DNA vaccines for aquacultured fish, N Lorenzen, SE LaPatra

Tags: DNA vaccines, vaccines, DNA, DNA vaccine, plasmid DNA, vaccination, fish pathogens, viral diseases, Atlantic salmon, infectious haematopoietic necrosis virus, fish farming, ADN, S.E. LaPatra, rainbow trout, Kurath G. & LaPatra S.E., les animaux, infectious hematopoietic necrosis virus, intramuscular injection, Anderson E.D. & Kurath G., disease outbreaks, commercial fish, British Columbia, commercial fish farming, Norwegian Biotechnology Advisory Board, farmed fish, vaccine, les poissons
Content: Rev. sci. tech. Off. int. Epiz., 2005, 24 (1), 201-213
DNA vaccines for aquacultured fish N. Lorenzen (1) & S.E. LaPatra (2) (1) Danish Institute for Food and Veterinary Research, Hangovej 2, DK-8200 Aarhus N, Denmark (2) Clear Springs Foods, Inc., Research Division, P.O. Box 712, Buhl, Idaho 83316, United States of America Summary Deoxyribonucleic acid (DNA) vaccination is based on the administration of the gene encoding the vaccine antigen, rather than the antigen itself. Subsequent expression of the antigen by cells in the vaccinated hosts triggers the host immune system. Among the many experimental DNA vaccines tested in various animal species as well as in humans, the vaccines against rhabdovirus diseases in fish have given some of the most promising results. A single intramuscular (IM) injection of microgram amounts of DNA induces rapid and long-lasting protection in farmed salmonids against economically important viruses such as infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). DNA vaccines against other types of fish pathogens, however, have so far had limited success. The most efficient delivery route at present is IM injection, and suitable delivery strategies for mass vaccination of small fish have yet to be developed. In terms of safety, no adverse effects in the vaccinated fish have been observed to date. As DNA vaccination is a relatively new technology, various theoretical and long-term safety issues related to the environment and the consumer remain to be fully addressed, although inherently the risks should not be any greater than with the commercial fish vaccines that are currently used. Present classification systems lack clarity in distinguishing DNA-vaccinated animals from genetically modified organisms (GMOs), which could raise issues in terms of licensing and public acceptance of the technology. The potential benefits of DNA vaccines for farmed fish include improved animal welfare, reduced environmental impacts of aquaculture activities, increased food quality and quantity, and more sustainable production. Testing under commercial production conditions has recently been initiated in Canada and Denmark. Keywords Animal welfare ­ Consumer perceptions ­ Cost-benefit ­ Delivery ­ Deoxyribonucleic acid vaccine ­ Farmed fish ­ Field-testing ­ Glycoprotein ­ Plasmid ­ Protective mechanisms ­ Regulatory issues ­ Safety ­ Viral diseases.
Introduction The first vaccines against infectious bacterial diseases in farmed fish were developed in the 1970s, and introduced into commercial aquaculture in the early 1980s. Overall there has been a significant reduction in the use of antibiotics following the introduction of vaccines, particularly in the farmed Atlantic salmon industry (56). This has contributed significantly to the growth of the industry and to consumer acceptance of farm-raised fish. The latter is due to the reduced environmental impact and improved food quality obtained by minimising antibiotic
use. In addition, animal welfare has been improved by the implementation of vaccination. The successful bacterial vaccines that are now routinely used in aquaculture were developed largely through empirical observations and are usually based on inactivated bacteria. Despite extensive research over many years, very few anti-viral vaccines are available and there are no commercial vaccines against fish parasites. There have been several attempts to develop traditional vaccines against viral diseases based on inactivated or attenuated viruses (9, 48, 77), and both types of vaccines
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have been shown to induce a certain level of protection against some of the important salmonid viruses, including viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV). Since viruses must be replicated in cultures of fish cells, the cost of producing vaccines based on inactivated viruses is usually too high to make this strategy economically viable. In comparison, attenuated virus vaccines have several advantages. These vaccines can be delivered via the water route, which is optimal in terms of minimal stress and cost, and because a certain amount of replication takes place in the vaccinated fish, the dose required for protection is small compared to inactivated virus. However, attenuated virus vaccines occasionally cause disease, and the release of live vaccines into the water bodies is often not compatible with veterinary and environmental control strategies. Viral vaccines in the form of a recombinant viral protein produced in genetically engineered Escherichia coli have also been attempted. For IPNV, a recombinant viral protein (VP2) is mixed in an oiladjuvanted multivalent bacterin vaccine for Atlantic salmon smolts. The vaccine is expected to have a protective effect against infectious pancreatic necrosis (IPN) (9). At the experimental stage, similar effects have been demonstrated for Atlantic halibut nodavirus (AHNV), where recombinant virus capsid protein in an oiladjuvanted vaccine has mediated some protection against disease in turbot (71). For the rhabdoviruses VHSV and IHNV, the protective effect of recombinant protein vaccines has been limited or inconsistent (48, 77). The most efficient vaccines against viral diseases in fish to date at the experimental level are deoxyribonucleic acid (DNA) vaccines against the salmonid rhabdoviruses, VHSV and IHNV. These vaccines are based on naked plasmid DNA, which following uptake in cells of the vaccinated fish mediates expression of the viral glycoprotein (3, 49). Several reviews on DNA vaccines for fish are available (4, 29, 32, 37, 46). Much of the early research in fish involved the use of genes encoding reporter proteins such as luciferase, -galactosidase and green fluorescent protein to study the magnitude of expression levels under different conditions, the tissue distribution, the duration of expression, and to some extent also the immune response (2, 24, 26, 28, 66). More recently, work on DNA vaccines containing genes that encode antigens from fish pathogens has expanded to explore immune responses and protection against pathogen challenge in fish (7, 44, 45, 52, 54, 61, 63, 73, 76). In humans a number of clinical trials with DNA vaccines against diseases such as acquired immune deficiency syndrome, hepatitis and malaria have been initiated. Although the results have been promising in terms of safety, the results have indicated that prime-boost strategies combining DNA vaccines with other types of vaccines
and/or adjuvants are needed to obtain an adequate immune response (16, 43). No veterinary or human DNA vaccines have been licensed yet, but recently, a prototype DNA vaccine against West Nile virus was used to vaccinate wild condors in California. A similar vaccine has proved efficient in protecting horses against the same virus and is likely to become the first commercially licensed DNA vaccine (62). However, despite many promising results in mice models, the majority of the DNA vaccines tested in veterinary target species so far have ­ as with DNA vaccines tested in humans ­ had relatively low efficacy (75). The main technical hurdle appears to be inefficient uptake of the administered DNA by the host cells (75). This article considers the principles and perspectives related to application of DNA vaccines in fish that are commercially cultured for food production, focusing on the DNA vaccines against fish rhabdoviruses. The advantages and disadvantages of DNA vaccines are summarised in Table I. Characteristics of the DNA vaccines against fish rhabdoviruses Although development of DNA vaccines has been attempted for various pathogens in a number of different fish species, the DNA vaccines against the salmonid rhabdoviruses IHNV and VHSV remain the most efficient and also the most extensively analysed to date. These vaccines are highly effective under a variety of conditions, including different fish life stages and different salmonid host species, and against challenge with different virus strains (13, 23, 38, 39, 50, 51, 74). The first step in producing a DNA vaccine is to identify and clone a protective antigen from the pathogen. For VHSV and IHNV, earlier work had shown that protective antibodies were directed against the viral surface glycoprotein G (31, 47). The gene encoding the G protein, in combination with regulatory sequences that allow expression in eukaryotic cells, was therefore also an obvious candidate for a DNA vaccine (Fig. 1). The viral genome includes five other genes, but none of these have proven useful for induction of immunity when delivered as DNA vaccines (11). Prior to vaccination, the vaccine plasmid is produced in Bacterial culture, purified and quality-assured. Following administration of a DNA vaccine, certain cells of the host take up the vaccine and utilise the machinery of the cell to produce the G protein. When detected by the fish immune system, such cells will appear like virus-infected cells with G-protein on their surface (Fig. 1). This leads to activation of both humoral
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Table I Advantages and disadvantages of deoxyribonucleic acid (DNA) vaccines
Advantages
Disadvantages/current problems
Generic and simple principle High level of safety ­ no risk of infectious disease Combination of advantages of traditional killed and attenuated vaccines Can be successful when traditional vaccine strategies fail Possibility of incorporating molecular adjuvants such as CpG motifs Activation of both humoral and cellular mechanisms * Multivalent vaccination possible by simple mixing of DNA vaccines * Good effect when given at an early life stage * Protection induced shortly after vaccination and is also long lasting * Protection induced at both low and high temperatures * Protection efficient across serotype variations * Ability to prepare vaccines for new pathogen variants quickly at low cost High stability of purified product Relatively low cost; easy production/quality assurance
Difficulty/cost of delivery; need for new strategies for mass vaccination of small fish Not efficient for all pathogens New concept ­ long-term safety issues remain to be analysed Official distinction between DNA-vaccinated animals and genetically modified organism (GMO)ґs not always clear Public aversion to ingredients from GMOs in food products, which might influence consumers' acceptance of veterinary DNA vaccines No regulatory precedents yet available for DNA vaccines for husbandry animals Possible complications of Intellectual property rights affecting commercialisation of veterinary DNA vaccines
*Specifically demonstrated in the case of DNA vaccines for fish
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and cellular defence mechanisms in the fish (2, 7, 8, 28, 49, 63, 73). One interesting feature of the immune response to the VHSV and IHNV G gene DNA vaccines is that the specific protection is preceded by an early nonspecific antiviral protection (Fig. 2), possibly related to interferon-induced mechanisms (35, 40, 52, 53, 54).
b) Prom.
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In the vaccine plasmid, the eukaryotic promoter (Prom.), antibiotic resistance selection marker (Antibiotic) and the inserted fish virus glycoprotein gene (G gene) are indicated (b). The G protein is a transmembrane molecule with oligosaccharide side chains and stabilised by disulphide bonds (s--s) (c). The G protein appears on the surface of virus infected cells as well as on the surface of virus particles. Once the vaccine plasmid has reached the nucleus of a cell in the vaccinated fish, expression of G protein will be initiated and G protein molecules will appear inside the cell and on the cell's surface, as if the cell had been naturally infected with virus (52) Fig. 1 Schematic drawing of a rhabdovirus particle (a), the vaccine plasmid (b), and the viral G protein (c)
Delivery and efficacy For mammals the preferred delivery strategies have been intramuscular (IM) injection or particle-mediated delivery by gene gun. The latter entails coating small gold particles with vaccine DNA, followed by air-pressure-mediated intradermal delivery. Although such DNA vaccination by gene gun is effective in fish (12, 24, 72), this technology is too expensive to be cost effective in commercial aquaculture. Interestingly, simple IM injection of purified plasmid DNA in a neutral buffer has proven to be more efficient in fish than in any other type of animal tested to date. Dose­response experiments have shown that a single injection of nanogram levels of plasmid DNA is sufficient to induce protective immunity against viral haemorrhagic septicaemia (VHS) and IHN in rainbow trout fingerlings (Fig. 3) (13, 44). The protection is not only rapidly induced but also long lasting (Fig. 2), (38, 44). It appears beneficial to vaccinate the fish when they are small, since larger fish require a higher dose of vaccine to be protected (39, 51).
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Fig. 2 Schematic illustration of the assumed complementary roles of early non-specific mechanisms and subsequent specific mechanisms in the protection induced by vaccination of rainbow trout with the fish rhabdovirus G gene DNA vaccines at 12°C to 15°C Protection is indicated as relative percentage of survival (52)
Delivery has always been an important issue for the practical application of fish vaccines. The need to develop mass immunisation methods that can be used in aquaculture has been recognised, and so various different administration routes are being investigated; these include immersion and ultrasound using DNA-coated microspheres and DNA formulated in liposomes, but none of these alternatives has yet provided comparable efficacy to that of IM injection (12, 18, 19, 64, 65). In Atlantic salmon farming, the fish are presently injected intraperitoneally with oil-adjuvanted bacterial vaccines. The addition of DNA vaccines to these vaccines would seem to be a rational strategy, but intraperitoneal delivery of DNA vaccines has appeared to require considerably higher amounts of DNA than IM delivery (54).
DNA vaccines against other fish pathogens The DNA vaccines developed for fish rhabdoviruses other than IHNV and VHSV, such as spring viraemia of carp virus and hirame rhabdovirus, have also shown promise (67, 73, 76), but developing an effective DNA vaccine has been more of a challenge for other fish pathogens. Initial work with DNA vaccines encoding the outer protein of IPNV, which has a significant impact on Atlantic salmon smolts in their first few months in seawater, did not show protection. However, a recent report indicated that a high level of protection was induced in Atlantic salmon by using a plasmid encoding the whole polyprotein of IPNV (57). In the case of channel catfish herpesvirus, the protective ability of DNA vaccines appears inconsistent (27, 60). Similarly, none of the DNA vaccines tested to date for ISAV has given significant protection (E. Anderson,
personal communication). The DNA vaccines for AHNV have also been thoroughly tested and again do not appear to provide protection (71). Interestingly, however, the VHSV DNA vaccine induced a high level of protection against AHNV in turbot when the challenge was performed shortly after vaccination, thus demonstrating that the early protection phenomenon described above is not limited to rhabdovirus infections in salmonids (70). One of the first bacterial fish pathogens for which DNA vaccines were tested was Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout (24), but no protective effect has been reported. A more generic approach has been attempted for Piscirickettsia salmonis, against which fish were vaccinated with a full expression library of plasmid DNA. A pathogenspecific antibody response was subsequently detected, but the level of protection was relatively low (58). Very recently, a DNA vaccine encoding the secreted mycobacterial antigen Ag85A has been shown to induce protection against Mycobacterium marinum in hybrid striped bass (61). The only DNA vaccine tested thus far for a fish parasite encoded the immobilisation antigen of Ichthyophthirius multifiliis and did not show protection when tested in rainbow trout (68).
Safety As with other veterinary vaccines, three aspects must be addressed when it comes to safety: the vaccinated animals, the environment and the consumer. In all the experimental and clinical DNA vaccination experiments performed so far, in animal models as well as in humans, no serious side
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Rainbow trout with an average weight of 3 g to 4 g were given an intramuscular injection of plasmid DNA and exposed to waterborne VHSV seven weeks later. Plasmid without the G-gene (pcDNA3) conferred no protection whereas very significant protection was obtained with even 0.01 µg of plasmid including the G-gene (pcDNA3-vhsG) (44)
Fig. 3 Dose-response vaccination trial with a DNA vaccine against viral haemorrhagic septicaemia virus (VHSV)
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effects on the vaccinated individual have been reported. A comprehensive review of safety aspects related to DNA vaccination of food-producing animals has been prepared by Holm (30). Since DNA vaccines based on purified plasmid DNA carry only a single gene from the pathogen, are non-infectious and are unable to replicate within the vaccinated host, there is no risk of transferring the actual disease with the vaccine. Nucleic acid vaccines are therefore considered safer than conventional vaccines, i.e. inactivated whole virus, with or without oil adjuvant, or attenuated live virus (6). In contrast to most conventional vaccines based on inactivated pathogens, DNA vaccines for fish are not formulated with an oil adjuvant, which is known to cause post-vaccination side effects such as peritonitis (42, 56). Other factors that make DNA vaccines preferable are that inactivated whole virus vaccines may contain unknown impurities and trace amounts of inactivating agents, while live attenuated vaccines pose a risk of infection by mutants or may revert to virulence. Moreover, where DNA vaccines are used, side effects due to contaminants are negligible. This is because plasmid DNA can be prepared to a very high level of purity, and DNA consists of a precise sequence of nucleotide residues. Quality assurance of DNA vaccines is therefore less complicated than with traditional or live recombinant vaccine types. A number of theoretical safety concerns may be considered for DNA vaccines. These include: ­ the fate of the plasmid in the vaccinated animals ­ the risk of the integration of vaccine DNA sequences into the genome of the host, and subsequent negative side effects such as development of disease or integration into the germ line followed by vertical transfer ­ the risk of inducing an anti-DNA immune response. Thorough discussions of these aspects have been made accessible via the Internet by the Norwegian Biotechnology Advisory Board (21) and by the Danish Institute for Food and Veterinary Research (30). The distribution of the DNA vaccine depends on the delivery route. For the purposes of this discussion the focus will be on IM injection, since this is the only route that has consistently been shown to provide significant protection. Shortly after IM injection of fish, the plasmid can be found in small amounts in various tissues (3, 28). However, the vast majority of the injected plasmid DNA remains in the muscle tissue at the injection site. As with mammals, more than 99% of the injected DNA disappears within the first weeks after vaccination, leaving small amounts of long-term persisting plasmid (J. Rasmussen, personal communication). As discussed by Holm (30), this is probably because only a small fraction of the injected
DNA is taken up by the host cells, whereas extracellular DNA is rapidly degraded by nucleases. Persistence of host cells with reporter gene constructs has been demonstrated up to two years following vaccination (15), but vaccine constructs encoding pathogen antigens most likely persist for a shorter period due to the elimination of transfected cells by the fish immune system (Fig. 4) (28, 45). Investigations to date suggest that the injected plasmid DNA does not integrate into the genome of the host cells (3, 34). However, from a theoretical standpoint, it must be expected that such integration will occur, although probably very rarely. Calculations suggest that the chances of integration of vaccine DNA are considerably smaller than the chances of natural mutations (41). The risk of negative side effects due to integration of vaccine sequences into the host genome therefore appears negligible, compared to the many benefits of DNA vaccines. The chance of integration into the germ line is in all probability an even rarer event. In this context it should be kept in mind that several natural infections, such as those of DNA-viruses (e.g. papilloma, herpes, hepatitis and pox viruses), result in considerable exposure of the organism to foreign DNA. This is also true for vaccines based on attenuated/
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The fish were anaesthetised and injected with 20 µg of plasmid in the epaxial muscle (a). In fish injected with a plasmid encoding the VHSV G-gene, expression of the G protein (red staining) by myocytes along the needle track induced a local inflammatory reaction (many infiltrating leucocytes with blue nuclei) which reached a maximum 21 days post vaccination (b). At 31 days post vaccination the majority of the G-positive myocytes had been eliminated and muscle regeneration at the needle track was in progress (c). In fish injected with a plasmid encoding the VHSV N-gene, no inflammation was seen 21 days post vaccination and myocytes containing N protein were still present 31 days post vaccination (d). The fish examined in b-d were all given extraordinary high doses of DNA in order to allow visualisation of the expressed VHSV proteins by immuno-histochemistry as well as the inflammatory reaction induced near muscle cells expressing the G-protein Fig. 4 Intramuscular delivery of a DNA vaccine against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout and immuno-histoChemical analysis of the injection site (Based on 45 and 52)
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nonpathogenic DNA viruses. Apart from a beneficial adjuvant-like effect of so-called CpG motifs in the bacterial genes included in the DNA vaccine plasmids (33, 36), no adverse effects in terms of an immune response to the vaccine DNA itself have been reported (34). What about the consumers eating DNA-vaccinated fish? Since consumers will generally only eat the fish months or even years after vaccination, very small amounts of vaccine are likely to be left at the time of consumption. Compared with the total amount of DNA in the food, the vaccine DNA will constitute a negligible amount. Should vaccine DNA be taken up via the intestine by cells of the consumer, the chances of negative side effects are expected to be very small, based on the fact that no such effects have been seen in numerous human volunteers who were given milligram doses of plasmid DNA in previous and ongoing safety testing of DNA vaccines against human pathogens (16, 43). Scientific data in this field are limited, however, and experiments, including feeding mammals flesh from DNA-vaccinated fish, should be conducted. This would also address concerns about the potential spread of a DNA vaccine in the environment by predatory animals that eat vaccinated fish. Part of the analysis should include testing the intestinal flora of the predators as well as the microbial flora in the immediate environment of the vaccinated fish. Although the chances are most probably minimal, other bacteria can theoretically take up the vaccine plasmid. However, E. coli, the most likely organism that could be implicated in transmitting the plasmid outside the target species, is not considered a natural component of the gut flora of salmonids under culture conditions (14) and is absent from the intestinal content of cultured fishes (25). In order to achieve the highest possible level of precaution, DNA vaccine plasmids for fish should be limited to include only the strictly necessary genes and regulatory elements, and be devoid of gene elements such as genes that mediate resistance to important antibiotics. In terms of veterinary regulations, use of marker vaccines is often desirable in order to allow differentiation between vaccinated and non-vaccinated animals on the basis of their antibody response. Although inclusion of a gene encoding a marker antigen should be fairly straightforward in the case of the DNA vaccines, such inclusion would go against the precautionary strategy of keeping the number of genes and regulatory elements to a minimum. Furthermore, since the antibody response in fish often varies considerably, depending on temperature as well as other parameters, the use of marker vaccines may be of limited value. Sensitive DNAamplification assays based on polymerase chain reaction allow detection of the vaccine plasmid in vaccinated fish up to at least six months post vaccination with
