Elsevier

Vaccine

Volume 29, Issue 16, 5 April 2011, Pages 3043-3054
Vaccine

A computationally optimized broadly reactive antigen (COBRA) based H5N1 VLP vaccine elicits broadly reactive antibodies in mice and ferrets

https://doi.org/10.1016/j.vaccine.2011.01.100Get rights and content

Abstract

Pandemic outbreaks of influenza are caused by the emergence of a pathogenic and transmissible virus to which the human population is immunologically naïve. Recent outbreaks of highly pathogenic avian influenza (HPAI) of the H5N1 subtype are of particular concern because of the high mortality rate (60% case fatality rate) and novel subtype. In order to develop a vaccine that elicits broadly reactive antibody responses against emerging H5N1 isolates, we utilized a novel antigen design technique termed computationally optimized broadly reactive antigen (COBRA). The COBRA HA sequence was based upon HA amino acid sequences from clade 2 H5N1 human infections and the expressed protein retained the ability to bind the receptor, as well as mediate particle fusion. Non-infectious recombinant VLP vaccines using the COBRA HA were purified from a mammalian expression system. Mice and ferrets vaccinated with COBRA HA H5N1 VLPs had protective levels of HAI antibodies to a representative isolates from each subclade of clade 2. Furthermore, VLP vaccinated animals were completely protected from a lethal challenge of the clade 2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005. This is the first report describing the use of COBRA-based antigen design. The COBRA HA H5N1 VLP vaccine elicited broadly reactive antibodies and is an effective influenza vaccine against HPAI virus.

Introduction

The swine-origin H1N1 pandemic of 2009 reminded the worldwide community of the ever-present threat of pandemic influenza. Pandemic outbreaks of influenza are caused by the emergence and spread of a pathogenic and transmissible virus to which the human population is immunologically naïve [1]. Although predicting an emerging pandemic subtype of influenza is difficult, outbreaks of highly pathogenic avian influenza of the H5N1 subtype are of particular concern because of the high mortality rate (60% case fatality rate) and novel subtype [2]. To date, H5N1 influenza has not transmitted efficiently from person to person, but accumulation of mutations or reassortment with a human transmissible virus could result in a highly transmissible H5N1 virus [3]. H5N1 and contemporary H3N2 seasonal influenza viruses are able to generate stable reassortant viruses although the pathogenic potential of the reassortant viruses is less than that of the highly pathogenic H5N1 virus [4], [5]. Additionally, a recent report has demonstrated that not only can H1N1 pandemic viruses and highly pathogenic H5N1 viruses efficiently reassort, but these reassortants can replicate to higher titers than the source H5N1 virus [6]. The genetic compatibility between the influenza viruses, combined with the continued spread of both novel H1N1 in humans and highly pathogenic H5N1 in wild birds, highlights the potential for a new emerging H5N1 influenza infecting the human population and therefore, the need to develop effective vaccines against H5N1 isolates.

One of the challenges to developing effective H5N1 vaccines is the antigenic diversity within the subtype. H5N1 viruses are separated into distinct clades based upon phylogenetic distance among the hemagglutinin (HA) genes [7]. The clades are geographically diverse and are evolving under unique pressures specific to each respective location [8]. The majority of human infections were identified within the antigenically distinct clades 1 and 2, with clade 2 infections spanning over 60 countries and moving westward from Asia into Africa and the Middle East [9]. Genetic diversity within clade 2 has resulted in distinct subclades including 2.1, 2.2, 2.3, 2.4 and 2.5 with some subclades being further divided into additional sub-subclades [7]. Despite high levels of HA protein sequence homology between clades (>90%), there is little receptor blocking antibody cross-reactivity across clades and even within subclades [7]. Developing vaccines that are able to overcome the challenge of H5N1 antigenic diversity is a crucial step in pandemic preparedness.

The antigenic diversity of all subtypes of influenza is a challenge to influenza vaccine development in general. The current seasonal influenza vaccine uses a polyvalent formulation to address the issue of multiple subtypes simultaneously circulating in the human population. Even though a representative vaccine strain is selected and is expected to represent the most common strain for each subtype in a given season, vaccine escape occurs and yearly epidemics continue to happen. A recent report has demonstrated that a polyvalent H5N1 vaccine with components derived from various clades can elicit cross-clade antibody cross-reactivity and protective efficacy [10]. Alternative strategies that have been investigated for addressing the challenge of antigenic diversity include targeting conserved viral proteins, such as the M2 ion channel or nucleoprotein (NP) and targeting conserved domains of HA [11], [12], [13], [14]. Additionally, engineering synthetic antigens that capture common immune epitopes from a population of primary viruses has the potential to overcome antigenic diversity.

