Dna Vaccine Fish

Rev. sci. tech. Off. int. Epiz., 2005, 24 (1), 201-213

DNA vaccines for aquacultured fishN. 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

SummaryDeoxyribonucleic acid (DNA) vaccination is based on the administration of thegene encoding the vaccine antigen, rather than the antigen itself. Subsequentexpression of the antigen by cells in the vaccinated hosts triggers the hostimmune system. Among the many experimental DNA vaccines tested in variousanimal species as well as in humans, the vaccines against rhabdovirus diseasesin fish have given some of the most promising results. A single intramuscular (IM)injection of microgram amounts of DNA induces rapid and long-lastingprotection in farmed salmonids against economically important viruses such asinfectious haematopoietic necrosis virus (IHNV) and viral haemorrhagicsepticaemia virus (VHSV). DNA vaccines against other types of fish pathogens,however, have so far had limited success. The most efficient delivery route atpresent is IM injection, and suitable delivery strategies for mass vaccination ofsmall fish have yet to be developed. In terms of safety, no adverse effects in thevaccinated fish have been observed to date. As DNA vaccination is a relativelynew technology, various theoretical and long-term safety issues related to theenvironment and the consumer remain to be fully addressed, although inherentlythe risks should not be any greater than with the commercial fish vaccines thatare currently used. Present classification systems lack clarity in distinguishingDNA-vaccinated animals from genetically modified organisms (GMOs), whichcould raise issues in terms of licensing and public acceptance of the technology.The potential benefits of DNA vaccines for farmed fish include improved animalwelfare, reduced environmental impacts of aquaculture activities, increasedfood quality and quantity, and more sustainable production. Testing undercommercial production conditions has recently been initiated in Canada andDenmark.

KeywordsAnimal welfare – Consumer perceptions – Cost-benefit – Delivery – Deoxyribonucleicacid vaccine – Farmed fish – Field-testing – Glycoprotein – Plasmid – Protectivemechanisms – Regulatory issues – Safety – Viral diseases.

IntroductionThe first vaccines against infectious bacterial diseases infarmed fish were developed in the 1970s, and introducedinto commercial aquaculture in the early 1980s. Overallthere has been a significant reduction in the use ofantibiotics following the introduction of vaccines,particularly in the farmed Atlantic salmon industry (56).This has contributed significantly to the growth of theindustry and to consumer acceptance of farm-raised fish.The latter is due to the reduced environmental impact andimproved food quality obtained by minimising antibiotic

use. In addition, animal welfare has been improved by theimplementation of vaccination.

The successful bacterial vaccines that are now routinelyused in aquaculture were developed largely throughempirical observations and are usually based oninactivated bacteria. Despite extensive research over manyyears, very few anti-viral vaccines are available and thereare no commercial vaccines against fish parasites.

There have been several attempts to develop traditionalvaccines against viral diseases based on inactivated orattenuated viruses (9, 48, 77), and both types of vaccines

have been shown to induce a certain level of protectionagainst some of the important salmonid viruses, includingviral haemorrhagic septicaemia virus (VHSV), infectioushaematopoietic necrosis virus (IHNV), infectiouspancreatic necrosis virus (IPNV) and infectious salmonanaemia virus (ISAV). Since viruses must be replicated incultures of fish cells, the cost of producing vaccines basedon inactivated viruses is usually too high to make thisstrategy economically viable. In comparison, attenuatedvirus vaccines have several advantages. These vaccines canbe delivered via the water route, which is optimal in termsof minimal stress and cost, and because a certain amountof replication takes place in the vaccinated fish, the doserequired for protection is small compared to inactivatedvirus. However, attenuated virus vaccines occasionallycause disease, and the release of live vaccines into the waterbodies is often not compatible with veterinary andenvironmental control strategies. Viral vaccines in the formof a recombinant viral protein produced in geneticallyengineered Escherichia coli have also been attempted. ForIPNV, a recombinant viral protein (VP2) is mixed in an oil-adjuvanted multivalent bacterin vaccine for Atlanticsalmon smolts. The vaccine is expected to have a protectiveeffect against infectious pancreatic necrosis (IPN) (9). Atthe experimental stage, similar effects have beendemonstrated for Atlantic halibut nodavirus (AHNV),where recombinant virus capsid protein in an oil-adjuvanted vaccine has mediated some protection againstdisease in turbot (71). For the rhabdoviruses VHSV andIHNV, the protective effect of recombinant protein vaccineshas been limited or inconsistent (48, 77).

