protect against various infectious diseases. The worldwide outbreaks of low pathogenic and highly pathogenic avian influenza highlight the pressing need for the development of similar mass vaccination strategies against avian influenza viruses. We have previously shown that a genetically modified live attenuated avian influenza virus (LAIV) was amenable for in ovo vaccination and provided optimal protection against H5 HPAI viruses. However, in ovo vaccination against other subtypes resulted in poor hatchability and, therefore, seemed impractical. In this study, we modified the H7 and H9 hemagglutinin (HA) proteins by substituting the amino acids at the cleavage site for those found in the H6 HA subtype. We found that with this modification, a single dose in ovo vaccination of 18-day old eggs provided complete protection against homologous challenge with low pathogenic virus in ≥70% of chickens at 2 or 6 weeks post-hatching. Further, inoculation of 19-day old egg embryos with 10(6 )EID(50 )of LAIVs improved hatchability to ≥90% (equivalent to unvaccinated controls) with similar levels of protection. Our findings indicate that the strategy of modifying the HA cleavage site combined with the LAIV backbone could be used for in ovo vaccination against avian influenza. Importantly, with protection conferred as early as 2 weeks post-hatching, with this strategy birds would be protected prior to or at the time of delivery to a farm or commercial operation.