Mucosal administration of raccoonpox virus expressing highly pathogenic avian H5N1 influenza neuraminidase is highly protective against H5N1 and seasonal influenza virus challenge

We previously generated recombinant poxviruses expressing influenza antigens and studied their efficacy as potential highly pathogenic avian influenza (HPAI) vaccines in mice. While both modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN) expressing hemagglutinin (HA) provided strong protection when administered by parenteral routes, only RCN-neuraminidase (NA) showed promise as a mucosal vaccine. In the present study we evaluated the efficacy of RCN-NA constructs by both intradermal (ID) and intranasal (IN) routes. Surprisingly, while RCN-NA completely protected mice when administered by the IN route, it failed to protect mice when administered by the ID route. After challenge, significantly less virus induced pathology was observed in the lungs of mice vaccinated with RCN-NA by the IN route as compared to the ID route. Furthermore, IN administration of RCN-NA elicited neutralizing antibodies detected in bronchoalveolar lavage (BAL) samples. We also determined the role of cellular immune responses in protection elicited by RCN-NA by depleting CD4 and CD8 T cells prior to challenge. Finally, we demonstrated for the first time that antibodies against NA can block viral entry in addition to viral spread in vitro. These studies demonstrate the importance of mucosal administration of RCN viral vectors for eliciting protective immune responses against the NA antigen.