A Computationally Optimized Hemagglutinin Virus-Like Particle Vaccine Elicits Broadly Reactive Antibodies that Protect Nonhuman Primates from H5N1 Infection

MAJOR ARTICLE A Computationally Optimized Hemagglutinin Virus-Like Particle Vaccine Elicits Broadly Reactive Antibodies that Protect Nonhuman Primates from H5N1 Infection 1 Center for Vaccine Research, 2Department of Microbiology and Molecular Genetics, 3Graduate Program in Immunology, and 4Division of Neuropathology, Department of Pathology, University of Pittsburgh, Pennsylvania; and 5Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee Background. Highly pathogenic H5N1 avian influenza viruses continue to spread via waterfowl, causing lethal infections in humans. Vaccines can prevent the morbidity and mortality associated with pandemic influenza isolates. Predicting the specific isolate that may emerge from the 10 different H5N1 clades is a tremendous challenge for vaccine design. Methods. In this study, we generated a synthetic hemagglutinin (HA) on the basis of a new method, computationally optimized broadly reactive antigen (COBRA), which uses worldwide sequencing and surveillance efforts that are specifically focused on sequences from H5N1 clade 2 human isolates. Results. Cynomolgus macaques vaccinated with COBRA clade 2 HA H5N1 virus-like particles (VLPs) had hemagglutination-inhibition antibody titers that recognized a broader number of representative isolates from divergent clades as compared to nonhuman primates vaccinated with clade 2.2 HA VLPs. Furthermore, all vaccinated animals were protected from A/Whooper Swan/Mongolia/244/2005 (WS/05) clade 2.2 challenge, with no virus detected in the nasal or tracheal washes. However, COBRA VLP–vaccinated nonhuman primates had reduced lung inflammation and pathologic effects as compared to those that received WS/05 VLP vaccines. Conclusions. The COBRA clade 2 HA H5N1 VLP elicits broad humoral immunity against multiple H5N1 isolates from different clades. In addition, the COBRA VLP vaccine is more effective than a homologous vaccine against a highly pathogenic avian influenza virus challenge. Since the first identified cases of H5N1 influenza in Hong Kong [1], the World Health Organization (WHO) influenza surveillance network increased its intensive monitoring of both human and avian populations for influenza virus infections [2]. Following the resurgence and subsequent spread of H5N1 influenza Received and accepted 13 October 2011; electronically published 23 March 2012. Correspondence: Ted M. Ross, PhD, University of Pittsburgh, School of Medicine, Center for Vaccine Research, 9047 Biomedical Science Tower 3, 3501 Fifth Ave, Pittsburgh, PA 15261 (). The Journal of Infectious Diseases 2012;205:1562–70 © The Author 2012. 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/jis232 1562 • JID 2012:205 (15 May) • Giles et al in 2004, there has been an intense need for vaccine strategies that elicit broadly reactive immunity against H5N1 isolates, in light of its pandemic potential. Vaccination is a potent and cost-effective countermeasure to the threat of seasonal or pandemic outbreaks of influenza [3]. However, there are several challenges for designing an effective vaccine against H5N1 influenza, including the phylogenetic characterization and sequence homology in hemagglutinin (HA) that groups H5N1 viruses into 10 clades as defined by the WHO/ OIE/FAO H5N1 Evolution Working Group [4], with many clades having additional subclades and subsubclades [5]. Human infections have been limited to clades 0, 1, 2, and 7. To address the sequence diversity inherent in H5N1 isolates circulating in avian populations, consensus Brendan M. Giles,1,2,3 Corey J. Crevar,1,2 Donald M. Carter,1,2 Stephanie J. Bissel,4 Stacey Schultz-Cherry,5 Clayton A. Wiley,4 and Ted M. Ross1,2,3 nonhuman primate model that is based on an avian clade 2 H5N1 isolate and demonstrates the pathologic or immune responses induced following infection of vaccinated or unvaccinated macaques. In this report, COBRA HA proteins were displayed on the surface of a virus-like particle (VLP) and used to vaccinate cynomolgus macaques. The elicited immune responses were directly compared to a VLP with the HA from a clade 2.2 isolate, A/Whooper swan/Mongolia/244/2005, to analyze the protective responses to H5N1 challenge because the clade 2.2 viruses are the most geographically widespread of the H5N1 isolates [22–25]. The COBRA HA VLP not only elicited broader antibody responses against H5N1 but also protected nonhuman primates from clade 2.2 H5N1 challenge more efficiently than the homologous vaccine. MATERIAL AND METHODS Expression and Purification of VLPs The COBRA HA sequence has been described [26]. Briefly, the sequence was generated by aligning amino acid sequences of all available HA proteins isolated from clade 2 H5N1 human infections (2004–2007) and deposited in the Influenza Virus Resource database available through the National Center for Biotechnology Information. 293T cells were transiently transfected with plasmids expressing HA, M1, and neuraminidase (NA) in low-serum media, incubated for 72 hours at 37°C, and purified by ultracentrifugation through a 20% glycerol cushion as previously described [26]. HA content was quantified as previously described [26] with WS/05 VLPs at 0.38 μg/μL HA and COBRA VLPs at 0.27 μg/μL. Vaccines were normalized for HA content (15 μg), and HA titer was measured to confirm quality. HA titers of both vaccines were between 1:256 and 1:512 at all time points. Nonhuman Primate Immunizations and H5N1 Challenges Figure 1. Phylogenetic diversity of H5N1 influenza virus. The unrooted phylogenetic tree was inferred from hemagglutinin (HA) amino acid sequences derived from 8–10 representative isolates in all clades and subclades and also the computationally optimized broadly reactive antigen (COBRA) HA, using the maximum likelihood method. Clade/subclade clusters were identified and are indicated in the shaded ovals. The star identifies where the COBRA antigen is located relative to the various representative isolates. Sequences were aligned with MUSCLE 3.7 software, and the alignment was refined by Gblocks 0.91b software. Phylogeny was determined using the maximum likelihood method with PhyML software. Trees were rendered using TreeDyn 198.3 software [44]. The National Center for Biotechnology Information accession numbers for the HA sequences used in phylogeny inference were obtained through the Influenza Virus Resource [45]. Cynomolgus macaques (Macaca fascicularis, male, 3–5 years old) were vaccinated with 15 μg (on the basis of HA content) of purified COBRA HA VLPs (n = 7) or WS/05 VLPs (n = 7) via intramuscular injection at weeks 0, 3, and 6. Vaccines at each dose were formulated with alum adjuvant (Imject Alum, Pierce Biotechnology, Rockford, IL) immediately prior to use. Imject Alum adjuvant and vaccine schedule were chosen on the basis of findings from a previous study involving ferrets (...truncated)