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

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Use of cell culture technology to minimize the need for animal trials in development and production of fish vaccines Helle Pilgaard Kristiansen, Helle Frank Skall & Niels Lorenzen Department of Animal Science, Aarhus University

Background Aquaculture is a rapidly expanding industry which gradually substitutes fisheries on the decreasing wildlife fish populations as a provider of healthy animal food products for human consumption. Like other husbandry animals, farmed fish face problems with infectious diseases. Losses due to disease outbreaks and needs for treatment with antibiotics and other compounds represent a major challenge for the growing industry. Prophylaxis is better than cure, and considerable research is invested in development of efficient vaccines for farmed fishes. Successful vaccines against important bacterial diseases have since 1987 implicated a more than 10-fold reduction of antibiotics usage in salmon farming in Norway1. This has further promoted researchers and vaccine companies to expand their efforts in development and production of more and better vaccines for farmed fish. However, vaccine development as well as potency testing of already commercialized vaccines rely heavily on empirical vaccination and challenge (infection) trials with live fish2. This implies use of thousands of experimental fish every year. Although animal (in vivo) experiments in vaccine development/production is justified by the benefit of improved animal health and welfare in aquaculture, we believe that cell culture (in vitro) techniques can be implemented for certain analyses. Focus will be on rainbow trout, the dominating fish species in Danish aquaculture. However, it is expected that the concept can be transferred/adapted to other animal species. Two important rainbow trout diseases will be included in the work namely viral haemorrhagic septicaemia (VHS) caused by VHS virus and furunculosis caused by the bacterium Aeromonas salmonicida.

Project aim The project aims at substituting the use of live animals with cell cultures for the initial screening and optimizing of two central parameters in fish vaccine development, namely selection of adjuvants and delivery strategy. Also, the project aims at establishing laboratory tests for confirmation of vaccine efficacy including batch potency testing. The proposed project is based on the hypothesis that vaccine uptake and innate immune response reactions to vaccine components can be studied using cell cultures thereby allowing initial selection/optimising of vaccine candidates. The outcome of the project will partly replace the use of live fish with cell cultures in vaccine development and production. This will reduce the number of experimental fish needed within these fields. Refinement will be addressed by initial screening of vaccine components on cell cultures for potential toxic/negative side effects, hereby avoiding the exposure of fish to potentially harmful elements.

Materials and methods Cell lines: The interplay between the host organism (fish) and pathogen is very complex, and this also counts for the host response to vaccines. To establish the best cell culture that correlates with the interactions in live animals, a panel of established cell lines3 derived from different host tissues will be employed (Table 1). This panel will be used to establish an in vitro platform for initial examination of how host cells respond to vaccine components. Monitoring cell line responses: The response of the different cell lines to vaccine components will be analysed by: 1) toxicity test including microscopical examination for cytopathogenic effects and a dye-based viability assay, 2) gene expression profiles for an array of immune-related genes as measured by Q-PCR4,5 (Table 2), and 3) antigen uptake analysed by confocal immunofluorescence microscopy (Figure 1). Finally 4), cells exposed to vaccine components will subsequently be exposed to fish pathogens to determine whether antimicrobial effect of initiated innate immune reactions can be detected at cell culture level. Vaccines: The work will include two previously developed vaccines with known protective effect when delivered/formulated according to specific conditions, but for both vaccines improvements are required to improve applicability. 1) An experimental DNA based vaccine against VHS virus providing high protection following intramuscular injection6, will be used in delivery studies aiming at developing a mucosal delivery strategy, while 2) a commercial bacterin based oil adjuvanted vaccine against A. salmonicida7 protecting the fish by i.p. injection will be used in studies of alternative adjuvants causing reduced side effects. Vaccination and challenge trials in rainbow trout using the same vaccines and pathogens as in the cells will be performed in associated projects.

Cell line

Description

Gene(s)

Description of encoded protein

RTG-2

Rainbow trout gonad cells

IFN type I

RTL-W1

Rainbow trout liver cells

IFNs (Interferons) are key proteins in the immune response against viruses.

