Short Communication Protective immunity elicited by a pseudotyped baculovirus-mediated bivalent H5N1 influenza vaccine Qunfeng Wu a , Shaobo Xiao a , Huiying Fan b , Yang Li a , Jinfang Xu a , Zhen Li a , Wei Lu a , Xiaoyue Su a , Wei Zou a , Meilin Jin a , Huanchun Chen a , Liurong Fang a,⇑ a Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China b College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China article info Article history: Received 3 August 2011 Revised 4 October 2011 Accepted 4 October 2011 Available online 12 October 2011 Keywords: Influenza virus Vaccine Hemagglutinin Bivalent vaccine abstract The development of novel H5N1 influenza vaccines to elicit a broad immune response is a priority in vet- erinary and human public health. In this report, a baculovirus vector was used to construct bivalent recombinant baculovirus vaccine encoding H5N1 influenza virus hemagglutinin proteins (BV-HAs) from clade 2.3.4 and clade 9 influenza viruses. Mice immunized with 5 Â 10 7 IFU BV-HAs developed signifi- cantly high levels of H5-specific neutralizing antibodies and cellular immunity that conferred 100% pro- tection against infection with H5N1 influenza viruses. This study suggests that baculovirus-delivered multi-hemagglutinin proteins might serve as a candidate vaccine for the prevention of pre-pandemic and pandemic H5N1 influenza viruses. Ó 2011 Elsevier B.V. All rights reserved. Since 2003, repeated outbreaks of highly pathogenic avian influenza (HPAI) H5N1 virus infections have occurred in poultry in eastern Asia and have had significantly devastating effects on the poultry industries of these regions (Li et al., 2004). More impor- tantly, H5N1 influenza virus can be transmitted directly from in- fected poultry to humans, causing serious respiratory diseases and even fatalities (Abdel-Ghafar et al., 2008). The wide geographic distribution and spread of H5N1 influenza virus in avian species and in humans is unprecedented (Neumann et al., 2010). Together with the ongoing genetic evolution of this virus, H5N1 influenza virus has a high potential for future pandemics. Therefore, it is an urgent need to develop effective vaccines to control the spread of H5N1 influenza virus, and the prevention of transmission of the virus between poultry and humans is a global priority. In our previous study, baculovirus pseudotyped with vesicular stomatitis virus (VSV) glycoprotein was used as a vector to express the hemagglutinin (HA) protein of HPAI, A/Chicken/Hubei/327/ 2004 (HB/327). Mice immunized with the resultant recombinant baculovirus (BV-G-HA) developed effective immune responses and afforded complete protection against infection by homologous virus (Wu et al., 2009). However, only moderate protection was provided against an evolutionarily distant strain (unpublished data). It is well known that the main barrier in influenza virus vaccine development is the high degree of genetic variability of the virus (Chen et al., 2008). For instance, current monovalent inactivated whole virus vaccine could induce a substantial neutralizing anti- body response against the homologous strain, but a decreased re- sponse to heterologous strains (Ehrlich et al., 2008). One way to minimize the antigenic diversity between the vaccine strain and circulating viruses is to perhaps create a vaccine expressing anti- gens covering the major circulating virus strains (Prabakaran et al., 2010). To attempt to broaden the protective effect of the BV-G-HA vaccine, we constructed a bivalent recombinant baculovi- rus vaccine expressing two HA proteins derived from two different HPAI H5N1 viruses, clade 9 and clade 2.3.4 (known as ‘Fujian-like’ viruses), representing the circulating strain in central China and in southeast Asia in recent years (Li et al., 2010; Zhou et al., 2010; Zou et al., 2009). The protective immune elicited by BV-HAs was evaluated and compared with those induced by a mono-HA-based BV-HA in a mouse challenge model. To construct the recombinant baculovirus encoding multiple HA proteins from different strains of H5N1 viruses, the HA gene coding sequences from clade 2.3.4 and clade 9 viruses were opti- mized for mammalian expression and synthesized (GenScript). The synthesized HA gene fragments were inserted into the pFast- Bac-VSV/G (Wu et al., 2009), under the control of the immediate early promoter of cytomegalovirus (CMV). Recombinant BV-HAs (Fig. 1A) were subsequently generated using the Bac-to-Bac Ò system (Invitrogen). A recombinant BV-HA expressing the codon- optimized HA gene of clade 2.3.4 was also constructed (Fig. 1A). 0166-3542/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2011.10.001 ⇑ Corresponding author. Address: Division of Animal Infectious Diseases, College of Veterinary Medicine, Huazhong Agricultural University, 1 Shi-zi-shan Street, Wuhan 430070, China. Tel.: +86 27 8728 6884; fax: +86 27 8728 1795. E-mail address: fanglr@mail.hzau.edu.cn (L. Fang). Antiviral Research 92 (2011) 493–496 Contents lists available at SciVerse ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral