Download DNA vaccines for aquacultured fish

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
use. In addition, animal welfare has been improved by the
implementation of vaccination.
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
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
202
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, β-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
Rev. sci. tech. Off. int. Epiz., 24 (1)
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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Rev. sci. tech. Off. int. Epiz., 24 (1)
Table I
Advantages 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
60 nm
a)
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).
180 nm
G~glycoprotein (trimer)
Genomic RNA (G gene in green)
Delivery and efficacy
c)
G gene
b)
Prom.
Vaccine
plasmid
Expression
in host cell
G protein
Membrane
Antibiotic r
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)
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. Dose–response 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).
204
Rev. sci. tech. Off. int. Epiz., 24 (1)
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).
80
60
40
20
0
0
4
8
Non-specific mechanisms
12
16
Weeks post vaccination
Specific mechanisms
20
24
Total protection
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,
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
100
Cumulative mortality (%)
Relative survival rate (%)
100
80
60
40
20
0
1
3
5
7
9
11 13 15 17 19 21 23 25 27 29 31 33 35
Days after challenge with VHS virus
1 µg pcDNA3
0.001 µg pcDNA3-vhsG
0.1 µg pcDNA3-vhsG
1 µg pcDNA3-vhsG
0.01 µg pcDNA3-vhsG
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)
205
Rev. sci. tech. Off. int. Epiz., 24 (1)
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.
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/
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
a
b
100 µm
d
100 µm
– 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
c
100 µm
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)
206
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
Rev. sci. tech. Off. int. Epiz., 24 (1)
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
Rev. sci. tech. Off. int. Epiz., 24 (1)
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
207
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
208
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
Rev. sci. tech. Off. int. Epiz., 24 (1)
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
209
Rev. sci. tech. Off. int. Epiz., 24 (1)
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-Å02-00042 FØTEK4).
Vaccins à ADN destinés aux poissons d’élevage
N. Lorenzen & S.E. LaPatra
Résumé
La vaccination à acide désoxyribonucléique (ADN) consiste à administrer le
gène codant pour l’antigène vaccinal et non l’antigène lui-même. L’expression de
cet antigène par les cellules du sujet vacciné stimule son système immunitaire.
Parmi les nombreux vaccins expérimentaux à ADN testés sur différentes
espèces animales ainsi que chez l’homme, ce sont les vaccins contre les
maladies à rhabdovirus chez les poissons qui ont donné les résultats les plus
prometteurs. Une injection intramusculaire unique de quantités d’ADN de l’ordre
du microgramme confère aux salmonidés d’élevage une protection rapide et
durable contre les virus qui produisent un impact économique important, tel que
les virus de la nécrose hématopoïétique infectieuse (VNHI) et de la septicémie
hémorragique virale (VSHV). Les vaccins à ADN dirigés contre les autres types
d’agents pathogènes touchant les poissons n’ont connu à ce jour qu’un succès
limité. L’administration la plus efficace à l’heure actuelle est l’injection
intramusculaire, et des stratégies d’administration adaptées restent à
développer pour la vaccination massive des petits poissons. Sur le plan de la
tolérance, aucun effet indésirable n’a été observé à ce jour chez les poissons
vaccinés. Étant donné que les vaccins à ADN constituent une technologie
relativement récente, certains aspects théoriques, de même que la sécurité à
long terme pour l’environnement et le consommateur, n’ont pas encore été
totalement résolus. Les risques ne devraient cependant pas être plus importants
qu’avec les vaccins actuellement commercialisés pour les poissons. Les
systèmes de classification dont on dispose aujourd’hui ne permettent pas de
distinguer clairement les animaux ayant reçu un vaccin à ADN des organismes
génétiquement modifiés, ce qui risque de poser des problèmes en termes
d’approbation et d’acceptation de cette nouvelle technologie. Parmi les
avantages potentiels des vaccins à ADN chez les poissons d’élevage, il faut citer
210
Rev. sci. tech. Off. int. Epiz., 24 (1)
les progrès en matière de bien-être animal, d’impact environnemental de
l’aquaculture, d’une meilleure qualité et quantité d’aliments et de production
durable. Des essais à échelle industrielle ont été récemment lancés au Canada
et au Danemark.
Mots-clés
Aspect réglementaire – Bien-être animal – Essai sur le terrain – Glycoprotéine – Maladie
virale – Mécanisme protecteur – Perception du consommateur – Plasmide – Poisson
d’élevage – Rapport coût/bénéfice – Sécurité – Vaccin à ADN – Voie d’administration.
Vacunas de ADN para peces de vivero
N. Lorenzen & S.E. LaPatra
Resumen
La vacunación con ácido desoxirribonucleico (ADN) consiste en administrar al
organismo receptor el gen que codifica el antígeno inmunógeno en lugar del
propio antígeno. La subsiguiente expresión del gen en las células 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 más prometedores son las vacunas contra
enfermedades rhabdovíricas de los peces. Una sola inyección intramuscular de
unos pocos microgramos de ADN induce, en salmónidos de vivero, una
protección rápida y duradera contra los agentes de enfermedades de gran
importancia económica como el virus de la necrosis hematopoyética infecciosa
(VNHI) o el de la septicemia hemorrágica viral (VSHV). Hasta la fecha, sin
embargo, las vacunas de ADN contra otros patógenos de los peces no han dado
mucho fruto. De momento la vía de administración más eficaz es la inyección
intramuscular, pero todavía no se han elaborado estrategias adecuadas para la
vacunación masiva de peces pequeños. Por lo que respecta a la inocuidad, no
se ha observado hasta ahora ningún efecto adverso en los peces vacunados.
Toda vez que la vacunación con ADN es una técnica relativamente nueva, aún
no se han estudiado a fondo, desde el punto de vista teórico 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 lógica los
riesgos no deberían ser mayores que con las vacunas comerciales que se están
administrando hoy en día a los peces. Los actuales sistemas de clasificación
resultan poco claros a la hora de distinguir entre animales vacunados con ADN
y organismos modificados genéticamente, hecho que podría tener
consecuencias en cuanto a las licencias de comercialización y a la aceptación
de esta técnica por parte de la opinión pública. La vacunación con ADN de
peces de vivero podría deparar, entre otros, los siguientes beneficios: mayor
nivel de bienestar animal; menores efectos ambientales de las actividades
acuícolas; obtención de alimentos de mejor calidad y en mayor cantidad; y
producción más sostenible. Hace poco tiempo han empezado a ensayarse estas
vacunas en condiciones de producción industrial en Canadá y Dinamarca.
Palabras clave
Administración de vacunas – Aspecto reglamentario – Bienestar animal – Enfermedad
vírica – Glucoproteína – Inocuidad – Mecanismo de protección – Pez de vivero –
Plásmido – Prueba de terreno – Punto de vista del consumidor – Costo-beneficio –
Vacuna de ácido desoxirribonucleico.
Rev. sci. tech. Off. int. Epiz., 24 (1)
211
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