1 µg of DNA (J. Rasmussen, personal communication), and would in many cases be sufficient to fulfil the veterinary requirements. Regulation of veterinary DNA vaccines Due to the rapid progress in the development of DNA vaccines, which only started experimentally in the early 1990s, there is limited experience with potential long-term effects. Since no DNA vaccines have been licensed yet, one remaining major challenge is to develop an appropriate set of regulatory requirements for these vaccines (16, 69). Administrative organisations such as the Food and Drug Administration in the United States of America and the European Agency for Evaluation of Medical Products have prepared some guidelines concerning DNA vaccines in general and veterinary DNA vaccines in particular (17, 20, 69). Several relevant issues, such as requirements on composition and safety testing, are covered, but no specific restrictions in terms of use/application of DNA vaccines are given. As discussed by Foss and Rogne (22), one central issue is differentiation between an animal that has been treated with a medical product containing manipulated gene(s) and a GMO. The delineation between these two classifications is not clear, but if the medical product results in stable integration of foreign DNA into the germ line of a treated animal then, by definition, the latter can become a GMO. However, with traditional DNA vaccines, the probability of turning the vaccinated animal into a GMO should be considered to be negligible, as discussed above. The various national regulatory organisations treat this issue in differing ways. The British Agriculture and Environment Biotechnology Commission considers that as long as the foreign DNA is not integrated into the host's genome, a DNA-vaccinated animal is not to be considered as a GMO (1). A similar standpoint has been taken by the Danish Medical Authorities in the case of the VHS DNA vaccine described above. In contrast, the Norwegian Directorate for Nature Management has suggested that a DNA-vaccinated fish should be considered genetically modified as long as the foreign DNA is present in the fish (22). This definition is based on the precautionary principle but could have a negative impact by diluting the GMO concept. For instance, how should animals that have eaten feed containing DNA from GMO-crops be classified? Under the Norwegian definition, DNA-vaccinated companion animals and wild animals vaccinated with genetically modified viruses, such as the vaccinia-virusbased rabies vaccine used in Europe and Canada, would also be defined as GMOs. Such a definition would further complicate regulatory issues. As recommended by the
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Norwegian Biotechnology Advisory Board (21, 22), new medical products based on the transfer of genes should be evaluated on a case-by-case basis, and gene-medicated animals should only be termed GMOs if the foreign DNA is likely to be inherited by the offspring or if the genetic material is expected to cause negative side effects of some kind if integrated. Field-testing Efficacy and safety While the salmonid rhabdovirus DNA vaccines have proved excellent under experimental conditions, testing under commercial fish farming conditions is needed before the real potential of these vaccines can be determined. Higher stress levels, different growth conditions and exposure to other pathogens are some of the parameters that could affect vaccine efficacy in the field. Field-testing should preferably include not only exposure of vaccinated fish to natural outbreaks of disease, but also a thorough examination of the health and growth performance of the vaccinated fish compared to non-vaccinated controls. Testing under field conditions has recently been initiated for IHNV in Atlantic salmon in Canada and is also scheduled for VHSV in Denmark. Infectious haematopoietic necrosis virus is endemic to the Pacific Northwest, but has varying effects on different Pacific salmonids. The virus first appeared in farmed Atlantic salmon in British Columbia, Canada, in 1992 (5). Four waves of outbreaks (1995, 1996, 1997 and 2001) have occurred since that time, resulting in the destruction of millions of smolts as a disease management measure. Mortality rates in older fish (2 kg to 3 kg) tend to range from 10% to 20%; in smolts the rate often exceeds 85%. Consequently, IHNV is having a serious impact on salmon aquaculture in British Columbia. The estimated economic loss from recent disease outbreaks was US$40 million, which represents US$200 million in lost sales. These mortalities not only have significant adverse economic impacts on the British Columbia aquaculture industry, preventing its growth, but also affect other socio-economic factors such as job creation in remote coastal communities. A clinical safety trial of a DNA vaccine against IHNV in Atlantic salmon under commercial production conditions in British Columbia is currently in progress. The vaccine has been approved for investigational use by the Animal Health and Production Division of the Canadian Food Inspection Agency. At the hatchery, three million Atlantic salmon with an average size of 25 g were each given an IM injection of 10 µg of vaccine at least 400 degree-days prior to seawater transfer (degree-days: sum of daily mean temperatures for a given time period). All hatchery effluent water in British
