Use of cell culture technology to minimize the need for animal trials in development and production of fish vaccines

Transcription

1 Use of cell culture technology to minimize the need for animal trials in development and production of fish vaccines S. Shih, D. Sepulveda, H. Kristiansen, H. F. Skall, V. Vakhari, N. Bols, and N. Lorenzen.

2 Background- Fish Diseases Viralviral hemorrhagic septicemia virus (VHSV) Parasitic- Sea lice Bacterial- Aeromonas salmonicida Fungal- Branchiomyces demigrans

3 Fish vaccination by ip injection

4 Dip-vaccination

5 Background- Impact of vaccine in Norway The use of antimicrobial has declined by about 99% ( ) Vibriosis vaccine Furunculosis vaccine Oil-based Furunc. vaccine Combination vaccines Ref.1 Usage of antimicrobial agents and occurrence of antimicrobial resistance in Norway Oslo and Tromsø Ref.2 Fish Vaccination A brief overview. Dr Marian McLoughlin

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7 Aims 1. Replace the use of live animals with cell cultures for the initial screening of vaccine components. 2. Gain a better understanding of the fish immune system to improve vaccine efficacy.

8 Egtved disease (Viral haemorrhagic septicaemia, VHS) Photo by N.H.Henriksen, Danish Aquaculture

9 Monoklonale muse-antistoffer mod VHS virus -proteiner

10 Passive immunization against VHSV Y Antibody to VHSV

11 Virus challenge

12 Monoclonal antibodies to VHSV-proteins Only antibodies to G are protective

13 Mimicking antibody mediated protection against virus in transfected cells p G pcdna Cell cultures were inoculated with virus 3 days after transfection with a plasmid encoding a virus-neutralizing antibody (p G) or a control (pcdna3). The antibodies produced by the p G-cells protected the cell culture against the cytopathogenic virus

14 RPS Time-course of protection following DNA vaccination against VHSV and IHNV Mx Ab Ab Weeks Non-specific post vaccination mechanisms Specific mechanisms Total protection Weeks post Vaccination 106 Lorenzen et al. (2002) Fish & Shellfish immunology 12,

15 Workflow

16 Fold change Fold change Result - Fish cell lines mimic the immune response Immune gene expression analysis of fish cells treated with poly(i:c) for 24 hours Poly(I:C)= synthetic dsrna (potential vaccine adjuvant) MX1 Vig3 Vig4 IFNa IL-1B MHCI MHCII IFNy RTG RTS RTgill RTgut RTL 0 RTG RTS RTgut RTL RTgill RTL (rainbow trout liver), RTgut (rainbow trout gut), RTgill (rainbow trout gill), RTG (rainbow trout gonad) and RTS (rainbow trout spleen)

17 (200X) Result - Fish cell lines mimic the immune system defense Monitoring VHSV replication by confocal microscopy 48 hpi. 96 hpi. VHSV-EGFP PIC5_VHSV-EGFP PIC10_VHSV -EGFP VHSV-EGFP PIC5_VHSV-EGFP PIC10_VHSV -EGFP BF EGFP

18 Fold chnage Fold change Result - Fish cell lines mimic the immune system defense Quantitative analysis of the time-course of poly(i:c)-mediated antiviral assay 1000 P-5+VHSV P-10+VHSV P-5 P-10 VHSV 48h posttreatment ,1 IFNa IFNy IL-1B MX1 Vig3 Vig4 TNFa C3 IL-8 MBL-1 COX2a IgM IL-10 MHCII EF1a 96h posttreatment P-5+VHSV P-10+VHSV P-5 P-10 VHSV ,1 0,01 IFNa IFNy IL-1B MX1 Vig3 Vig4 TNFa C3 IL-8 MBL-1 COX2a IgM IL-10 MHCII EF1a P-5: 5ug/ml of poly(i:c); P-10: 10 ug/ml of poly(i:c)

19 Fold change Fold change Result - Fish cell lines mimic the immune system defense Quantitative analysis of the time-course of poly(i:c) induced gene expression RTL cell IFNa MX1 Vig3 Vig4 IL-1B 100 Liver IFNa MX1 Vig3 Vig4 IL-1B ,1 RTL-P10 24h P-5-48h P-10-48h P-5-96h P ,1 DAY1 DAY4

20 Oil-adjuvanted bacterial vaccines can induce adherances following ip vaccination

21 Fold change (normalized to EF1a) Gene expression profiles of RTL cells stimulated with mineral or vegetable oil adjuvant -correlation with vaccine induced side effects (?) 100 RTL cell, 24h Mineral oil+nacl Vegetable oil+nacl Mineral oil+antigen Vegetable oil+antigen ,1

22 Conclusions 1. Vaccine-immunostimulant could protect the fish cell cultures against virus infection in a dose and time dependent manner, mimicking the innate immune response in the animal (fish) host. 2. Established cell lines can be used to address aspects of vaccine induced adaptive immunity, but panels of different cell lines are required to address the complexity of the immune system. 3. Cell culture technology has potential to partly substitute use of live fish in vaccine development.

23 Replacement Reduction Refinement Thanks for your attention!

24 OD 450 nm Result - Fish cell lines mimic the immune system defense Detection of viral hemorrhagic septicemia virus (VHSV) using ELISA (enzyme-linked immunosorbent assay) - 48 hpi. 1/8 dilution 1/16 dilution 1/32 dilution 1/64 dilution 3 2,4 1,8 1,2 0,6 0 VHSV Poly(I:C) VHSV TCID50/ml - VHSV-stock TCID50/ml - mock - - PIC TCID50/ml 5 ug/ml PIC-5-mock - 5 ug/ml PIC TCID50/ml 10 ug/ml PIC-10-mock - 10 ug/ml

25 DNA vaccination: the concept Virus particle Surface protein (G) Gene cloning Vaccine plasmid pcdna3-vhsg Fermentation + Purification Vaccination by Intramuscular injection