Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. Whilst vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic that is highly efficacious at low doses and is broadly specific against antigenically-drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific-Fc fusion protein (FcDART). In mice, a single 2.5mg/kg therapeutic or prophylactic dose of either monoclonal antibody provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically-drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, based on antibody escape mutations. Whilst it was possible to generate escape mutants in vitro, they were neutralized in vivo by the antibodies, as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutant in vitro. In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses.
