A Critical HA1 Neutralizing Domain of H5N1 Influenza in an Optimal Conformation Induces Strong Cross-Protection

et al. (2013) A Critical HA1 Neutralizing Domain of H5N1 Influenza in an Optimal Conformation Induces Strong Cross-Protection. PLoS ONE 8(1): e53568. doi:10.1371/journal.pone.0053568 A Critical HA1 Neutralizing Domain of H5N1 Influenza in an Optimal Conformation Induces Strong Cross- Protection Lanying Du 0 Guangyu Zhao 0 Shihui Sun 0 Xiujuan Zhang 0 Xiaojun Zhou 0 Yan Guo 0 Ye Li 0 Yusen Zhou 0 Shibo Jiang 0 Jianqing Xu, Fudan University, China 0 1 Lindsley F. Kimball Research Institute, New York Blood Center , New York , New York, United States of America, 2 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology , Beijing , China , 3 School of Medical Laboratory Science, Wenzhou Medical College , Wenzhou, Zhejiang , China , 4 Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University , Shanghai , China 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-13-263Fc 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. - Funding: This work was supported by grants from National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (NIAID) of the United States (R03AI088449) to LD, from the National 973 Basic Research Program of China (2005CB523001) to YZ, and from the Chinese Ministry of Science & Technology, Hong Kong, Macau, and Taiwan Collaborative Programs (201200007673) to SJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The highly pathogenic avian influenza (HPAI) A/H5N1 is considered a significant threat for the next influenza pandemic. The genetic variability of this virus makes it an unprecedented risk for the global spread of the new virus strains. Although 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. Among all influenza virus proteins, HA, a major antigen on the viral surface, serves as an important protein in inducing neutralizing antibodies and cross-protection [3]. The HAspecific antibodies could neutralize infectivity of the HPAI N5N1 viruses by interacting with the receptor binding domain (RBD) or blocking conformational rearrangement associated with membrane fusion [4,5]. It has been reported that antibodies to virus HA protein mediate heterosubtype neutralizing responses to A/H5N1 viruses in healthy volunteers exposed to H5N1 [6]. Animals vaccinated with HA DNA also show higher neutralizing antibody responses and/or better protection than NA, NP, or M2 DNA vaccines against challenges with homologous or heterologous H5N1 viruses [7]. A tri-clade DNA vaccine encoding HA of clade 0, 2.3.2.1 and 7.2 elicits broadly neutralizing antibody responses against H5 clades and subclades and protects mice against heterologous H5N1 challenge [8]. Therefore, based on its strong ability to induce neutralizing antibodies and protection, HA is considered a primary target for designing effective vaccines against H5N1 virus infection. The HA protein is a homotrimer. Each of its single-chain monomers initially synthesizes as a precursor polypeptide, HA0, which is then cleaved by host proteases into two subunits, HA1 and HA2 [9]. The RBD of H5N1 viruses is located at the Nterminal HA1 region, covering amino acid residues from around 112 to 263 [1012]. A reassortant virus, comprising four mutations (N158D/N224K/Q226L/T318I) of H5 HA (three of which are in RBD) and seven gene segments from a 2009 pandemic H1N1 virus, may preferentially recognize human-type receptors and transmit efficiently in ferrets, emphasizing the importance of HA, particularly RBD, in receptor binding specificity, virus infection and transmission. The success of laboratory-generated transmissible mutant virus and continual evolvement of H5N1 viruses in the nature significantly increase the possibility for emerging receptor-binding variants of H5N1 viruses with pandemic potential [1]. Therefore, identification of the critical neutralizing domain (CND) of HA, particularly RBD, will be of great importance to develop efficacious and safe vaccines against variant H5N1 virus. It should be noted that (...truncated)