Journal of Virological Methods 194 (2013) 280–288 Contents lists available at ScienceDirect Journal of Virological Methods jou rn al hom epage: www.elsevier.com/locate/jviromet The highly conserved HA2 protein of the influenza a virus induces a cross protective immune response Jong-Soo Lee a , Mohammed Y.E. Chowdhury a , Ho-Jin Moon a , Young-Ki Choi b , Melbourne R. Talactac a , Jae-Hoon Kim a , Min-Eun Park a , Hwa-Young Son a , Kwang-Soon Shin a , Chul-Joong Kim a,∗ a College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea b College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea Article history: Received 26 March 2013 Received in revised form 23 July 2013 Accepted 21 August 2013 Available online 1 September 2013 Keywords: Influenza vaccine Cross protection Immunization Hemagglutinin a b s t r a c t Existing influenza vaccines protect mostly homologous subtypes and acted most effectively only when well matched to the circulating strain. Immunization with an updated vaccine is therefore necessary to maintain long-term protection and the development of a broadly protective influenza vaccine against the threat of pandemic outbreak. The highly conserved HA2 glyco-polypeptide (HA2 gp) is a promising new candidate for such an influenza vaccine. Helical domain and the fusion peptide (residues 15–137) of surface antigen from influenza A subtype A/EM/Korea/W149/06 (H5N1) was used to assess the potential- ity of HA2 vaccination against multiple subtypes of the influenza viruses. The construct, named H5HA2 was expressed in Escherichia coli and allowed to refold from inclusion bodies. Purified proteins were used to investigate the immunogenicity of H5HA2 and its potential for cross protection. The immu- nization of mice with H5HA2 induced HA2 antibodies, HA2 specific T-cell responses, and protection against homologous A/EM/Korea/W149/06 (H5N1) influenza. Immunized mice were also protected from two distinct heterosubtypes of influenza: A/Puerto Rico/1/34(H1N1) and bird/Korea/w81/2005(H5N2). Results suggest that recombinant proteins based on the highly conserved residues within HA2 are candidates for the development of vaccines against pandemic outbreaks of emergent influenza variants. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Acute respiratory illness caused by the influenza A virus is a major global health concern. The severity of infection varies with the virulence of the strain as well as the age and immune status of the host (Glezen and Couch, 1978). Vaccination is the only effective way to prevent influenza virus infection as this virus easily evolves resistance to antiviral drugs, such as the neuraminidase inhibitors, oseltamivir and zanamivir and the M2 ion channel blockers, riman- tidin and amantidine, (Beigel et al., 2005). Current trivalent vaccines Abbreviations: HA2, hemagglutinin-2; gp, glycol-polypeptide; IgG, immunoglobulin G; HRP, horseradish peroxidase; MLD50, 50% mouse lethal dose; dpi, day post infection; TCID50, median tissue culture infectious dose; OD, optical density; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunosorbent spot; BSL, bio-safety level. ∗ Corresponding author. Present address: College of Veterinary Medicine, Chung- nam National University, 220 Gung-Dong, Yuseong-Gu, Daejeon 305-764, Republic of Korea. Tel.: +82 42 821 7910; fax: +82 42 825 7910. E-mail addresses: dalim2000@gmail.com, dalim 2000@yahoo.com (M.Y.E. Chowdhury), cjkim@cnu.ac.kr (C.-J. Kim). confer protection only against the homologous vaccine strain as well as closely related variants and rarely provide cross protec- tion against divergent strains within and across subtypes (Ekiert et al., 2009). Therefore, the development of a vaccine that protects against a wide range of influenza subtypes is a priority for pandemic preparedness. The hemagglutinin (HA) of the influenza virus is a polypeptide consisting of the HA1 and HA2 sub-domains (Wang et al., 2010). HA2 is considerably more conserved than HA1 (Both et al., 1983) and is able to induce a specific antibody response during natural infection in human (Styk et al., 1979) and mice (Kostolansky et al., 2002). Antibodies induced by HA2 are highly cross reactive within a given subtype and even among various subtypes of influenza virus (Gocník et al., 2008). N-terminal fusion peptides (the first 11 residues), are highly conserved among all known influenza sub- types with a few minor substitutions (Du et al., 2010). Recently, the immunogen produced by the expression of the stalk region of HA on the surface of mammalian cell (Steel et al., 2010) and in Escherichia coli (Bommakanti et al., 2010) was found to be effective against pathogenic influenza. In both studies, the HA2 stalk domain and fusion peptide along with some residues from the HA1 domain were used to construct the immunogen. 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.08.022