Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection

In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9) virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.
Author Summary Several hundred human avian H7N9 virus infections with a case fatality rate of approximately 37% have occurred in China since 2013. The emergence of this virus has raised concerns about its pandemic potential and has triggered the development of H7N9 vaccines. Using the traditional correlate of protection for influenza viruses—the hemagglutination inhibition assay—H7N9 vaccines prove to be poorly immunogenic. However, once antibody levels are high enough substantial crossreactivity is observed. Here we characterize several monoclonal antibodies against the H7N9 hemagglutinin. One set of antibodies is active in the hemagglutination inhibition assay across a panel of distant H7 virus strains and the common conserved epitope of these antibodies might explain the broad crossreactivity observed with H7N9 vaccines. The second set of antibodies shows no antiviral activity in vitro but is highly protective in vivo, partially through Fc-mediated functions. Targeting of the non-neutralizing but protective epitope of these antibodies by the immune system might explain the 'low immunogenicity' observed in H7N9 vaccine trials. In conclusion our data suggests that current H7N9 vaccines could protect against divergent H7 strains with pandemic potential in the future and that the traditional correlates of protection for influenza vaccines might not capture the protective immunity to H7 viruses.