Influence of Host Cell Defence during Influenza Vaccine Production in MDCK Cells
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1 Vaccine Technology III, 07 June 2010 Puerto Vallarta/Mexico Influence of Host Cell Defence during Influenza Vaccine Production in MDCK Cells T. Frensing 1, C. Seitz 1, B. Heynisch 2 and U. Reichl 1/2 1 Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany 2 Chair of Bioprocess Engineering, Otto von Guericke University Magdeburg, Germany MAX-PLANCK-INSTITUT DYNAMIK KOMPLEXER TECHNISCHER SYSTEME MAGDEBURG
2 Influenza Virus NA (Neuraminidase) Lipid bilayer HA (Hemagglutinin) M2 (Ion channel) Orthomyxoviridea: segmented single-stranded RNA genome divided into influenza A, B and C viruses annually 3-5 million cases of severe illness worldwide and 250, ,000 deaths (WHO) best protection by vaccination annually with trivalent seasonal influenza vaccine Egg-based vaccine production M1 (matrix protein) Ulmer et al., 2006 Innate immune response in vaccine production June
3 Bioprocess Engineering Group Cell culture-based vaccine production MDCK (Madin-Darby canine kidney) cells Cytodex 1 (dextran) microcarriers -O-CHCH 2 CH 2 -N CH 2 CH 3 CH 2 CH 3 4 days of cell growth to approx. 2.0 x 10 6 cells/ml medium exchange and virus infection with MOI 25 virus propagation for 2-3 days Genzel et al., h 10 h 22 h 100 µm Innate immune response in vaccine production June
4 Influenza A/PuertoRico/8/34 Influenza virus strain PR8 variants: A/PR/8/34 RKI (Robert Koch Institute) A/PR/8/34 NIBSC (National Institute for Biological Standards and Control) PR8 variants differ in: yield replication kinetics apoptosis induction Schulze-Horsel et al., 2009 amount of differentially expressed host proteins Vester et al., 2010 Innate immune response in vaccine production June
5 1 2 3 Limitations by Innate Immune Response? 4 8 Mock Infection Mock Infection Mx1 12 Mock Infection average ratio MDCK cells Julkunen et al., Vester et al., 2009 Innate immune response in vaccine production June
6 Signalling Pathways infection at a low MOI NS1 P P P NS1 Western blots P qrt-pcr for IFN-β and cmx1 Haller et al., 2006 Innate immune response in vaccine production June
7 Interferon Induction IκBα IRF-3 IFN-mediated signalling / activation of the antiviral state relative induction relative induction time () time () PR8-NIBSC - - PR8-RKI T-flask experiments with an MOI 5; n=4 * * * relative induction * * time () Björn Heynisch Heynisch et al., 2009 submitted Innate immune response in vaccine production June
8 Interferon Induction IκBα relative induction IRF-3 relative induction T-flask T-flask MOI 5; n=4 MOI 25; n= time () * * time () * relative induction time () - - PR8-NIBSC - - PR8-RKI relative induction time () relative induction Bioreactor (DasGip) MOI 25; n= time () relative induction time () Innate immune response in vaccine production June
9 Interferon Induction Claudius Seitz IFN-β (fold induction) M1/NS1 mrna copies / 18s RNA copy Interferon induction PR8-delNS1 PR8-NIBSC PR8-RKI (NS WSN33 (NS Intracellular viral mrna PR8-delNS1 (M1) PR8-NIBSC (NS1) PR8-RKI (NS1) WSN33 (NS1) Mx1 (fold induction) MOI Antiviral state marker cmx PR8-delNS1 PR8-NIBSC PR8-RKI (NS WSN33 (NS loss of function: - Does the inhibition of host defence increase the virus titre? gain of function: - Does the activation of the antiviral state reduce the virus yield? Seitz & Frensing et al., 2010 Innate immune response in vaccine production June