The most efficient vaccines against viral diseases in fish todate at the experimental level are deoxyribonucleic acid(DNA) vaccines against the salmonid rhabdoviruses, VHSVand IHNV. These vaccines are based on naked plasmidDNA, which following uptake in cells of the vaccinated fishmediates 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 fishinvolved the use of genes encoding reporter proteins suchas luciferase, β-galactosidase and green fluorescent proteinto study the magnitude of expression levels under differentconditions, the tissue distribution, the duration ofexpression, and to some extent also the immune response(2, 24, 26, 28, 66). More recently, work on DNA vaccinescontaining genes that encode antigens from fish pathogenshas expanded to explore immune responses and protectionagainst pathogen challenge in fish (7, 44, 45, 52, 54, 61,63, 73, 76).

In humans a number of clinical trials with DNA vaccinesagainst diseases such as acquired immune deficiencysyndrome, hepatitis and malaria have been initiated.Although the results have been promising in terms ofsafety, the results have indicated that prime-boost strategiescombining DNA vaccines with other types of vaccines

and/or adjuvants are needed to obtain an adequateimmune response (16, 43). No veterinary or human DNAvaccines have been licensed yet, but recently, a prototypeDNA vaccine against West Nile virus was used to vaccinatewild condors in California. A similar vaccine has provedefficient in protecting horses against the same virus and islikely to become the first commercially licensed DNAvaccine (62). However, despite many promising results inmice models, the majority of the DNA vaccines tested inveterinary target species so far have – as with DNAvaccines tested in humans – had relatively low efficacy(75). The main technical hurdle appears to be inefficientuptake of the administered DNA by the host cells (75).

This article considers the principles and perspectivesrelated to application of DNA vaccines in fish that arecommercially cultured for food production, focusing onthe DNA vaccines against fish rhabdoviruses. Theadvantages and disadvantages of DNA vaccines aresummarised in Table I.

Characteristics of the DNAvaccines against fishrhabdovirusesAlthough development of DNA vaccines has beenattempted for various pathogens in a number of differentfish species, the DNA vaccines against the salmonidrhabdoviruses IHNV and VHSV remain the most efficientand also the most extensively analysed to date. Thesevaccines are highly effective under a variety of conditions,including different fish life stages and different salmonidhost species, and against challenge with different virusstrains (13, 23, 38, 39, 50, 51, 74).

The first step in producing a DNA vaccine is to identify andclone a protective antigen from the pathogen. For VHSVand IHNV, earlier work had shown that protectiveantibodies were directed against the viral surfaceglycoprotein G (31, 47). The gene encoding the G protein,in combination with regulatory sequences that allowexpression in eukaryotic cells, was therefore also anobvious candidate for a DNA vaccine (Fig. 1). The viralgenome includes five other genes, but none of these haveproven useful for induction of immunity when delivered asDNA vaccines (11). Prior to vaccination, the vaccineplasmid is produced in bacterial culture, purified andquality-assured. Following administration of a DNAvaccine, certain cells of the host take up the vaccine andutilise the machinery of the cell to produce the G protein.When detected by the fish immune system, such cells willappear like virus-infected cells with G-protein on theirsurface (Fig. 1). This leads to activation of both humoral

Rev. sci. tech. Off. int. Epiz., 24 (1)202

and cellular defence mechanisms in the fish (2, 7, 8, 28,49, 63, 73). One interesting feature of the immuneresponse to the VHSV and IHNV G gene DNA vaccines isthat the specific protection is preceded by an earlynonspecific antiviral protection (Fig. 2), possibly related tointerferon-induced mechanisms (35, 40, 52, 53, 54).

Delivery and efficacyFor mammals the preferred delivery strategies have beenintramuscular (IM) injection or particle-mediated deliveryby gene gun. The latter entails coating small gold particleswith vaccine DNA, followed by air-pressure-mediatedintradermal delivery. Although such DNA vaccination bygene gun is effective in fish (12, 24, 72), this technology istoo expensive to be cost effective in commercialaquaculture. Interestingly, simple IM injection of purifiedplasmid DNA in a neutral buffer has proven to be moreefficient in fish than in any other type of animal tested todate. Dose–response experiments have shown that a singleinjection of nanogram levels of plasmid DNA is sufficientto induce protective immunity against viral haemorrhagicsepticaemia (VHS) and IHN in rainbow trout fingerlings(Fig. 3) (13, 44). The protection is not only rapidlyinduced but also long lasting (Fig. 2), (38, 44). It appearsbeneficial to vaccinate the fish when they are small, sincelarger fish require a higher dose of vaccine to be protected(39, 51).

