DNA Prime and Virus-like Particle Boost From a Single H5N1 Strain Elicits Broadly Neutralizing Antibody Responses Against Head Region of H5 Hemagglutinin

MAJOR ARTICLE DNA Prime and Virus-like Particle Boost From a Single H5N1 Strain Elicits Broadly Neutralizing Antibody Responses Against Head Region of H5 Hemagglutinin 1 Unit of Antiviral Immunity and Genetic Therapy, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China; 2Institut Pasteur in Cambodia, Phnom Penh; 3Comprehensive AIDS Research Center, School of Medicine, Tsinghua University, Beijing; and 4Shanghai Institute of Biological Products, Shanghai, China Since 1996, highly pathogenic avian influenza (HPAI) H5N1 virus has presented a persistent threat to public health. Its high degree of genetic diversity also poses enormous challenges in developing effective vaccines. To search for vaccine regimens that could elicit broadly neutralizing antibody responses against diverse HPAI H5N1 strains, in the present study we tested H5 hemagglutinin (HA) from an A/Thailand/1(KAN)-1/2004 strain in a heterologous prime-boost vaccination. We demonstrated that priming mice with DNA and boosting with virus-like particle induced antibody responses that cross-neutralize all reported clades and subclades of HPAI H5N1 viruses and protect mice from high lethal dose HPAI H5N1 challenge in both active and passive immunizations. Unexpectedly, cross-divergent H5 neutralizing antibodies are directed to the HA head and block both attachment and postattachment of virus entry. Thus, we conclude that as a promising pan-H5 vaccine candidate this prime-boost regimen could be further developed in ferrets and in humans. Keywords. HPAI H5N1; vaccination; neutralizing antibody responses. Since 1996, the highly pathogenic avian influenza (HPAI) H5N1 virus has spread in a variety of domestic and wild birds and was sporadically transmitted to humans in Asia, Europe, and Africa. As of 7 March 2013, the World Organization for Animal Health had highlighted thousands of HPAI H5N1 outbreaks in poultry and wild birds in 63 countries [1]. As of 15 February 2013, 620 human H5N1 infections had been confirmed, resulting in 367 deaths [2]. Although to date Received 2 April 2013; accepted 1 July 2013; electronically published 2 August 2013. Presented in part: WHO Influenza Vaccine Meeting, 24 January 2013, Hong Kong. Correspondence: Paul Zhou, PhD, Unit of Antiviral Immunity and Genetic Therapy, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai, China 200025 (). The Journal of Infectious Diseases 2014;209:676–85 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: . DOI: 10.1093/infdis/jit414 676 • JID 2014:209 (1 March) • Wang et al the human cases have largely been infected by close contact with sick poultry and the isolated viruses still show characteristics of avian influenza viruses, H5N1 strains with enhanced binding affinity to the humantype receptor have been reported [3]. In addition, 2 highly publicized studies show that laboratory-adapted H5N1 variants with few amino acid residue mutations in hemagglutinin (HA) were able to spread through aerosol from infected to uninfected ferrets [4, 5]. Moreover, database search identified 2 H5N1 strains whose HA needs only 2 additional mutations to create variants with the transmissible feature identified in one of the above studies [6]. These findings raised serious concerns about the possible evolving of HPAI H5N1 virus to become transmissible among people and resulting in a global pandemic. When spreading over much of the eastern hemisphere, HA of HPAI H5N1 viruses has evolved into 10 clades in various host species [7]. Among them, clade 2 is divided into 5 subclades (2.1, 2.2, 2.3, 2.4, and 2.5), Guiqin Wang,1 Fan Zhou,1 Philippe Buchy,2 Teng Zuo,3 Hongxing Hu,1 Jingjing Liu,1 Yufeng Song,1 Heng Ding,1 Cheguo Tsai,1 Ze Chen,4 Linqi Zhang,3 Vincent Deubel,2 and Paul Zhou1 virus-like particles (VLPs) from the same strain. We demonstrated that this prime-boost regimen induces antibody responses that cross-neutralize all reported clades and subclades of HPAI H5N1 viruses and HA peptide–specific CD8 T-cell responses and protects mice from high lethal dose of divergent HPAI H5N1 challenge by both active and passive immunizations. Moreover, we demonstrated that the cross-divergent H5 neutralizing antibodies are directed to the head region of HA and block both virus attachment and postattachment. Thus, this is the first report that a heterologous prime-boost regimen with HA from a single HPAI H5N1 strain could elicit antibody responses that cross-neutralize all reported clades and subclades of HPAI H5N1 viruses and that such antibody responses are directed to the head region of HA. MATERIALS AND METHODS Active and Passive Immunization and Challenge Female BALB/c mice (6–8 weeks old) were primed twice intramuscularly with codon optimized H5 HA DNA and boosted once intraperitoneally with VLPs expressing H5 HA from the A/Thailand/1(KAN)-1/04 strain. Control mice were primed twice with empty vector and boosted with human immunodeficiency virus type 1 (HIV-1) gag alone VLPs. For the sake of brevity in the remaining text, we use the terms “TH-DDV” for 2 DNA injections followed by 1 VLP injection of H5 HA from an A/Thailand/1(KAN)-1/2004 strain and “control-DDV” for 2 injections of empty vector followed by 1 injection of HIV-1 Figure 1. Phylogenetic analysis of hemagglutinin gene of H5 avian influenza viruses. Using the neighbor-joining method, the bootstrap consensus tree inferred from 1000 replicates was analyzed. Most strains were chosen from the World Health Organization–suggested vaccine strains. The A/Thailand/1 (KAN-1)/2004 strain and consensus sequence are shown in blue. *Challenge strains. Broad Antibodies Induced by Prime Boost • JID 2014:209 (1 March) • 677 and clade 7 is divided into 2 subclades (7.1 and 7.2). Subclade 2.1 is further divided into 2.1.1, 2.1.2, 2.1.3, 2.1.3.1, 2.1.3.2, and 2.1.3.3; subclade 2.2 into 2.2.1, 2.2.2, and 2.2.1.1; and subclade 2.3 into 2.3.1, 2.3.2, 2.3.2.1, 2.3.3, 2.3.4, 2.3.4.1, 2.3.4.2, and 2.3.4.3. To deal with this high level of genetic diversity, 2 general vaccine approaches are currently explored. The first approach is based on using particular strains or combinations of strains (multivalent) selected from a geographic region where the vaccine is intended for use [8–13]. For example, the World Health Organization (WHO) has already created 22 seed vaccine strains for use in different geographic regions. These strains cover (sub)clades 1.1, 1, 2.1, 2.1.3.2, 2.2, 2.2.1.1, 2.2.1, 2.3.2.1, 2.3.4, 4, and 7.1 [8]. The second approach is to construct either a consensus or an ancestral HA sequence reconstructed on the basis of an evolutionary model [14, 15]. Such sequences have the advantage of being central and most similar to currently circulating strains (...truncated)