DOI:10.1111/j.1750-2659.2009.00104.x www.blackwellpublishing.com/influenza Original Article Development of a new candidate H5N1 avian influenza virus for pre-pandemic vaccine production Jie Dong,a Yumiko Matsuoka,b Taronna R. Maines,b David E. Swayne,c Eduardo O’Neill,b C. Todd Davis,b Neal Van-Hoven,b Amanda Balish,b Hong-jie Yu,a Jacqueline M. Katz,b Alexander Klimov,b Nancy Cox,b De-xin Li,a Yu Wang,a Yuan-ji Guo,a Wei-zhong Yang,a Ruben O. Donis,b, Yue-long Shua aChinese National Influenza Center, State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, China CDC, Xuanwu District Beijing, China. bInfluenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA. cSoutheast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Athens, GA, USA Correspondence: Dr. Ruben O. Donis, Influenza Division, NCIRD, CCID, Centers for Disease Control and Prevention, 1600 Clifton Road - Mail Stop G-16 Atlanta, GA 30333, USA. E-mail: [email protected] Accepted 19 August 2009. Published Online 9 October 2009. Background Highly pathogenic H5N1 avian influenza viruses Methods A reassortant virus termed, Anhui ⁄ PR8, was produced currently circulating in birds have caused hundreds of human by reverse genetics in compliance with WHO pandemic vaccine infections, and pose a significant pandemic threat. Vaccines are a development guidelines and contains six genes from A ⁄ Puerto major component of the public health preparedness for this likely Rico ⁄ 8 ⁄ 34 as well as the neuraminidase and hemagglutinin (HA) event. The rapid evolution of H5N1 viruses has resulted in the genomic segments from the A ⁄ Anhui ⁄ 01 ⁄ 2005 virus. The multi- emergence of multiple clades with distinct antigenic basic cleavage site of HA was removed to reduce virulence. characteristics that require clade-specific vaccines. A variant Results The reassortant Anhui ⁄ PR8 grows well in eggs and is H5N1 virus termed clade 2.3.4 emerged in 2005 and has caused avirulent to chicken and ferrets but retains the antigenicity of the multiple fatal infections. Vaccine candidates that match the parental A ⁄ Anhui ⁄ 01 ⁄ 2005 virus. antigenic properties of variant viruses are necessary because inactivated influenza vaccines elicit strain-specific Conclusion These results indicate that the Anhui ⁄ PR8 reassortant protection. lost a major virulent determinant and it is suitable for its use in vaccine manufacturing and as a reference vaccine virus against the Objective To address the need for a suitable seed for H5N1 clade 2.3.4 viruses circulating in eastern China, Vietnam, manufacturing a clade 2.3.4 vaccine, we developed a new H5N1 Thailand, and Laos. pre-pandemic candidate vaccine by reverse genetics and evaluated its safety and replication in vitro and in vivo. Keywords Pandemic influenza, H5N1 vaccine, seed virus. Please cite this paper as: Dong et al. (2009) Development of a new candidate H5N1 avian influenza virus for pre-pandemic vaccine production. Influenza and Other Respiratory Viruses 3(6), 287–295. Introduction The pandemic of 1957 was caused by an H2N2 reassortant virus containing avian HA, neuraminidase (NA), and poly- Influenza viruses cause seasonal epidemics of acute respira- merase basic protein 1(PB1) genes in the background of tory disease with most severe consequences in pediatric and genes belonging to a circulating human H1N1 virus and geriatric populations. Sporadically, influenza A viruses with rapidly displaced the circulating H1N1 viruses.2–4 In 1968, a novel hemagglutinin (HA) subtype cause pandemics with an H3N2 reassortant virus containing avian HA and PB1 serious public health consequences. Pandemics are caused genes with the remaining genes from the contemporary by influenza viruses containing surface protein(s) for which H2N2 virus emerged to cause a pandemic.2,4,5 Another the human population has little or no immunity, and influenza pandemic could occur at any time and effective endowed with the ability to spread in the human popula- public health interventions based on appropriate prepared- tion. The three influenza pandemics of the 20th century ness are needed to diminish morbidity and mortality. involved the transmission of influenza viruses from animals Numerous different subtypes of the influenza A virus to humans.1 The most severe pandemic of the previous cen- exist in nature, resulting from combinations of 16 serotypes tury was the ‘Spanish Influenza’ of 1918–1919, which spread of HA (H1–H16) and nine serotypes of NA (N1–N9).6 The globally in just 1 year and caused at least 40 million deaths.1 human population has immunity to only a few of the ª 2009 Blackwell Publishing Ltd, Influenza and Other Respiratory Viruses, 3, 287–295 287 Dong et al. known serotypes. To date, viruses with HA subtypes (H1, ture Collection (Manassas, VA) and cultured in Dulbecco’s H2, H3, H5, H6, H7, and H9) and four NAs (N1, N2, N3, modified Eagle’s Medium (DMEM) containing 10% fetal and N7) have been isolated (or antibodies against them bovine serum (FBS). Vero cells from a working cell bank have been detected) from