Columbia is treated with ultraviolet light, so the risk of transfer of the plasmid to freshwater and marine invertebrates and other non-target aquatic species is minimal. Studies have further demonstrated that uptake of plasmid DNA via the water route is highly inefficient (12). Since the disease agent IHNV is endemic to the British Columbia coast, expression of the IHNV G protein already occurs naturally in the environment. If an adverse event occurs during the field vaccination trials, containment procedures will be implemented. After seawater transfer, the risk of shed and spread is considered negligible. This study is the first clinical safety trial of a DNA vaccine in fish under commercial production conditions. In Europe, VHS is the most important viral disease in farmed rainbow trout. Outbreaks of this virus can result in very high mortality among rainbow trout of all sizes, and at present the only possible control measure is stampingout animals on infected farms in combination with intensive surveillance and control programmes. In Denmark, intensive stamping-out programmes over the past 30 years have reduced the percentage of infected farms from 90% in the early 1970s to 5% to 10% today. However, the remaining farms are situated in an endemically infected zone and disease eradication has been very difficult, possibly due to the size and complexity of the water bodies as well as the intensity of the fish farming activities (60). An effective vaccine could be a very valuable tool to supplement the stamping-out process. After one or two seasons with DNA-vaccinated fish, horizontal transmission of the virus would decrease and stamping-out would probably have a much higher chance of success in terms of eradicating the virus. Restocking should then include non-vaccinated fish only, since vaccination will not be allowed in zones that are to be declared free from VHSV. Regular use of vaccination could also be beneficial in larger endemically infected areas in other European countries. Since IHNV and VHSV are both present in several regions, co-administration of the DNA vaccines could be an option. Under laboratory conditions the two vaccines do not affect one another (unpublished observations) and simple mixing of the two plasmids before IM injection would be a reasonable strategy. A small-scale preliminary DNA vaccine field test in Denmark has been initiated as a collaborative project of the Danish Institute for Food and Veterinary Research and the Danish fish farmers association (Danish Aquaculture). Fingerling-size fish will be vaccinated with 1 µg of plasmid DNA and kept in farms that are free from VHSV but situated outside the VHSV-free certified zone. Once VHS outbreaks occur (in these or in other fish farms), net-cages with vaccinated and non-vaccinated control fish will be transferred to ponds affected by the disease. Upon termination of the trials, all experimental fish will be humanely euthanized and destroyed. As well as examining
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the protective effect of the vaccine, the study will include animal safety aspects and track the persistence and fate of the vaccine plasmid. Permission/acceptance has been obtained from the relevant public authorities, including the Danish Medicines Agency, the Danish Forest and Nature Agency, the Danish Agency for Animal Experiments and the Danish Veterinary and Food Administration. Animal welfare Compared with other forms of animal farming, finfish aquaculture has both advantages and disadvantages in terms of animal welfare. Fish have specific physical and chemical requirements relating to the aquatic environment, and when these requirements are not met, the health and survival of the animals can be jeopardised by the resulting stress. Culturing animals in water requires stricter attention to detail than terrestrial animal culture does. In terms of animal welfare, one benefit of this attention to detail is that aquatic producers recognise that controlling animal stress is essential for economic success, and that the development of specific stress management protocols is vital for aquatic animal health and survival (10). The utilisation of appropriate vaccines can be a very effective stress management technique, since infected/diseased fish are considerably more susceptible to stress. A North American research project was recently initiated to determine the effect of the IHNV DNA vaccine on the Health and Welfare of Atlantic salmon. The study will make a comprehensive examination to compare physiological, immunological and haematological factors in vaccinated and unvaccinated fish, by sampling the fish in the freshwater hatchery both prior to and after vaccination, and every three months following seawater entry. Studies of this type will provide information on the safety of the vaccine from the perspective of fish health and welfare. Cost-benefit The technology for the production of plasmid DNA for medical purposes has continuously been improved over the past decade, and the cost has simultaneously been reduced. As only tiny amounts of DNA are needed to vaccinate fish, the pure production cost of fish DNA vaccines will probably be low enough to make the technology viable in commercial aquaculture. Automatic devices for IM delivery of the vaccines to small fish (or some alternative methods) will have to be developed, but this is considered feasible, taking into account that vaccination machines for intraperitoneal delivery are already used commercially. However, the cost of licensing could inhibit the use of vaccines in commercial fish farming. There are a considerable number of patents and other types of intellectual Property Rights within the field of
DNA vaccines, and it will be important that royalty fees and similar costs are set at levels that reflect the relatively small profit levels obtained from manufacturing fish vaccines. The benefits of efficacious vaccines against viral diseases in fish will include: ­ improved health and welfare of aquacultured fish ­ reduced environmental impact of fish farming activities, by decreasing the discharge of medical substances, disinfectants, plant nutrients and organic feed/waste residuals into the water-bodies ­ improved quality and safety of food products, based on healthy fish that are free from medical/chemical residuals ­ improved economic efficiency in fish farming activities and related industries. Moreover, by potentially being among the first approved DNA vaccines for veterinary use, DNA vaccines for fish could help to move such treatment into clinical use in general. Public perception Introduction of vaccines into aquaculture