Rev. sci. tech. Off. int. Epiz., 24 (1) 203

Table IAdvantages and disadvantages of deoxyribonucleic acid (DNA) vaccines

Advantages Disadvantages/current problems

Generic and simple principle Difficulty/cost of delivery; need for new strategies for mass vaccination of small fish

High level of safety – no risk of infectious disease Not efficient for all pathogens

Combination of advantages of traditional killed and attenuated vaccines New concept – long-term safety issues remain to be analysed

Can be successful when traditional vaccine strategies fail Official distinction between DNA-vaccinated animals and genetically modified organism

(GMO)´s not always clear

Possibility of incorporating molecular adjuvants such as CpG motifs Public aversion to ingredients from GMOs in food products, which might influence

consumers’ acceptance of veterinary DNA vaccines

Activation of both humoral and cellular mechanisms * No regulatory precedents yet available for DNA vaccines for husbandry animals

Multivalent vaccination possible by simple mixing of DNA vaccines * Possible complications of intellectual property rights affecting commercialisation of

veterinary 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

*Specifically demonstrated in the case of DNA vaccines for fish

In the vaccine plasmid, the eukaryotic promoter (Prom.), antibiotic resistance selectionmarker (Antibiotic) and the inserted fish virus glycoprotein gene (G gene) are indicated (b).The G protein is a transmembrane molecule with oligosaccharide side chains andstabilised by disulphide bonds (s—s) (c). The G protein appears on the surface of virusinfected cells as well as on the surface of virus particles. Once the vaccine plasmid hasreached the nucleus of a cell in the vaccinated fish, expression of G protein will be initiatedand G protein molecules will appear inside the cell and on the cell’s surface, as if the cellhad been naturally infected with virus (52)

Fig. 1Schematic drawing of a rhabdovirus particle (a), the vaccineplasmid (b), and the viral G protein (c)

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G~glycoprotein (trimer)

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Delivery has always been an important issue for thepractical application of fish vaccines. The need to developmass immunisation methods that can be used inaquaculture has been recognised, and so various differentadministration routes are being investigated; these includeimmersion and ultrasound using DNA-coatedmicrospheres and DNA formulated in liposomes, but noneof these alternatives has yet provided comparable efficacyto that of IM injection (12, 18, 19, 64, 65). In Atlanticsalmon farming, the fish are presently injectedintraperitoneally with oil-adjuvanted bacterial vaccines.The addition of DNA vaccines to these vaccines wouldseem to be a rational strategy, but intraperitoneal deliveryof DNA vaccines has appeared to require considerablyhigher amounts of DNA than IM delivery (54).

DNA vaccines against other fishpathogensThe DNA vaccines developed for fish rhabdoviruses other thanIHNV and VHSV, such as spring viraemia of carp virus andhirame rhabdovirus, have also shown promise (67, 73, 76), butdeveloping an effective DNA vaccine has been more of achallenge for other fish pathogens. Initial work with DNAvaccines encoding the outer protein of IPNV, which has asignificant impact on Atlantic salmon smolts in their first fewmonths in seawater, did not show protection. However, a recentreport indicated that a high level of protection was induced inAtlantic salmon by using a plasmid encoding the wholepolyprotein of IPNV (57). In the case of channel catfishherpesvirus, the protective ability of DNA vaccines appearsinconsistent (27, 60). Similarly, none of the DNA vaccines testedto date for ISAV has given significant protection (E. Anderson,

personal communication). The DNA vaccines for AHNV havealso been thoroughly tested and again do not appear to provideprotection (71). Interestingly, however, the VHSV DNA vaccineinduced a high level of protection against AHNV in turbot whenthe challenge was performed shortly after vaccination, thusdemonstrating that the early protection phenomenon describedabove is not limited to rhabdovirus infections in salmonids (70).

One of the first bacterial fish pathogens for which DNAvaccines were tested was Renibacterium salmoninarum, thecausative agent of bacterial kidney disease in salmon andtrout (24), but no protective effect has been reported. Amore generic approach has been attempted forPiscirickettsia salmonis, against which fish were vaccinatedwith a full expression library of plasmid DNA. A pathogen-specific antibody response was subsequently detected, butthe level of protection was relatively low (58). Veryrecently, a DNA vaccine encoding the secretedmycobacterial antigen Ag85A has been shown to induceprotection against Mycobacterium marinum in hybridstriped bass (61). The only DNA vaccine tested thus far fora fish parasite encoded the immobilisation antigen ofIchthyophthirius multifiliis and did not show protectionwhen tested in rainbow trout (68).

Safety As with other veterinary vaccines, three aspects must beaddressed when it comes to safety: the vaccinated animals,the environment and the consumer. In all the experimentaland clinical DNA vaccination experiments performed sofar, 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 injectionof plasmid DNA and exposed to waterborne VHSV seven weeks later. Plasmid without theG-gene (pcDNA3) conferred no protection whereas very significant protection was obtainedwith even 0.01 µg of plasmid including the G-gene (pcDNA3-vhsG) (44)

Fig. 3Dose-response vaccination trial with a DNA vaccine againstviral haemorrhagic septicaemia virus (VHSV)

Fig. 2Schematic illustration of the assumed complementary roles ofearly non-specific mechanisms and subsequent specificmechanisms in the protection induced by vaccination ofrainbow trout with the fish rhabdovirus G gene DNA vaccines at12°C to 15°CProtection is indicated as relative percentage of survival (52)

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effects on the vaccinated individual have been reported. A comprehensive review of safety aspects related to DNAvaccination of food-producing animals has been preparedby Holm (30).

