Abstract
The highly pathogenic avian influenza (HPAI) H5N1 viruses, especially the laboratory-generated H5N1 mutants, have demonstrated the potential to cross the species barrier and infect mammals and humans. Consequently, the design of an effective and safe anti-H5N1 vaccine is essential. We previously demonstrated that the full-length hemagglutinin 1 (HA1) could induce significant neutralizing antibody response and protection. Here, we intended to identify the critical neutralizing domain (CND) in an optimal conformation that can elicit strong cross-neutralizing antibodies and protection against divergent H5N1 strains. We thus constructed six recombinant proteins covering different regions of HA1 of A/Anhui/1/2005(H5N1), each of which was fused with foldon (Fd) and Fc of human IgG. We found that the critical fragment fused with Fd/Fc (HA-13–263-Fdc, H5 numbering) that could elicit the strongest neutralizing antibody response is located in the N-terminal region of HA1 (residues 13–263), which covers the receptor-binding domain (RBD, residues 112–263). We then constructed three additional recombinants fused with Fd plus His tag (HA-13–263-Fd-His), Fc only (HA-13–263-Fc), and His tag only (HA-13–263-His), respectively. We found that the HA-13–263-Fdc, which formed an oligomeric conformation, induced the strongest neutralizing antibody response and cross-protection against challenges of two tested H5N1 virus strains covering clade 1: A/VietNam/1194/2004 (VN/1194) or clade 2.3.4: A/Shenzhen/406H/06 (SZ/406H), while HA-13–263-Fc dimer and HA-13–263-Fd-His trimer elicited higher neutralizing antibody response and protection than HA-13–263-His monomer. These results suggest that the oligomeric form of the CND containing the RBD can be further developed as an effective and safe vaccine for cross-protection against divergent strains of H5N1 viruses.
Highlights
The highly pathogenic avian influenza (HPAI) A/H5N1 is considered a significant threat for the influenza pandemic
The expressed proteins were shown as monomers in the boiled sample lines, but the molecular weight shown in the non-boiled proteins is around 2fold higher than that of the boiled samples after addition of the sample buffer containing 2% SDS and 1% 2-mercaptoethanol reducing agent and migration of the gels in the presence of 0.1% SDS, indicating that all expressed proteins fused with Fd and Fc formed suitable conformational structures similar to dimers in the reducing SDS-PAGE (Fig. 2A)
Western blot results revealed strong bands corresponding to the relative molecular weight of the recombinant proteins, demonstrating that all proteins were recognized by a hemagglutinin 1 (HA1)-specific mAb (Fig. 2B)
Summary
The highly pathogenic avian influenza (HPAI) A/H5N1 is considered a significant threat for the influenza pandemic. Human-tohuman transmission of this virus has been very rare, this phenomenon is challenged by recent successful transmission of the laboratory-generated mutant H5N1 virus [1,2]. Either insertion of mutated hemagglutinin (HA) gene of H5N1 into a 2009 pandemic H1N1 strain or selection of a H5N1 virus strain with five mutations results in the generation of viruses able to confer efficient transmissibility among ferrets, an animal model closely resembling humans in flu studies [1,2]. Since the H5N1 virus has shown case fatality rate around 60% with 359 deaths among a total 608 human infections reported to WHO as of August 10, 2012 (http://www.who.int/influenza/ human_animal_interface/ EN_GIP_20120810CumulativeNumberH5N1cases.pdf), suitable measures and novel strategies are urgently needed to prevent the potential threat caused by H5N1 viruses with divergent strains. Effective vaccines would play a key role in preventing the dire predictions noted above
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