A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells

et al. (2011) A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells. PLoS ONE 6(2): e17297. doi:10.1371/journal.pone.0017297 A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells Hanxin Lu 0 Surender Khurana 0 Nitin Verma 0 Jody Manischewitz 0 Lisa King 0 John H. Beigel 0 Hana Golding 0 Ralph Tripp, University of Georgia, United States of America 0 1 Division of Viral Products, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA) , Bethesda , Maryland, United States of America, 2 Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases , SAIC-Frederick, NCI-Frederick, Frederick, Maryland , United States of America Background: Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing. Methodology/Principal Findings: We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/ 2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/ 2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine. Conclusions/Significance: Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains. - Funding: This project was funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. HHSN261200800001E and in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The recent global spread of swine-origin H1N1 highlighted the need for rapid development of effective vaccines against pandemic influenza viruses. Much of our recent knowledge was derived from studies with the highly pathogenic (HP) H5N1 avian influenza A viruses (AIV) [1]. The H5N1 viruses still cause severe human disease, and may undergo adaptation for human-to-human transmission. As of October 18, 2010 there have been 507 human cases of H5N1 resulting in 302 deaths (fatality rate = 59%) (http:// www.who.int/csr/disease/avian_influenza). Production of hemagglutinin using recombinant technology could overcome the constraints of traditional inactivated influenza vaccine manufacturing that require several months for generation of vaccine viruses using reassortment/reverse genetics, and adaptation for high growth in eggs. Most of the influenza protective antigenic sites are conformation dependent and map primarily to HA1 globular head [2]. In previous reports, codonoptimized HA ectodomain with mutated cleavage site (to prevent processing of HA1-HA2) and an added exogenous foldon sequence at the C-terminus was expressed transiently in 293 cells in order to produce stable oligomers [3,4,5]. Technologies that can be easily translated into well controlled large scale manufacturing process will have a great advantage. Thus far, various influenza vaccine prototypes produced in a baculovirus-insect cell expression system have undergone pre-clinical and clinical development [6,7], but it is not well understood if the baculovirus produced HA products are identical in terms of antigenicity and immunogenicity to the egg grown or mammalian cells based vaccines. Recombinant proteins produced from mammalian cells are expected to have the same extent of posttranslational modifications as egg grown influenza viruses. Transient transfection of mammalian cells (often HEK293T cells) followed by selection of stable tranfectants, usually result in random integration, clone-toclone variability and upredicatable level of expression due to position effects based on the site of integration in the host genome. Therefore, one of the important parameters of recombinant protein production for manufacturing is the ability to derive stable mammalian cell lines with defined integration site(s) and reproducible level of protein expression. Previously, the Flp-In system has been used to express proteins for basic research purposes and in one instance, to produce monoclonal antibodies in CHO cells [8,9]. In the current study, we have constructed the first vector system for HA protein expression in mammalian cells using the FRT/FLP strategy to overcome position effects and for rapid derivation of stable cell lines expressing HA. As a proof-of-concept, we have cloned the HA0 and HA1 proteins from the highly pathogenic H5N1, A/ Vietnam/1203/2004. No codon optimization or modifications to the polybasic cleavage site were made in order to exactly match the HA sequence of the transmitted avian influenza strain [10]. We provide data demonstrating that both proteins are properly folded and can elicit homologous and heterologous H5N1 neutralizing antibodies in rabbits. Importantly, in the face of impending pandemic, cloning, transfection, and stable cell line generation can be done within 23 weeks. Cloning of HA1 and HA0 from H5N1 A/Vietnam/1203/ 2004 in 293Flp-In system The Flp-In system, which offers a single targeted integration site was found to be more efficient than the conventional transient transfection/selection protocols. Based on a site-specific recombination strategy, a modified Flp-In system was successfully developed to generate stably expressing cell lines. The recombination occurs at a single site and eliminates clonal variability. Expression from the same integration site resulted in reproducible stable levels of insert expression and protein secretion [10,11]. Therefore such a system is amenable to controlled manufacturing with built in quality assurance steps. To enhance HA protein expression and protein secretion (...truncated)