Glycosylations in the Globular Head of the Hemagglutinin Protein Modulate the Virulence and Antigenic Properties of the H1N1 Influenza Viruses

Hemagglutinin glycosylations modulate the virulence and antigenicity of H1N1 influenza viruses. Hitting Flu Head-On Viruses and the immune system are engaged in a battle royale—each constantly trying to upstage the other. One way that flu viruses try to combat the immune response is through a series of mutations—known as antigenic drift. One such type of mutation is the introduction of a glycosylation site on the antigenic globular head of influenza hemagglutinin (HA) proteins. Glycosylation is thought to shield regions from targeting antibodies, but it has remained unclear how exactly HA glycosylation affects the virus–immune system interaction. Now, Medina et al. report that glycosylation not only affected influenza virus pathogenicity and allowed escape from polyclonal antibodies elicited by previous influenza virus strains but also affected their ability to induce cross-reactive antibodies against drifted antigenic variants. The authors introduced glycosylations onto the HA head of pandemic 2009 H1N1 influenza in a manner reflecting the timing of their appearance in previous seasonal viral drifted strains. They found that glycosylations could protect a strain from preexisting immunity against a wild-type version of the virus. However, previous infection with viruses glycosylated at particular residues led to protection against both glycosylated and wild-type viruses, as well as elicited cross-reactive immunity against other influenza viruses containing multiple glycosylations. These data suggest that altering glycosylations could be a potential strategy to improve upon the flu vaccine. With the global spread of the 2009 pandemic H1N1 (pH1N1) influenza virus, there are increasing worries about evolution through antigenic drift. One way previous seasonal H1N1 and H3N2 influenza strains have evolved over time is by acquiring additional glycosylations in the globular head of their hemagglutinin (HA) proteins; these glycosylations have been believed to shield antigenically relevant regions from antibody immune responses. We added additional HA glycosylation sites to influenza A/Netherlands/602/2009 recombinant (rpH1N1) viruses, reflecting their temporal appearance in previous seasonal H1N1 viruses. Additional glycosylations resulted in substantially attenuated infection in mice and ferrets, whereas deleting HA glycosylation sites from a pre-pandemic virus resulted in increased pathogenicity in mice. We then more directly investigated the interactions of HA glycosylations and antibody responses through mutational analysis. We found that the polyclonal antibody response elicited by wild-type rpH1N1 HA was likely directed against an immunodominant region, which could be shielded by glycosylation at position 144. However, rpH1N1 HA glycosylated at position 144 elicited a broader polyclonal response able to cross-neutralize all wild-type and glycosylation mutant pH1N1 viruses. Moreover, mice infected with a recent seasonal virus in which glycosylation sites were removed elicited antibodies that protected against challenge with the antigenically distant pH1N1 virus. Thus, acquisition of glycosylation sites in the HA of H1N1 human influenza viruses affected not only their pathogenicity and ability to escape from polyclonal antibodies elicited by previous influenza virus strains but also their ability to induce cross-reactive antibodies against drifted antigenic variants.