humans.4,7 Among these, subtype qualified for vaccine production were described previ- H5, H6, H7, and H9 viruses of avian origin have infected ously.16 Vero cells were cultured in DMEM (Invitrogen, humans and are considered potential pandemic threats. Carlsbad, CA, USA) with 10% irradiated FBS (Cambrex, Highly pathogenic avian influenza (HPAI) H5N1 viruses East Rutherford, NJ, USA) and 50 lg ⁄ ml Neomycin currently circulating in birds have resulted in more than (Sigma, St. Louis, MO, USA). Primary chicken embryo 400 instances of H5N1 transmission to humans since 2004, fibroblast (CEF) and primary chicken kidney (PCK) cells suggesting that this virus could initiate the next pandemic.8 were obtained from Charles River Laboratories, Wilming- The HA sequences of avian H5N1 viruses have diverged ton, MA, USA. Influenza virus A ⁄ Anhui ⁄ 1 ⁄ 2005 (H5N1) is into ten distinct phylogenetic clades (genetic groups).9 a HPAI virus isolated from a fatal case of a pregnant Genetic divergence into clades is also correlated with anti- woman in the Anhui province, China during 2005.17 The genic drift and often antibodies generated against one clade virus was isolated from a tracheal aspirate, propagated in do not efficiently inhibit hemagglutination or neutralize a 10-day-old embryonated chicken eggs and its viral RNA virus from another clade.10,11 As an additional complica- extracted in a biosafety level (BSL)-3-enhanced laboratory. tion, some clades have evolved further into second and Harvested virus was quantified with a standard HA assay third order clades (subclades). Most currently circulating using a 0Æ5% turkey erythrocyte cell suspension18 and also H5N1 strains that have infected humans belong to four in eggs and CEF cells. serologically distinct antigenic groups (clades 1, 2.1, 2.2, and 2.3.4).9 Sequence analysis of HA and NA genes, PCR The impact of viral evolution on the antigenic structure of amplification, mutagenesis, and cloning HA becomes evident upon comparing the HA amino acid Influenza viral genomes were sequenced as previously sequences of the major H5N1 clades. A large proportion of described.19 Briefly, RNA was extracted with the QIAamp the amino acids that constitute putative antigenic sites in the Viral RNA Mini Kit (Qiagen, Valencia, CA), and used for HA are polymorphic suggesting that the antigenicity of the RT-PCR (OneStep RT ⁄ PCR Kit; Qiagen). The sequencing four major clades has drifted.12,13 The consequence of such reaction was performed using BigDye Terminator Cycle structural evolution is reflected in the reactivity of these Sequencing kit (Applied Biosystems, Foster City, CA). Auto- viruses with panels of ferret antisera which are used to assess mated sequencing was performed using an ABI 3100 Genetic antigenic differences which may trigger the need to prepare a Analyzer. To clone the HA and at the same time remove the more closely matched vaccine candidate virus.10,14 multi-basic cleavage site, overlapping PCR was performed In this study, we analyzed the genetic and antigenic using two sets of primers; one set corresponding to HA1 properties of a clade 2.3.4, subtype H5N1 virus which was (5¢-GATCGCTCTTCAGGGAGCAAAAGCAGGGG-3¢ and then utilized for the derivation of a vaccine candidate by 5¢-CTAGGCTCTTCTTCGTTCTCGTAGAGGACTATTTCT- reassortment with the A ⁄ Puerto Rico ⁄ 8 ⁄ 1934 (PR8) strain, GAG-3¢) and other corresponding to HA2 (5¢-AGTCGC- which provided the polymerase, NP, M, and NS genes. This TCTTCGCGAGGACTATTTGGAGCTATAGCAGG-3¢ and virus was expected to be antigenically representative of the 5¢-ACTGGCTCTTCTATTAGTAGAAACAAGGGTGTTTTT- entire clade 2.3.4 and to provide some cross-reactive AACTAC-3¢). For the amplification of the NA gene, we used immunity outside this clade. In addition, since it has the the following universal primers which were modified from internal genes from PR8 which should confer high growth Hoffmann et al.20 5¢-GATCGCTCTTCAGGGAGCAAAAG- in chicken embryo and partial attenuation in humans, it CAGGAGT-3¢ and 5¢-ACTGGCTCTTCTATTAGTAGAAAC- was expected to grow to reasonably high viral titers and to AAGGAGTTTTTT-3¢. The amplified PCR products for HA lack virulence.15 These properties were evaluated to deter- and NA genes were digested with restriction enzyme SapI mine the suitability of this reassortant to develop a seed and ligated into the bidirectional transcription plasmid virus for the preparation of an inactivated subunit or split pCIPolISapIT (modified from pPolISapIT).19 vaccine, the only type of H5N1 vaccine currently approved by the FDA, for use in the event of a pandemic. Plasmid DNA The plasmids containing the PR8 internal genes were modi- Materials and methods fied from the pPolISapIT-derived PR8 plasmids described previously21 by transferring regulatory elements including Cells and viruses the human polymerase I promoter, mouse terminator, and Madin–Darby canine kidney and 293T human embryonic PR8 genome sequences into the pCDNA3.1+ mammalian kidney cells were obtained from the American Type Cul- expression vector (Invitrogen).