Consensus-based H5N1 have been generated for several influenza proteins including HA, neuraminidase (NA) and matrix (M1) and each has elicited cross-reactive immune responses [15], [16], [17], [18]. Consensus sequences are traditionally generated by aligning a population of sequences and selecting the most common residue at each position. These sequences are expected to effectively capture conserved linear epitopes and elicit cross-reactive cellular immune responses [15], [19], [20], [21]. Furthermore, consensus-based H5N1 HA immunogens expressed from DNA plasmids elicit broad antibody responses [16], [18]. However, consensus-based antigen design is inherently influenced by the input sequences used to generate the synthetic molecule and as such is subject to sampling bias. Using the NCBI Influenza Virus Resource database, the vast majority of HA sequences from both human and environmental isolates are from clades 1 and 2 [22]. Furthermore, within clade 2 the majority of human isolates are from infections in Indonesia and the subclade 2.1. The predominance of certain isolates, i.e. a majority of clade 2.1 isolates from Indonesia, can bias the output consensus sequence generated and may not accurately reflect the genetic diversity of H5N1 influenza viruses. In order to overcome these limitations, we report a new methodology of antigen design using multiple rounds of consensus generation termed computationally optimized broadly reactive antigen (COBRA). This method was designed to address the diversity specifically within clade 2 and utilized global surveillance efforts to generate a vaccine with the potential to elicit increased breadth of antibody responses within this antigenically diverse clade.

In this study, COBRA HA antigens are expressed on virus-like particles (VLPs) and purified as vaccine immunogens. VLPs are self-assembling, nonpathogenic, genomeless particles that are similar in size and morphology to intact virions [23]. VLPs can be produced in a variety of eukaryotic expression systems including yeast, insect and mammalian cells. Importantly, A VLP-based vaccine which protects against human papillomavirus has been approved for human use [24]. Influenza virus VLP vaccines are attractive alternatives to traditional split vaccines because they do not require the use of any live virus at any step of the vaccine production process [25]. Additionally, VLPs present surface antigens in their native oligomeric structures which are important for maintaining conformational epitopes.

We hypothesized that the COBRA HA delivered in the context of a VLP will elicit an antibody response that demonstrates increased breadth when compared to that of a VLP containing an HA molecule derived from a primary isolate. Here, we report the COBRA HA is a functional molecule, elicits broadly reactive antibody responses, and completely protects mice and ferrets from a heterologous, highly pathogenic H5N1 virus challenge.

Section snippets

Antigen construction and synthesis

Influenza A HA nucleotide sequences isolated from human H5N1 infections were downloaded from the NCBI Influenza Virus Resource database (see supporting materials for complete list of accession numbers and isolate descriptions) [22]. Nucleotide sequences were translated into protein sequences using the standard genetic code. Full length sequences from H5N1 clade 2 human infections from 2004 to 2006 were acquired and used for subsequent consensus generations. For each round of consensus

Computationally optimized broadly reactive antigen design

To address the challenge of antigenic diversity present in H5N1 influenza, we designed a computationally optimized broadly reactive antigen (COBRA). For the first step of antigen generation, 129 unique hemagglutinin (HA) sequences were downloaded from the NCBI Influenza Virus Resource (IVR) sequence database [22] representing clade 2 H5N1 viruses isolated from human infections between 2004 and 2006. The sequences were first grouped into phylogenetic subclades and then further divided into

Discussion

In this study, we investigated the efficacy of a computationally optimized broadly reactive antigen (COBRA) approach designed for influenza HA as a H5N1 vaccine candidate. Centralized vaccines are a potential strategy for inducing broadly reactive immune responses and are comprised of synthetic antigens that represent a population of sequences. These vaccine antigens are generated by three different methods: center-of-the-tree, ancestral, and consensus [38]. Center-of-the-tree (COT) sequences

Acknowledgments

This work was supported by an NIH training grant award T32AI060525 to BMG, NIH/NIAID award U01AI077771 to TMR, the Center for Vaccine Research and a collaborative research grant from PATH Vaccine Solutions. This project is funded, in part, under a grant with the Pennsylvania Department of Health. The department specifically disclaims responsibility for any analyses, interpretations or conclusions. The authors would like to thank Brianne Stein and Corey Crevar for technical assistance. In

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