a rapid flp-in system for expression of secreted h5n1 influenza hemagglutinin vaccine immunogen in mammalian cells快速flp-in分泌h5n1流感病毒血凝素的表达系统在哺乳动物细胞疫苗免疫原.pdfVIP

A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells 1 1 1 1 1 2 Hanxin Lu , Surender Khurana , Nitin Verma , Jody Manischewitz , Lisa King , John H. Beigel , Hana Golding1* 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 Abstract 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 immunizat