Since DNA vaccines based on purified plasmid DNA carryonly a single gene from the pathogen, are non-infectiousand are unable to replicate within the vaccinated host,there is no risk of transferring the actual disease with thevaccine. Nucleic acid vaccines are therefore consideredsafer than conventional vaccines, i.e. inactivated wholevirus, with or without oil adjuvant, or attenuated live virus(6). In contrast to most conventional vaccines based oninactivated pathogens, DNA vaccines for fish are notformulated with an oil adjuvant, which is known to causepost-vaccination side effects such as peritonitis (42, 56).Other factors that make DNA vaccines preferable are thatinactivated whole virus vaccines may contain unknownimpurities and trace amounts of inactivating agents, whilelive attenuated vaccines pose a risk of infection by mutantsor may revert to virulence. Moreover, where DNA vaccinesare used, side effects due to contaminants are negligible.This is because plasmid DNA can be prepared to a veryhigh level of purity, and DNA consists of a precise sequenceof nucleotide residues. Quality assurance of DNA vaccinesis therefore less complicated than with traditional or liverecombinant vaccine types.

A number of theoretical safety concerns may be consideredfor DNA vaccines. These include:

– the fate of the plasmid in the vaccinated animals

– the risk of the integration of vaccine DNA sequencesinto the genome of the host, and subsequent negative sideeffects such as development of disease or integration intothe germ line followed by vertical transfer

– the risk of inducing an anti-DNA immune response.

Thorough discussions of these aspects have been madeaccessible via the Internet by the Norwegian BiotechnologyAdvisory Board (21) and by the Danish Institute for Foodand Veterinary Research (30).

The distribution of the DNA vaccine depends on thedelivery route. For the purposes of this discussion thefocus will be on IM injection, since this is the only routethat has consistently been shown to provide significantprotection. Shortly after IM injection of fish, the plasmidcan be found in small amounts in various tissues (3, 28).However, the vast majority of the injected plasmid DNAremains in the muscle tissue at the injection site. As withmammals, more than 99% of the injected DNA disappearswithin the first weeks after vaccination, leaving smallamounts of long-term persisting plasmid (J. Rasmussen,personal communication). As discussed by Holm (30), thisis probably because only a small fraction of the injected

DNA is taken up by the host cells, whereas extracellularDNA is rapidly degraded by nucleases. Persistence of hostcells with reporter gene constructs has been demonstratedup to two years following vaccination (15), but vaccineconstructs encoding pathogen antigens most likely persistfor a shorter period due to the elimination of transfectedcells by the fish immune system (Fig. 4) (28, 45).

Investigations to date suggest that the injected plasmidDNA does not integrate into the genome of the host cells(3, 34). However, from a theoretical standpoint, it must beexpected that such integration will occur, althoughprobably very rarely. Calculations suggest that the chancesof integration of vaccine DNA are considerably smallerthan the chances of natural mutations (41). The risk ofnegative side effects due to integration of vaccinesequences into the host genome therefore appearsnegligible, compared to the many benefits of DNAvaccines. The chance of integration into the germ line is inall probability an even rarer event.

In this context it should be kept in mind that severalnatural infections, such as those of DNA-viruses (e.g. papilloma, herpes, hepatitis and pox viruses), resultin considerable exposure of the organism to foreign DNA.This is also true for vaccines based on attenuated/


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 postvaccination (b). At 31 days post vaccination the majority of the G-positive myocytes hadbeen eliminated and muscle regeneration at the needle track was in progress (c). In fishinjected 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 postvaccination (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. 4Intramuscular delivery of a DNA vaccine against viralhaemorrhagic septicaemia virus (VHSV) in rainbow trout andimmuno-histochemical analysis of the injection site(Based on 45 and 52)

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1 µg of DNA (J. Rasmussen, personal communication), andwould in many cases be sufficient to fulfil the veterinaryrequirements.

Regulation of veterinary DNAvaccinesDue to the rapid progress in the development of DNAvaccines, which only started experimentally in the early1990s, there is limited experience with potential long-termeffects. Since no DNA vaccines have been licensed yet, oneremaining major challenge is to develop an appropriate setof regulatory requirements for these vaccines (16, 69).