has, to our knowledge, not had any negative impact on the way consumers perceive aquacultured fish. Whether this is due to a lack of information about vaccination procedures or a general acceptance remains to be determined. However, as a result of the current debate about the use of GMOs in food production, the potential relationship between GMOs and DNA-vaccinated fish could become a sensitive issue, at least in countries where consumers are reluctant to accept food containing GMO-derived products. In such countries it is therefore important to have a clear regulatory strategy as well as to keep the public well informed. A classification of DNA-vaccinated animals as GMOs, and related requests on GMO-labelling of food products derived from such animals, would be very likely to have a strong negative impact on the sales of ­ and thereby prevent the use of ­ DNA vaccines. The authors believe that the most fruitful strategies for society as a whole would be to adopt the individual examination of risk and benefit for each vaccine, as recommended by the Norwegian Biotechnology Advisory Board (22), and to exclude DNA-vaccinated animals from the GMO labelling requirements, except if there is scientific evidence of a real risk of the integration of vaccine DNA into the inherited germ-line DNA. Final remarks In contrast to many DNA vaccines tested in other animal species, the DNA vaccines against rhabdoviruses in
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aquacultured fish have proved to be very effective in the target species. A single 1 µg dose of plasmid DNA promptly stimulates immunity, which appears to persist throughout the normal lifespan of a cultured food fish. As traditional vaccines against fish rhabdoviruses have not been successful, the DNA vaccine technology could provide a valuable tool for more sustainable production of farmed fish. Although there has been preliminary testing using IM injection under field conditions, more suitable delivery methods need to be developed in order to make vaccination of small fish (below 5 g) economically feasible. Other requirements that will present an important challenge for authorities and scientists working in fish vaccinology are to achieve transparency of regulatory and safety issues, and to ensure public dissemination of information about the positive effects of DNA vaccines in aquaculture. As this issue went to press the FDA released a draft guidance note entitled `Guidance for industry: considerations for plasmid DNA vaccines for infectious disease indications' (www.fda.gov/cber/gdlns/plasdnavac.pdf). When finalised,
the document will represent an update to the guidelines published by FDA in 1996 (20). Acknowledgements The authors thank numerous colleagues for their assistance, in particular: K. Einer-Jensen and E. Lorenzen, who provided the material for the figures; J. Rasmussen and E. Anderson, who provided unpublished information about the persistence of DNA vaccine and about ISAV DNA vaccine experiments, respectively; G. Kurath, who gave access to literature `in press'; and A. Holm, G. Foss and H. Korsholm, who offered useful comments on the manuscript. The American Fisheries Society is acknowledged for allowing the use of figure material from reference 44. Elsevier is thanked for permission to include figure material from references 44, 45 and 52. This work was supported by a research grant from the Danish Ministry for Food, Agriculture and Fisheries (93s-24F4-Е02-00042 FШTEK4).
Vaccins а ADN destinйs aux poissons d'йlevage N. Lorenzen & S.E. LaPatra Rйsumй La vaccination а acide dйsoxyribonuclйique (ADN) consiste а administrer le gиne codant pour l'antigиne vaccinal et non l'antigиne lui-mкme. L'expression de cet antigиne par les cellules du sujet vaccinй stimule son systиme immunitaire. Parmi les nombreux vaccins expйrimentaux а ADN testйs sur diffйrentes espиces animales ainsi que chez l'homme, ce sont les vaccins contre les maladies а rhabdovirus chez les poissons qui ont donnй les rйsultats les plus prometteurs. Une injection intramusculaire unique de quantitйs d'ADN de l'ordre du microgramme confиre aux salmonidйs d'йlevage une protection rapide et durable contre les virus qui produisent un impact йconomique important, tel que les virus de la nйcrose hйmatopoпйtique infectieuse (VNHI) et de la septicйmie hйmorragique virale (VSHV). Les vaccins а ADN dirigйs contre les autres types d'agents pathogиnes touchant les poissons n'ont connu а ce jour qu'un succиs limitй. L'administration la plus efficace а l'heure actuelle est l'injection intramusculaire, et des stratйgies d'administration adaptйes restent а dйvelopper pour la vaccination massive des petits poissons. Sur le plan de la tolйrance, aucun effet indйsirable n'a йtй observй а ce jour chez les poissons vaccinйs. Йtant donnй que les vaccins а ADN constituent une technologie relativement rйcente, certains aspects thйoriques, de mкme que la sйcuritй а long terme pour l'environnement et le consommateur, n'ont pas encore йtй totalement rйsolus. Les risques ne devraient cependant pas кtre plus importants qu'avec les vaccins actuellement commercialisйs pour les poissons. Les systиmes de classification dont on dispose aujourd'hui ne permettent pas de distinguer clairement les animaux ayant reзu un vaccin а ADN des organismes gйnйtiquement modifiйs, ce qui risque de poser des problиmes en termes d'approbation et d'acceptation de cette nouvelle technologie. Parmi les avantages potentiels des vaccins а ADN chez les poissons d'йlevage, il faut citer
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les progrиs en matiиre de bien-кtre animal, d'impact environnemental de l'aquaculture, d'une meilleure qualitй et quantitй d'aliments et de production durable. Des essais а йchelle industrielle ont йtй rйcemment lancйs au Canada et au Danemark. Mots-clйs Aspect rйglementaire ­ Bien-кtre animal ­ Essai sur le terrain ­ Glycoprotйine ­ Maladie virale ­ Mйcanisme protecteur ­ Perception du consommateur ­ Plasmide ­ Poisson d'йlevage ­ Rapport coыt/bйnйfice ­ Sйcuritй ­ Vaccin а ADN ­ Voie d'administration.
