Cell Biology & Molecular Genetics
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Item Reverse Genetics of Influenza B and the Development of a Novel LAIV Vaccine(2014) Finch, Courtney LaPaglia; Perez, Daniel R; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Due to the disease burden of influenza virus types A and B, vaccines, which are manufactured as formalin-inactivated killed virus (KV) and live-attenuated virus (LAIV), are produced to provide coverage against currently circulating influenza A (IAV) and B (IBV) viruses. Although the licensed LAIV vaccine provides enhanced coverage over the KV vaccine, it is only licensed for immunocompetent individuals ages 2-49 years without pre-existing conditions, so individuals who are most at risk cannot receive it. Previously, our lab showed that incorporation of an 8 amino acid HA tag in frame at the C-terminus of the PB1 open-reading frame (ORF) in addition to the mutations found in the PB2 and PB1 segments of the licensed LAIV vaccine yielded a stable, efficacious alternative LAIV vaccine for IAV; however, to develop a complete vaccine, a corresponding IBV candidate is required. Towards this goal, a contemporary IBV strain, B/Brisbane/60/2008, was cloned and recovered by reverse genetics (RG-B/Bris). Subsequently, it was demonstrated that the parental and RG-B/Bris show similar growth kinetics in vitro. An initial vaccine attempt, which combined PB2 cap-binding mutants with the HA tag in PB1, was made but led to the realization of the PB2 cap-binding mutations, PB2 W359F and F406Y, as virulence factors. In a subsequent vaccine attempt, mutations analogous to those found only in segment 2 of the A/Ann Arbor/6/60 cold-adapted LAIV backbone were introduced into the homologous segment of RG-B/Bris. The following mutations were introduced into the PB1 gene segment of RG-B/Bris, either in the presence or absence of a C-terminal HA tag: K391E, E580G, and S660A. Two viruses were rescued, referred to as RG-B/Bris ts and RG-B/Bris att, both containing the set of three amino acid mutations but differing in the absence or presence of the HA tag, respectively. Both viruses showed ideal attenuation, safety, and immunogenicity in DBA/2 mice and conferred protection against lethal IBV challenge. More importantly, RG-B/Bris att, but not RG-B/Bris ts, showed ideal stability with no reverting mutations over 8 passages in eggs. Taken together, a stable, immunogenic, and live attenuated virus alternative to the current live influenza B virus vaccine was produced.Item REASSORTMENT AND GENE SELECTION OF INFLUENZA VIRUSES IN THE FERRET MODEL AND POTENTIAL PLATFORMS FOR IN VIVO REVERSE GENETICS(2014) Angel, Matthew Gray; Perez, Daniel R; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza A virus is a highly infectious agent that cause seasonal epidemics affecting 5-15% of the world population with mild to severe illness and possibly death. While this pathogen represents a significant disease burden to the human population, it can also infect a wide range of animals including swine and land-based poultry, which are thought to serve as intermediate hosts between the human and natural wild aquatic bird reservoir. Here, two viruses, a swine-origin pandemic H1N1 and a seasonal human H3N2 are examined for segment fitness during co-infection of in vivo animal models. In three independent co-infections, reassortment between seasonal and pandemic viruses resulted in the selection of an H1N2 virus with a seasonal PB1 with an otherwise pandemic internal gene constellation. Selection for the seasonal PB1 and NA as well as the pandemic M segment was observed to occur rapidly during segment resolution. As pandemic M gene reassortant strains are being consistently identified in the field, studies were performed to identify the genetic determinants in pandemic M gene selection. Research here shows that both the M1 capsid protein and M2 ion channel from the pandemic virus are sufficient to drive the selection of the entire M segment. As swine represent an important intermediate host for the adaptation of potentially pandemic viruses, including pandemic M gene reassortant strains, alternative DNA and recombinant baculovirus-based platforms are investigated for their ability to generate influenza viruses from porcine polymerase I promoters and serve as potential vaccine candidates. Research here shows that influenza A virus can be rescued de novo using the porcine polymerase I promoter in an eight plasmid system. Furthermore, a single bacmid can be constructed that rescues influenza virus or baculovirus encoding the influenza reverse genetic system in mammalian tissue culture or Sf9 cells, respectively. These represent a new generation of species-tailored vaccine platforms.