Administrative organisations such as the Food and DrugAdministration in the United States of America and theEuropean Agency for Evaluation of Medical Products haveprepared some guidelines concerning DNA vaccines ingeneral and veterinary DNA vaccines in particular (17, 20,69). Several relevant issues, such as requirements oncomposition and safety testing, are covered, but no specificrestrictions in terms of use/application of DNA vaccines aregiven. As discussed by Foss and Rogne (22), one central issueis differentiation between an animal that has been treatedwith a medical product containing manipulated gene(s) anda GMO. The delineation between these two classifications isnot clear, but if the medical product results in stableintegration of foreign DNA into the germ line of a treatedanimal then, by definition, the latter can become a GMO.However, with traditional DNA vaccines, the probability ofturning the vaccinated animal into a GMO should beconsidered to be negligible, as discussed above.

The various national regulatory organisations treat thisissue in differing ways. The British Agriculture andEnvironment Biotechnology Commission considers that aslong as the foreign DNA is not integrated into the host’sgenome, a DNA-vaccinated animal is not to be consideredas a GMO (1). A similar standpoint has been taken by theDanish Medical Authorities in the case of the VHS DNAvaccine described above. In contrast, the NorwegianDirectorate for Nature Management has suggested that aDNA-vaccinated fish should be considered geneticallymodified as long as the foreign DNA is present in the fish(22). This definition is based on the precautionaryprinciple but could have a negative impact by diluting theGMO concept. For instance, how should animals that haveeaten feed containing DNA from GMO-crops be classified?Under the Norwegian definition, DNA-vaccinatedcompanion animals and wild animals vaccinated withgenetically modified viruses, such as the vaccinia-virus-based rabies vaccine used in Europe and Canada, wouldalso be defined as GMOs. Such a definition would furthercomplicate regulatory issues. As recommended by the

nonpathogenic DNA viruses. Apart from a beneficialadjuvant-like effect of so-called CpG motifs in the bacterialgenes included in the DNA vaccine plasmids (33, 36), noadverse effects in terms of an immune response to thevaccine DNA itself have been reported (34).

What about the consumers eating DNA-vaccinated fish?Since consumers will generally only eat the fish months oreven years after vaccination, very small amounts of vaccineare likely to be left at the time of consumption. Comparedwith the total amount of DNA in the food, the vaccineDNA will constitute a negligible amount. Should vaccine DNA be taken up via the intestine by cells of theconsumer, the chances of negative side effects are expectedto be very small, based on the fact that no such effects have been seen in numerous human volunteers whowere given milligram doses of plasmid DNA in previousand ongoing safety testing of DNA vaccines against humanpathogens (16, 43). Scientific data in this field are limited,however, and experiments, including feeding mammalsflesh from DNA-vaccinated fish, should be conducted. Thiswould also address concerns about the potential spread ofa DNA vaccine in the environment by predatory animalsthat eat vaccinated fish. Part of the analysis should includetesting the intestinal flora of the predators as well as themicrobial flora in the immediate environment of thevaccinated fish.

Although the chances are most probably minimal, otherbacteria can theoretically take up the vaccine plasmid.However, E. coli, the most likely organism that could be implicated in transmitting the plasmid outside thetarget species, is not considered a natural component of thegut 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 shouldbe limited to include only the strictly necessary genes and regulatory elements, and be devoid of geneelements such as genes that mediate resistance toimportant antibiotics.

In terms of veterinary regulations, use of marker vaccinesis often desirable in order to allow differentiation betweenvaccinated and non-vaccinated animals on the basis of their antibody response. Although inclusion of a geneencoding a marker antigen should be fairly straightforwardin the case of the DNA vaccines, such inclusion would go against the precautionary strategy of keeping the number of genes and regulatory elements to aminimum. Furthermore, since the antibody response in fish often varies considerably, depending ontemperature as well as other parameters, the use of markervaccines may be of limited value. Sensitive DNA-amplification assays based on polymerase chain reactionallow detection of the vaccine plasmid in vaccinated fishup to at least six months post vaccination with

Norwegian Biotechnology Advisory Board (21, 22), newmedical products based on the transfer of genes should beevaluated on a case-by-case basis, and gene-medicatedanimals should only be termed GMOs if the foreign DNAis likely to be inherited by the offspring or if the geneticmaterial is expected to cause negative side effects of somekind if integrated.

Field-testing

Efficacy and safety

While the salmonid rhabdovirus DNA vaccines haveproved excellent under experimental conditions, testingunder commercial fish farming conditions is needed beforethe real potential of these vaccines can be determined.Higher stress levels, different growth conditions andexposure to other pathogens are some of the parametersthat could affect vaccine efficacy in the field. Field-testingshould preferably include not only exposure of vaccinatedfish to natural outbreaks of disease, but also a thoroughexamination of the health and growth performance of thevaccinated fish compared to non-vaccinated controls.Testing under field conditions has recently been initiatedfor IHNV in Atlantic salmon in Canada and is alsoscheduled for VHSV in Denmark.

