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Abstract Background Currently licensed human vaccines are subtype-specific and do not protect against pandemic H5N1 viruses. Previously, our group has reported on the construction of an influenza virus-like particle (VLP) as a new generation candidate vaccine. A mixture of influenza H5N1 VLPs representing clade 1 and 2 viruses were examined for the ability to elicit protective immunity against isolates from various clades and subclades of H5N1. Results Mice were vaccinated intramuscularly with each VLP individually, the mixture of VLPs, a mixture of purified recombinant hemagglutinin (rHA), or mock vaccinated. Elicited antibodies were assayed for the hemagglutination-inhibition (HAI) activity against clades 1 and clade 2 isolates. Mice vaccinated with each VLP individually or in a mixture had robust HAI responses against homologous viruses and HAI responses against the clade 2.3 virus, Anh/05. However, these vaccines did not induce an HAI response against the clade 2.2 virus, WS/05. Interestingly, clade 2 VLP vaccinated mice were protected against both clade 1 and 2 H5/PR8 viruses, but clade 1 VLP vaccinated mice were only protected against the clade 1 virus. Mice vaccinated with a mixture of VLPs were protected against both clade 1 and 2 viruses. In contrast, mice vaccinated with a mixture of rHA survived challenge, but lost ~15% of original weight by days 5–7 post-challenge. Conclusion These results demonstrate that a multivalent influenza VLP vaccine representing different genetic clades is a promising strategy to elicit protective immunity against isolates from emerging clades and subclades of H5N1.

Vaccination is the most effective way to prevent influenza virus infections. However, the diversity of antigenically distinct isolates is a challenge for vaccine development. In order to overcome the antigenic variability and improve the protective efficacy of influenza vaccines, our research group has pioneered the development of computationally optimized broadly reactive antigens (COBRA) for hemagglutinin (HA). Two candidate COBRA HA vaccines, P1 and X6, elicited antibodies with differential patterns of hemagglutination inhibition (HAI) activity against a panel of H1N1 influenza viruses. In order to better understand how these HA antigens elicit broadly reactive immune responses, epitopes in the Cb, Sa, or Sb antigenic sites of seasonal-like and pandemic-like wild-type or COBRA HA antigens were exchanged with homologous regions in the COBRA HA proteins to determine which regions and residues were responsible for the elicited antibody profile. Mice were vaccinated with virus-like particles (VLPs) expressing one of the 12 modified HA antigens (designated V1 to V12), COBRA HA antigens, or wild-type HA antigens. The elicited antisera was assessed for hemagglutination inhibition activity against a panel of historical seasonal-like and pandemic-like H1N1 influenza viruses. Primarily, the pattern of glycosylation sites and residues in the Sa antigenic region, around the receptor binding site (RBS), served as signatures for the elicitation of broadly reactive antibodies by these HA immunogens. Mice were vaccinated with VLPs expressing HA antigens that lacked a glycosylation site at residue 144 and a deleted lysine at position 147 residue were more effective at protecting against morbidity and mortality following infection with pandemic-like and seasonal-like H1N1 influenza viruses. IMPORTANCE There is a great need to develop broadly reactive or universal vaccines against influenza viruses. Advanced, next-generation hemagglutinin (HA) head-based vaccines that elicit protective antibodies against H1N1 influenza viruses have been developed. This study focused on understanding the specific amino acids around the receptor binding site (RBS) that were important in elicitation of these broadly reactive antibodies. Specific glycan sites and amino acids located at the tip of the HA molecule enhanced the elicitation of these broadly reactive antibodies. A better understanding of the HA structures around the RBS will lead to more effective HA immunogens. [ABSTRACT FROM AUTHOR]