10 Loss of function: viral antagonist NS1 relative expression IFN-β NS1 10% 8% 6% 4% 2% 0.2% 0.1% % delns1 NS1 IFN-β PR8-NIBSC relative expression Mx1 control NS1 100% 80% 60% 40% 10% 5% 0% delns1 cmx1 PR8-NIBSC control NS1 virus genome copies (ml -1 ) virus genome copies (ml -1 ) MOI PR8-delNS NS1 (qrt-pcr) 1.8 control (qrt-pcr) NS1 (log HA) control (log HA) PR8-NIBSC NS1 (qrt-pcr) 1.8 control (qrt-pcr) NS1 (log HA) control (log HA) but: NS1 promotes viral mrnas translation. Which NS1 function enhanced the virus titre? Innate immune response in vaccine production June log HA/100 µl log HA/100 µl
11 Loss of function: viral antagonist rp NS1 relative expression 100% 80% 60% 40% 20% 0% rp IFN IFN-β rp Mx cmx1 control rp virus genome copies (ml -1 ) MOI rp (qrt-pcr) control (qrt-pcr) rp (log HA) control (log HA) log HA/100 µl Inhibition of IFN signalling does not result in higher virus yields Innate immune response in vaccine production June
12 Gain of function: cytokine stimulation ultrafiltration 100 kda cut-off 1st infection: MOI 5 A 100 cmx1 induction 80 virus genome copies (ml -1 ) PR8-RKI MOI 25 2nd infection: MOI control (qrt-pcr) CM (qrt-pcr) control (log HA) CM (log HA) log HA/100 µl Mx1 (fold induction) Innate immune response in vaccine production June MOCK delns1 PR8-RKI PR8-NIBSC Virus replication is slowed down, but almost the same titre is reached - active virus titre (TCID50) - MOIs (25 / 025 / 0025) - PR8-NIBSC, A/WSN/33
13 Gain of function: cytokine stimulation 3.5 B/Malaysia/2506/2004 (MOI 25) 1600 IFN induction log HA / 100 µl control stimulated fold induction control stimulated cmx1 induction NS1 NS1 fold induction control stimulated Innate immune response in vaccine production June
14 Minireplicon Assay minigenome with reporter gene Pol-I promoter expression plasmids Pol-II promoter HEK 293 cells NCR FF-Luciferase (-) NCR Renilla PA PB1 FF-Luc PB2 NP vrna (-) Prof. Dr. Georg Kochs Mx luciferase assay Innate immune response in vaccine production June
15 Minireplicon Assay 140 relative activity [%] Mx 0 empty vector empty vector MxA MxA MxA mut MxA mut cmx1 cmx2 mmx1 mmx1 mut cmx1 cmx2 mmx1 mmx1 mut NP ERK2 No inhibitory effects of canine Mx proteins Seitz, C. & Frensing, T., Höper, D., Kochs, G., Reichl, U. (2010). High yields of Influenza A virus in MDCK cells are promoted by an insufficient IFN-induced antiviral state. Journal of General Virology, accepted Innate immune response in vaccine production June
16 Summary considerable IFN signalling in influenza virus infected MDCK cells no impact on final virus titres by inhibition or stimulation of IFN lack of inhibitory potential of canine Mx proteins against influenza virus IFN signalling has only a minor effect on influenza virus replication in MDCK cells, which makes these cells an ideal system for high yield vaccine production. Innate immune response in vaccine production June
17 Acknowledgment Signal transduction group: Claudius Seitz Nancy Wynserski Bianca Kaps Björn Heynisch Maria Rettkowski former students: Kristina Heinze Stefan Heldt Anja Fincke Henrike Götze Innate immune response in vaccine production June
18 Acknowledgment Bioprocess Engineering Group Prof. Dr.-Ing. Udo Reichl Cooperations: Virology Freiburg: Prof. Dr. Georg Kochs FLI Insel Riems: Dr. Dirk Höper PD. Dr. med. vet. Timm Harder Thanks for your attention! MPI-Infection Biology, Berlin: Dr. Alexander Karlas Innate immune response in vaccine production June
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