Infectious haematopoietic necrosis virus is endemic to thePacific Northwest, but has varying effects on differentPacific salmonids. The virus first appeared in farmedAtlantic 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 destructionof millions of smolts as a disease management measure.Mortality rates in older fish (2 kg to 3 kg) tend to rangefrom 10% to 20%; in smolts the rate often exceeds 85%.Consequently, IHNV is having a serious impact on salmonaquaculture in British Columbia. The estimated economicloss from recent disease outbreaks was US$40 million,which represents US$200 million in lost sales. Thesemortalities not only have significant adverse economicimpacts on the British Columbia aquaculture industry,preventing its growth, but also affect other socio-economicfactors such as job creation in remote coastal communities.

A clinical safety trial of a DNA vaccine against IHNV inAtlantic salmon under commercial production conditions inBritish Columbia is currently in progress. The vaccine hasbeen approved for investigational use by the Animal Healthand Production Division of the Canadian Food InspectionAgency. At the hatchery, three million Atlantic salmon withan average size of 25 g were each given an IM injection of 10µg of vaccine at least 400 degree-days prior to seawatertransfer (degree-days: sum of daily mean temperatures for agiven time period). All hatchery effluent water in British

Columbia is treated with ultraviolet light, so the risk oftransfer of the plasmid to freshwater and marineinvertebrates and other non-target aquatic species isminimal. Studies have further demonstrated that uptake ofplasmid DNA via the water route is highly inefficient (12).Since the disease agent IHNV is endemic to the BritishColumbia coast, expression of the IHNV G protein alreadyoccurs naturally in the environment. If an adverse eventoccurs during the field vaccination trials, containmentprocedures will be implemented. After seawater transfer, therisk of shed and spread is considered negligible. This studyis the first clinical safety trial of a DNA vaccine in fish undercommercial production conditions.

In Europe, VHS is the most important viral disease infarmed rainbow trout. Outbreaks of this virus can result invery high mortality among rainbow trout of all sizes, andat present the only possible control measure is stamping-out animals on infected farms in combination withintensive surveillance and control programmes. InDenmark, intensive stamping-out programmes over thepast 30 years have reduced the percentage of infectedfarms from 90% in the early 1970s to 5% to 10% today.However, the remaining farms are situated in anendemically infected zone and disease eradication has beenvery difficult, possibly due to the size and complexity ofthe water bodies as well as the intensity of the fish farmingactivities (60). An effective vaccine could be a very valuabletool to supplement the stamping-out process. After one ortwo seasons with DNA-vaccinated fish, horizontaltransmission of the virus would decrease and stamping-outwould probably have a much higher chance of success interms of eradicating the virus. Restocking should theninclude non-vaccinated fish only, since vaccination will notbe allowed in zones that are to be declared free from VHSV.

Regular use of vaccination could also be beneficial in largerendemically 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 affectone another (unpublished observations) and simplemixing of the two plasmids before IM injection would be areasonable strategy.

A small-scale preliminary DNA vaccine field test inDenmark has been initiated as a collaborative project of theDanish Institute for Food and Veterinary Research and theDanish fish farmers association (Danish Aquaculture).Fingerling-size fish will be vaccinated with 1 µg of plasmidDNA and kept in farms that are free from VHSV butsituated outside the VHSV-free certified zone. Once VHSoutbreaks occur (in these or in other fish farms), net-cageswith vaccinated and non-vaccinated control fish will betransferred to ponds affected by the disease. Upontermination of the trials, all experimental fish will behumanely euthanized and destroyed. As well as examining


the protective effect of the vaccine, the study will includeanimal safety aspects and track the persistence and fate ofthe vaccine plasmid. Permission/acceptance has beenobtained from the relevant public authorities, includingthe Danish Medicines Agency, the Danish Forest andNature Agency, the Danish Agency for Animal Experimentsand the Danish Veterinary and Food Administration.

Animal welfareCompared with other forms of animal farming, finfishaquaculture has both advantages and disadvantages interms of animal welfare. Fish have specific physical andchemical requirements relating to the aquatic environment,and when these requirements are not met, the health andsurvival of the animals can be jeopardised by the resultingstress. Culturing animals in water requires stricterattention to detail than terrestrial animal culture does. Interms of animal welfare, one benefit of this attention todetail is that aquatic producers recognise that controllinganimal stress is essential for economic success, and that thedevelopment of specific stress management protocols isvital for aquatic animal health and survival (10). Theutilisation of appropriate vaccines can be a very effectivestress management technique, since infected/diseased fishare considerably more susceptible to stress.

A North American research project was recently initiated todetermine the effect of the IHNV DNA vaccine on thehealth and welfare of Atlantic salmon. The study will makea comprehensive examination to compare physiological,immunological and haematological factors in vaccinatedand unvaccinated fish, by sampling the fish in thefreshwater hatchery both prior to and after vaccination,and every three months following seawater entry. Studiesof this type will provide information on the safety of thevaccine from the perspective of fish health and welfare.

