Cross-clade protective immunity of H5N1 influenza vaccines in a mouse model
Introduction
Since the outbreak of H5N1 influenza A virus in humans in 1997, these viruses have continued to exert a growing toll, with more than 240 confirmed fatal human cases (http://www.who.int/csr/disease/avian_influenza/en/). With the re-emergence of H5N1 virus in humans in 2003, the epidemic regions have expanded from Asia to Europe, the Middle-East and Africa, raising concerns over a possible influenza pandemic [1]. In the event of such a pandemic, vaccination is one of the most potent methods of protection against this deadly threat. Outbreaks and the pandemic potential of H5N1 viruses have, for this reason, led to stockpiling of H5N1 pre-pandemic inactivated vaccines for human use in many countries.
The considerable diversity in hemagglutinin (HA) antigenicity among the H5N1 viruses, which transpired as a result of naturally occurring genetic alterations, has led to the creation of distinct clades and subclades [2]. It is difficult to predict which H5N1 virus could become a pandemic virus. Moreover, current H5N1 inactivated vaccines show low immunogenicity in humans [3], such that antigenic matching of vaccine viruses to a pandemic virus would be required for efficient protection. Therefore, the World Health Organization (WHO) now recommends the stockpiling of a panel of vaccines with HA antigenic variations, including clade 1 viruses, which have circulated mainly in Southeast Asia, as well as clade 2.1, 2.2, and 2.3.4 viruses, which are circulating predominantly in Indonesia, Asia, Europe and Africa, and Asian countries including China, respectively, as pre-pandemic vaccines [4]. Previously, the HA antigenicities of H5N1 viruses were characterized by using polyclonal and monoclonal antibodies, revealing clade/subclade-dependent antigenic variations, which included cross-reactivity to each other to various extents [4], [5], [6], [7], [8], [9]. However, systemic evaluation of the immunogenic relationship of H5N1 vaccines, including cross-clade protection, has not been carried out using animal models, with the exception of some attributive cross-protection analyses between limited clades, such as clade 1 and 2.1 viruses [6], [10], [11], [12], [13], [14], [15].
In this study, we prepared four H5N1 test vaccines, associated with clade 1, 2.1, 2.2, and 2.3.4 viruses, immunized mice with each vaccine and then challenged them with homologous as well as heterologous virulent viruses of different clades to evaluate fundamental immunogenic variations between H5N1 viruses in a mouse model.
Section snippets
Cells
Madin-Darby canine kidney (MDCK) cells were grown in minimal essential medium (MEM) with 5% newborn calf serum. 293T human embryonic kidney cells were maintained in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) with 10% fetal calf serum. Cells were maintained at 37 °C in 5% CO2.
Viruses
The H5N1 A/Vietnam/30259/04 (VN30259; clade 1), A/Indonesia/3006/05 (Indo3006; clade 2.1), A/whooper swan/Mongolia/4/05 (Mongolia4; clade 2.2), and A/Vietnam/30850/05 (VN30850; clade 2.3.4) viruses were
Growth properties of PR8/H5N1 6:2 reassortants in embryonated chicken eggs
We successfully generated four PR8/H5N1 6:2 reassortants bearing modified avirulent-type HA (mHA) and NA derived from different phylogenetic clade H5N1 viruses, namely VN30259 (clade 1), Indo3006 (clade 2.1), Mongolia4 (clade 2.2), and VN30850 (clade 2.3.4), by reverse genetics. We assessed their growth properties in embryonated chicken eggs, which is the main substrate for influenza vaccine production (Fig. 1). The virus NIBRG-14 [bearing mHA and NA derived from VN1194 and its remaining genes
Discussion
In this study, we produced four H5N1 inactivated vaccines with PR8/H5N1 6:2 reassortants from isolates of different phylogenetic clades (clade 1, 2.1, 2.2, and 2.3.4), based on the WHO-recommended reverse genetics strategy, and assessed their cross-clade antigenicity/immunogenicity by using serological assays and a mouse vaccination/challenge model. Although we detected clear variations in viral HA antigenicity of the H5N1 viruses by serological analyses, any H5N1 test vaccine, except one clade
Acknowledgements
We thank Drs. J.M. Wood and J.S. Robertson (National Institute for Biological Standards and Control, UK), for NIBRG-14 virus, and S. Watson for editing the manuscript. This work was supported, in part, by Grants-in-Aid for Specially Promoted Research and for Scientific Research (B); by a Contract Research Fund for Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases; and by the Special Coordination Funds for Promoting Science and Technology from the Ministry of
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These authors contributed equally to this work.