Rtgut-GC

Rainbow trout intestine cells

Mx

Mx proteins have direct antiviral effects.

IRF3

IRF3 is an important transcription factor in the immune response against viruses.

Vig-1 (Viperin)

Viperin has direct antiviral effects.

PKR

PKR is a translation inhibitor and has an antiviral effect.

Rtgill-W1

Rainbow trout gill cells

RTHDF

Rainbow trout fibroblasts (Hypodermal cells) Rainbow trout monocyte cell line

RTS-11

Table 1: Cell lines used to establish an in vitro platform.

Table 2: List of Interferon stimulated genes (ISGs) (selected). The genes have been selected based on existing literature, and all have been reported to be induced following vaccination of rainbow trout.

Figure 1: Analysis of antigen uptake by confocal immunofluorescence microscopy. Cells were transfected with the DNA vaccine construct encoding VHSV G. The cells were incubated for 24 h and fixed and stained with a antivhsG specific antibody. The viral antigen vhsG is shown in green. Blue is the cell nuclei.

Relevance and perspective The project addresses the 3Rs in the following way: Reduction: By substituting use of experimental animals with cell culture in the initial steps of vaccine development, the project will contribute to a reduction in the number of experimental animals needed in vaccine research, and as stated below, potentially also in batch release testing in commercial vaccine production. Refinement: Apart from allowing selection of only the most promising compounds/formulations for in vivo testing, it is also expected that the cell array platform will provide important new hints as to how vaccines can be efficiently delivered by alternative routes to injection. As vaccines for injection are the ones implying the highest stress exposures upon delivery (netting, anaesthesia, injection) and with the most serious side effects (inflammation in the peritoneal cavity, peritonitis), the work is on sight expected to make both vaccine development work and commercial vaccine delivery more refined. Replacement: By using a cell culture platform comprising cell lines derived from different tissues of the relevant target species it is expected that in vitro correlates of basic reactions to adjuvants/vaccine formulations like level of toxicity, ability to stimulate innate immune reactions and possibly immune priming can be established and used to replace use of live fish in initial steps of examining new adjuvants/formulations in vaccine development. The same principles are expected to count for development of new delivery strategies including initial examination of toxicity and uptake in vitro, before going in vivo with a reduced number of approaches. When testing vaccines to go on the market, the European Pharmacopoiea 8.3 describes a minimum of 50 fish to be used for safety tests in the laboratory, a minimum of 30 fish per group for immunogenicity testing and a minimum of 25 fish per group for batch potency testing. With inclusion of appropriate controls, this implies use of hundreds of fish for quality assurance of each vaccine batch. According to the research literature, experimental vaccination trials often include up to 500 or more fish per trial. If the proposed project is successful we estimate that implementation of the results can contribute to a 25% reduction of these numbers. References

1. NORM/NORM-VET 2013. Usage of antimicrobial agents and occurrence of Antimicrobial resistance in Norway. Tromsø/Oslo 2014. 2. Alderman DJ (2009) Control of the use of veterinary drugs and vaccines in aquaculture in the European Union. In: Rogers C (ed.), Basurco B (ed.). The use of veterinary drugs and vaccines in Mediterranean aquaculture. Zaragoza: CIHEAM, p. 13-28 (Options Méditerranéen nes: Série A. Séminaires Méditerranéens; n. 86). 3. Bols NC et al. (2005). Use of fish cell lines in the toxicology and ecotoxicology of fish. Piscine cells lines in environmental toxicology. Biochemistry and Molecular Biology of Fishes, vol. 6 (Chapter 2). 4. Purcell MK et al. (2006). Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus. Molecular Immunology 43, 2089-2106. 5. Schoggins JW & Rice CM (2011). Interferon-stimulated genes and their antiviral effector functions. Current Opinion in Virology 1, 519-25. 6. Lorenzen N, et al. (2002). DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout. Fish & Shellfish Immunology 12: 439-453. 7. Håstein T et al. (2005). Bacterial vaccines for fish – an update of the current situation worldwide. Dev. Biol. (Basel).2005; 121: 55-74.

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