Cost-benefitThe technology for the production of plasmid DNA formedical purposes has continuously been improved over thepast 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 probablybe low enough to make the technology viable in commercialaquaculture. Automatic devices for IM delivery of thevaccines to small fish (or some alternative methods) will haveto be developed, but this is considered feasible, taking intoaccount that vaccination machines for intraperitonealdelivery are already used commercially. However, the cost oflicensing could inhibit the use of vaccines in commercial fishfarming. There are a considerable number of patents andother types of intellectual property rights within the field of

DNA vaccines, and it will be important that royalty fees andsimilar costs are set at levels that reflect the relatively smallprofit levels obtained from manufacturing fish vaccines.

The benefits of efficacious vaccines against viral diseases infish 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/wasteresiduals into the water-bodies

– improved quality and safety of food products, based onhealthy fish that are free from medical/chemical residuals

– improved economic efficiency in fish farming activitiesand related industries.

Moreover, by potentially being among the first approvedDNA vaccines for veterinary use, DNA vaccines for fishcould help to move such treatment into clinical use ingeneral.

Public perceptionIntroduction of vaccines into aquaculture has, to ourknowledge, not had any negative impact on the wayconsumers perceive aquacultured fish. Whether this is due toa lack of information about vaccination procedures or ageneral acceptance remains to be determined. However, as aresult of the current debate about the use of GMOs in foodproduction, the potential relationship between GMOs and DNA-vaccinated fish could become a sensitiveissue, at least in countries where consumers are reluctant toaccept food containing GMO-derived products. In suchcountries it is therefore important to have a clear regulatorystrategy as well as to keep the public well informed. Aclassification of DNA-vaccinated animals as GMOs, andrelated requests on GMO-labelling of food products derivedfrom such animals, would be very likely to have a strongnegative impact on the sales of – and thereby prevent the useof – DNA vaccines. The authors believe that the most fruitfulstrategies for society as a whole would be to adopt theindividual examination of risk and benefit for each vaccine,as recommended by the Norwegian Biotechnology AdvisoryBoard (22), and to exclude DNA-vaccinated animals fromthe GMO labelling requirements, except if there is scientificevidence of a real risk of the integration of vaccine DNA intothe inherited germ-line DNA.

Final remarksIn contrast to many DNA vaccines tested in other animalspecies, the DNA vaccines against rhabdoviruses in


aquacultured fish have proved to be very effective in thetarget species. A single 1 µg dose of plasmid DNA promptlystimulates immunity, which appears to persist throughoutthe normal lifespan of a cultured food fish. As traditionalvaccines against fish rhabdoviruses have not been successful,the DNA vaccine technology could provide a valuable toolfor more sustainable production of farmed fish. Althoughthere has been preliminary testing using IM injection underfield conditions, more suitable delivery methods need to bedeveloped in order to make vaccination of small fish (below5 g) economically feasible. Other requirements that willpresent an important challenge for authorities and scientistsworking in fish vaccinology are to achieve transparency ofregulatory and safety issues, and to ensure publicdissemination of information about the positive effects ofDNA vaccines in aquaculture.

As this issue went to press the FDA released a draft guidancenote entitled ‘Guidance for industry: considerations forplasmid DNA vaccines for infectious disease indications’(www.fda.gov/cber/gdlns/plasdnavac.pdf). When finalised,

the document will represent an update to the guidelinespublished by FDA in 1996 (20).

Acknowledgements

The authors thank numerous colleagues for their assistance,in particular: K. Einer-Jensen and E. Lorenzen, who providedthe material for the figures; J. Rasmussen and E. Anderson,who provided unpublished information about thepersistence of DNA vaccine and about ISAV DNA vaccineexperiments, respectively; G. Kurath, who gave access toliterature ‘in press’; and A. Holm, G. Foss and H. Korsholm,who offered useful comments on the manuscript. TheAmerican Fisheries Society is acknowledged for allowing theuse of figure material from reference 44. Elsevier is thankedfor permission to include figure material from references 44, 45 and 52. This work was supported by a research grantfrom the Danish Ministry for Food, Agriculture and Fisheries(93s-24F4-Å02-00042 FØTEK4).