Vacunas de ADN para peces de vivero N. Lorenzen & S.E. LaPatra Resumen La vacunaciуn con бcido desoxirribonucleico (ADN) consiste en administrar al organismo receptor el gen que codifica el antнgeno inmunуgeno en lugar del propio antнgeno. La subsiguiente expresiуn del gen en las cйlulas del animal vacunado activa su sistema inmunitario. Entre las muchas vacunas de ADN experimentales que se han ensayado en varias especies animales y en el hombre, las que ofrecen resultados mбs prometedores son las vacunas contra enfermedades rhabdovнricas de los peces. Una sola inyecciуn intramuscular de unos pocos microgramos de ADN induce, en salmуnidos de vivero, una protecciуn rбpida y duradera contra los agentes de enfermedades de gran importancia econуmica como el virus de la necrosis hematopoyйtica infecciosa (VNHI) o el de la septicemia hemorrбgica viral (VSHV). Hasta la fecha, sin embargo, las vacunas de ADN contra otros patуgenos de los peces no han dado mucho fruto. De momento la vнa de administraciуn mбs eficaz es la inyecciуn intramuscular, pero todavнa no se han elaborado estrategias adecuadas para la vacunaciуn masiva de peces pequeсos. Por lo que respecta a la inocuidad, no se ha observado hasta ahora ningъn efecto adverso en los peces vacunados. Toda vez que la vacunaciуn con ADN es una tйcnica relativamente nueva, aъn no se han estudiado a fondo, desde el punto de vista teуrico y de la inocuidad a largo plazo, una serie de aspectos relacionados con la influencia de las vacunas sobre el medio ambiente y la salud del consumidor, aunque en buena lуgica los riesgos no deberнan ser mayores que con las vacunas comerciales que se estбn administrando hoy en dнa a los peces. Los actuales sistemas de clasificaciуn resultan poco claros a la hora de distinguir entre animales vacunados con ADN y organismos modificados genйticamente, hecho que podrнa tener consecuencias en cuanto a las licencias de comercializaciуn y a la aceptaciуn de esta tйcnica por parte de la opiniуn pъblica. La vacunaciуn con ADN de peces de vivero podrнa deparar, entre otros, los siguientes beneficios: mayor nivel de bienestar animal; menores efectos ambientales de las actividades acuнcolas; obtenciуn de alimentos de mejor calidad y en mayor cantidad; y producciуn mбs sostenible. Hace poco tiempo han empezado a ensayarse estas vacunas en condiciones de producciуn industrial en Canadб y Dinamarca. Palabras clave Administraciуn de vacunas ­ Aspecto reglamentario ­ Bienestar animal ­ Enfermedad vнrica ­ Glucoproteнna ­ Inocuidad ­ Mecanismo de protecciуn ­ Pez de vivero ­ Plбsmido ­ Prueba de terreno ­ Punto de vista del consumidor ­ Costo-beneficio ­ Vacuna de бcido desoxirribonucleico.
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66. Russell P.H., Kanellos T., Negrou M. & Ambali A.G. (2000). ­ Antibody responses of goldfish (Carassius auratus L.) to DNA-immunisation at different temperatures and feeding levels. Vaccine, 18, 2331-2336. 67. Shchelkunov I.S., Oreshkova S.F., Shchelkunova T.I., Nikolenko G.N., Kupinskaya O.A. & Voronova O.S. (2001). ­ Comparative testing of experimental SVC-vaccines developed with the use of recombinant DNA technology (abstract). In Book of Abstracts, 10th International Conference of the European Association of Fish Pathologists (EAFP). 10-14 September, Dublin. EAFP, Denmark, 173. 68. Sign J. (2003). ­ The immune response in rainbow trout (Oncorhynchus mykiss) against the ciliate Ichthyophthirius multifiliis Fouquet, 1876: selected immune mechanisms and testing of DNA vaccination. PhD thesis, the Royal Veterinary and Agricultural University, Denmark. Frederiksberg Bogtrykkeri A/S, Denmark, 120 pp. 69. Smith H.A. (2000). ­ Regulation and review of DNA vaccine products. Dev. Biol., 104, 57-62. 70. Sommerset I., Lorenzen E., Lorenzen N., Bleie H. & Nerland A.H. (2003). ­ A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot. Vaccine, 21 (32), 4661-4667. 71. Sommerset I., Skern R., Biering E., Bleie H., Fiksdal I.U., Grove S. & Nerland A.H. (2005). ­ Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination. Fish Shellfish Immunol., 18 (1), 13-29. 72. Sudha P.M., Low S., Kwang J. & Gong Z. (2001). ­ Multiple tissue transformation in adult zebrafish by gene gun bombardment and muscular injection of naked DNA. Mar. Biotechnol., 3 (2), 119-125. 73. Takano T., Iwahori A., Hirono I. & Aoki T. (2004). ­ Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination. Fish Shellfish Immunol., 17 (4), 367-374. 74. Traxler G.S., Anderson E., LaPatra S.E., Richard J., Shewmaker B. & Kurath G. (1999). ­ Naked DNA vaccination of Atlantic salmon Salmo salar against IHNV. Dis. aquat. Organisms, 38 (3), 183-190. 75. Van Drunen Littel-van den Hurk S., Babiuk S.L. & Babiuk L.A. (2004). ­ Strategies for improved formulation and delivery of DNA vaccines to veterinary target species. Immunol. Rev., 199, 113-125. 76. Vesely T., Pokorova D., Einer-Jensen K. & Lorenzen N. (2004). ­ DNA vaccination of small carp (Cyprinus carpio) against SVCV: the protective effect depends on temperature. In Book of Abstracts, 6th International Symposium on Fish Immunology. 26-29 May, Turku, Finland, 43. 77. Winton J.R. (1997). ­ Immunization with viral antigens: infectious haematopoietic necrosis. In Fish vaccinology (R. Gudding, A. Lillehaug, P.J. Midtlyng & F. Brown, eds). Dev. biol. Standard., 90, 211-220.

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