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
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 250,000-500,000 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 2010 2
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., 2004 5 h 10 h 22 h 100 µm Innate immune response in vaccine production June 2010 3
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 2010 4
1 2 3 Limitations by Innate Immune Response? 4 8 Mock Infection Mock Infection Mx1 12 Mock Infection average ratio 6 5 4 3 2 1 MDCK cells Julkunen et al., 2001 0 4 8 12 Vester et al., 2009 Innate immune response in vaccine production June 2010 5
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 2010 6
Interferon Induction IκBα IRF-3 IFN-mediated signalling / activation of the antiviral state relative induction 1.2 0.8 0.4 relative induction 0 4 8 12 0 4 8 time () time () 12 - - PR8-NIBSC - - PR8-RKI 1.2 0.8 0.4 T-flask experiments with an MOI 5; n=4 * * * relative induction 1.2 0.8 * * 0.4 0 4 8 12 16 20 24 time () Björn Heynisch Heynisch et al., 2009 submitted Innate immune response in vaccine production June 2010 7
Interferon Induction IκBα relative induction IRF-3 relative induction 1.2 0.8 0.4 1.2 0.8 0.4 T-flask T-flask MOI 5; n=4 MOI 25; n=3 0 4 8 12 time () * * 0 4 8 12 time () * relative induction 0.8 0.4 0 4 8 12 16 20 24 28 32 time () - - PR8-NIBSC - - PR8-RKI relative induction1.2 1.2 0.8 0.4 0 4 8 12 16 20 24 28 32 time () relative induction Bioreactor (DasGip) 0.8 0.4 MOI 25; n=1 0 4 8 12 16 20 24 time () 1.2 0.8 0.4 relative induction1.2 0 4 8 12 16 20 24 time () Innate immune response in vaccine production June 2010 8
Interferon Induction Claudius Seitz IFN-β (fold induction) M1/NS1 mrna copies / 18s RNA copy 10 6 10 5 10 4 10 3 10 2 10 1 10 0 10-1 10 1 10 0 10-1 10-2 10-3 10-4 10-5 10-6 10-7 Interferon induction PR8-delNS1 PR8-NIBSC PR8-RKI (NS WSN33 (NS1 0 5 10 15 Intracellular viral mrna PR8-delNS1 (M1) PR8-NIBSC (NS1) PR8-RKI (NS1) WSN33 (NS1) 0 5 10 15 Mx1 (fold induction) MOI 5 120 100 80 60 40 20 15 Antiviral state marker cmx1 10 5 PR8-delNS1 PR8-NIBSC PR8-RKI (NS WSN33 (NS1 0 0 5 10 15 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 2010 9
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 25 10 12 6.0 10 PR8-delNS1 11 5.4 10 10 4.8 4.2 10 9 3.6 10 8 3.0 10 7 2.4 10 6 NS1 (qrt-pcr) 1.8 control (qrt-pcr) 1.2 10 5 NS1 (log HA) 0.6 10 4 control (log HA) 0 24 48 72 10 12 6.0 10 11 PR8-NIBSC 5.4 10 10 4.8 4.2 10 9 3.6 10 8 3.0 10 7 2.4 10 6 NS1 (qrt-pcr) 1.8 control (qrt-pcr) 1.2 10 5 NS1 (log HA) 0.6 10 4 control (log HA) 0 24 48 72 but: NS1 promotes viral mrnas translation. Which NS1 function enhanced the virus titre? Innate immune response in vaccine production June 2010 10 log HA/100 µl log HA/100 µl
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 25 6.0 5.4 4.8 4.2 10 9 3.6 10 8 3.0 10 7 2.4 10 6 1.8 1.2 10 5 0.6 10 4 0 24 48 10 12 10 11 10 10 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 2010 11
Gain of function: cytokine stimulation ultrafiltration 100 kda cut-off 1st infection: MOI 5 A 100 cmx1 induction 80 virus genome copies (ml -1 ) 10 12 10 11 10 10 10 9 10 8 10 7 PR8-RKI MOI 25 2nd infection: MOI 25 0025 control (qrt-pcr) CM (qrt-pcr) control (log HA) CM (log HA) 16 24 32 40 4.2 3.6 3.0 2.4 1.8 1.2 0.6 log HA/100 µl Mx1 (fold induction) 60 40 20 0 Innate immune response in vaccine production June 2010 12 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
Gain of function: cytokine stimulation 3.5 B/Malaysia/2506/2004 (MOI 25) 1600 IFN induction log HA / 100 µl 3 2.5 2 1.5 1 0.5 0 control stimulated 15 20 25 30 35 40 45 50 fold induction 1200 800 400 0 25 control stimulated 0 16 24 40 48 cmx1 induction NS1 NS1 fold induction 20 15 10 5 control stimulated 0 0 16 24 40 48 Innate immune response in vaccine production June 2010 13
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 2010 14
Minireplicon Assay 140 relative activity [%] 120 100 80 60 40 20 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 2010 15
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 2010 16
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 2010 17
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 2010 18