Vaccins à ADN destinés aux poissons d’élevage

N. Lorenzen & S.E. LaPatra

RésuméLa vaccination à acide désoxyribonucléique (ADN) consiste à administrer legène codant pour l’antigène vaccinal et non l’antigène lui-même. L’expression decet antigène par les cellules du sujet vacciné stimule son système immunitaire.Parmi les nombreux vaccins expérimentaux à ADN testés sur différentesespèces animales ainsi que chez l’homme, ce sont les vaccins contre lesmaladies à rhabdovirus chez les poissons qui ont donné les résultats les plusprometteurs. Une injection intramusculaire unique de quantités d’ADN de l’ordredu microgramme confère aux salmonidés d’élevage une protection rapide etdurable contre les virus qui produisent un impact économique important, tel queles virus de la nécrose hématopoïétique infectieuse (VNHI) et de la septicémiehémorragique virale (VSHV). Les vaccins à ADN dirigés contre les autres typesd’agents pathogènes touchant les poissons n’ont connu à ce jour qu’un succèslimité. L’administration la plus efficace à l’heure actuelle est l’injectionintramusculaire, et des stratégies d’administration adaptées restent àdévelopper pour la vaccination massive des petits poissons. Sur le plan de latolérance, aucun effet indésirable n’a été observé à ce jour chez les poissonsvaccinés. Étant donné que les vaccins à ADN constituent une technologierelativement récente, certains aspects théoriques, de même que la sécurité àlong terme pour l’environnement et le consommateur, n’ont pas encore ététotalement résolus. Les risques ne devraient cependant pas être plus importantsqu’avec les vaccins actuellement commercialisés pour les poissons. Lessystèmes de classification dont on dispose aujourd’hui ne permettent pas dedistinguer clairement les animaux ayant reçu un vaccin à ADN des organismesgénétiquement modifiés, ce qui risque de poser des problèmes en termesd’approbation et d’acceptation de cette nouvelle technologie. Parmi lesavantages potentiels des vaccins à ADN chez les poissons d’élevage, il faut citer


Vacunas de ADN para peces de vivero

N. Lorenzen & S.E. LaPatra

ResumenLa vacunación con ácido desoxirribonucleico (ADN) consiste en administrar alorganismo receptor el gen que codifica el antígeno inmunógeno en lugar delpropio antígeno. La subsiguiente expresión del gen en las células del animalvacunado activa su sistema inmunitario. Entre las muchas vacunas de ADNexperimentales que se han ensayado en varias especies animales y en elhombre, las que ofrecen resultados más prometedores son las vacunas contraenfermedades rhabdovíricas de los peces. Una sola inyección intramuscular deunos pocos microgramos de ADN induce, en salmónidos de vivero, unaprotección rápida y duradera contra los agentes de enfermedades de granimportancia económica como el virus de la necrosis hematopoyética infecciosa(VNHI) o el de la septicemia hemorrágica viral (VSHV). Hasta la fecha, sinembargo, las vacunas de ADN contra otros patógenos de los peces no han dadomucho fruto. De momento la vía de administración más eficaz es la inyecciónintramuscular, pero todavía no se han elaborado estrategias adecuadas para lavacunación masiva de peces pequeños. Por lo que respecta a la inocuidad, nose ha observado hasta ahora ningún efecto adverso en los peces vacunados.Toda vez que la vacunación con ADN es una técnica relativamente nueva, aúnno se han estudiado a fondo, desde el punto de vista teórico y de la inocuidad alargo plazo, una serie de aspectos relacionados con la influencia de las vacunassobre el medio ambiente y la salud del consumidor, aunque en buena lógica losriesgos no deberían ser mayores que con las vacunas comerciales que se estánadministrando hoy en día a los peces. Los actuales sistemas de clasificaciónresultan poco claros a la hora de distinguir entre animales vacunados con ADNy organismos modificados genéticamente, hecho que podría tenerconsecuencias en cuanto a las licencias de comercialización y a la aceptaciónde esta técnica por parte de la opinión pública. La vacunación con ADN depeces de vivero podría deparar, entre otros, los siguientes beneficios: mayornivel de bienestar animal; menores efectos ambientales de las actividadesacuícolas; obtención de alimentos de mejor calidad y en mayor cantidad; yproducción más sostenible. Hace poco tiempo han empezado a ensayarse estasvacunas en condiciones de producción industrial en Canadá y Dinamarca.

Palabras claveAdministración de vacunas – Aspecto reglamentario – Bienestar animal – Enfermedadvírica – Glucoproteína – Inocuidad – Mecanismo de protección – Pez de vivero –Plásmido – Prueba de terreno – Punto de vista del consumidor – Costo-beneficio –Vacuna de ácido desoxirribonucleico.

les progrès en matière de bien-être animal, d’impact environnemental del’aquaculture, d’une meilleure qualité et quantité d’aliments et de productiondurable. Des essais à échelle industrielle ont été récemment lancés au Canadaet au Danemark.

Mots-clésAspect réglementaire – Bien-être animal – Essai sur le terrain – Glycoprotéine – Maladievirale – Mécanisme protecteur – Perception du consommateur – Plasmide – Poissond’élevage – Rapport coût/bénéfice – Sécurité – Vaccin à ADN – Voie d’administration.

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