FISH VACCINE

Vaccination is one of the important means of

controlling disease.
In 1798, Edward Jenner worked on small pox. He
employed the term vaccine (vaccination for
protective inoculation).
Pasteur extended Jenners findings to other
infective diseases such as anthrax, rabies and
chicken cholera.
By vaccination it is possible to induce active
immunity to diseases.
Immunisation is brought about by the use of killed
or weakened (attenuated) bacteria.
The immune system recognizes and begins to
produce antibodies.
Control of diseases by vaccination has a number of
advantages over chemotherapeutic methods.
Vaccination is preventive measure.
The use of vaccines has entered in the field of
aquaculture recently.
Because of the intensive culture systems, many industries
have resorted to the routine use of vaccines which
confer a high degree of protection when correctly
used.
Use in salmon, trout, Mediterranean sea bass and even in
shrimp and lobsters is now a standard part of
husbandry North and South America and Asia.
The concept of vaccinating fish on a commercial scale has
now been realized with respect to Enteric Red Mouth
and Vibriosis.
Fish immunization began in 1942, with the
successful oral immunization of trout against
bacterium Aeromonas salmonicida by Duff.
Fish vaccines in general, fall into three major
categories, namely, killed whole cell vaccine, live-
attenuated vaccine and recombinant DNA based
vaccines.
Efficacy of these vaccines has been improved using
adjuvants, immunostimulants or vaccines
carriers.
However, it is still affected by the routes of vaccine
administration.
In general, injection is better than immersion and
oral administration.
 Killed whole cell vaccines
Killed whole cell vaccine is a suspension of heat or
chemical killed pathogens that are able to induce
specific protective immune response against those
pathogens when administered into the host.
They are used in controlling fish bacterial pathogens
such as, V. anguillarum, V. salmonicida, V. ordalli, Y.
ruckeri, and A. salmonicida.
These killed vaccines are formalin inactivated whole cell
vaccines administered with or without adjuvants and
are commercially available.
These bacterial vaccines are highly immune protective,
and are cheap to produce, but are not known at
present as to what specific antigens of these vaccines
are involved in offering protection.
 In many cases it is believed that the protective
substances are lipo polysaccharides.
Killed vaccines have been developed for some
pathogenic fish viruses such as infectious pancreatic
necrosis virus (IPNV), infectious haematopoietic
necrosis virus (IHNV), viral haemorrohagic septicaemia
virus (VHSV) and spring viremia of carp virus (SVCV).
Injection of rainbow trout fry with the inactivated
IPNV offers good protection in rainbow trout but when
administered in brook trout with Freunds complete
adjuvant it induces strong humoral response with poor
protection.
Successful use of killed VHSV in rainbow trout has also
been recorded.
 Formalin-inactivated IHNV has been found to protect
rainbow trout against lethal IHNV when immunized
at high concentration.
Although all these above vaccines look promising at
laboratory scale none of them has been
commercialized.
It is only the killed vaccine of spring viremia of carp
virus (SVCV) that was commercially available for
some years. This vaccine comprises of two
inactivated strains of SVCV emulsified in oil.
 Disadvantages of using killed virus vaccines

high cost of their production in cell culture,

cumbersome method of purification and
delivery.
In general, killed vaccines alone trigger only
the humoral immune response and not the
cell-mediated immune response.
Further, induces protective immunity, which
fades away over time and needs to be given in
booster doses.
 2.Live attenuated vaccines
It is a suspension of attenuated live pathogens that are
able to replicate inside the host and induce protective
immune response but unable to cause disease.
They mimic the actual infection by pathogens and
hence a small dose of vaccine is enough to induce long
lasting protective immune response.
Can induce both humoral and cell-mediated immune
responses.
These are strong stimulants of cell-mediated immune
response. These preferentially enhance T cell
proliferative response relative to B cell responses.
Some of the conventional live viral vaccines have been
produced against VHSV,IHSV and IPNV.
 Avirulent strains of IHNV are also used as live
vaccines.
Use of VHSV-attenuated strains obtained
through serial passage of VHSV in carp-cell line
under progressive increase of temperature has
been used as live vaccine.
Protection of goldfish against some common
ectoparasites has been observed by
intraperitoneal and immersion immunizations
with live tomites of Ichthyophthirius multifiliis
and Tetrahymena pyriformis.
 Although some of these vaccines are found
useful as live vaccines in laboratory, so far
none of them has been licensed for field trial.
This is because of some of the possible
disadvantages,
such vaccine strains becoming virulent in non-
target species,
possibility of reversion to pathogenic state
and problems associated with residual
virulence.
3. Recombinant DNA-based vaccines
R DNA technology has been widely used in development
of novel vaccines that are now collectively termed as
recombinant DNA-based vaccines or new generation
vaccines.
Different types of vaccines based on recombinant DNA
technology have been developed which include:
recombinant immunogenic protein vaccines or epitopes
purified from vectors carrying the gene of interest
produced in prokaryotic or eukaryotic expression systems,
peptide vaccines,
live vaccines produced by defined genetic manipulations
and microbial vectors carrying gene coding for
immunogenic protein, and
DNA vaccines.
 1. Recombinant protein vaccines
Production of a recombinant protein vaccine starts with
identification of the immunogenic subunit or protein from the
pathogen of interest and verification of its immunogenicity in
vivo and in vitro.
For example, purified glycoproteins from IHNV and VHSV have
been used as subunit vaccines in fish and shown to be
immunoprotective, and used widely for recombinant vaccine
production.
Similarly, an RNA-free subunit vaccine prepared from grass carp
haemorrhage virus (GCHV) treated with 1% NP40 in low salt
solution has been shown to induce more than 80% protection
in carp.
Once, the immunogenic proteins or subunits of pathogen are
identified, the gene(s) involved in coding for them can be
introduced into a vector, over-expressed in expression hosts
and can be used as recombinant protein vaccines.
 The vector systems used to express recombinant proteins
are viruses or bacterial plasmids.
Expression systems commonly used are prokaryotic and
eukaryotic cells.
Prokaryotic expression system comprises of bacteria such
as, Escherichia coli, and the eukaryotic expression system
comprises of yeast, insect cells and mammalian cells.
Disadvantages of prokaryotic systems
They lack the signals required for proper post-translational
modification and there lies the problem of improper
folding and lack of glycosylation.
This leads to production of proteins of unpredicted
antigenicities. in the form of inclusion bodies that need to
be treated biochemically before being used as vaccine.
This biochemical treatment of denaturation and
renaturation of recombinant protein reduced its
immunogenicity.
Advantages of prokaryotic expression system
High level expression of recombinant protein (often
more than 30%), well studied genetic and fermentation
system of E. coli and easy scaling up of vaccine
production.

In the case of eukaryotic expression system, although

the problem of folding and glycosylation does not exist,
the final yield of expressed protein remains low, and
hence the scaling up of the production process is
difficult.
Both prokaryotic as well as eukaryotic expression
systems have been used to produce fish viral, bacterial
and parasitic antigens, and prokaryotic system is most
widely used.
 b) Peptide vaccines
Peptide vaccines comprise of synthetic peptides that
are able to induce protective immune response when
administered into the host.
To produce peptide vaccines it is necessary to identify
immunogenic regions, also known as epitopes on the
antigenic protein.
The term epitope refers to a stretch of 6-8 amino acids
on antigens that specifically binds to antibodies or to
receptors on immune T cells.
Those epitopes that bind to the antibody produced by
specific B cells are called as B-cell epitopes while those
recognized by receptors on the surface of activated T-
cells are termed as T-cell epitopes.
Monoclonal antibodies are indispensable to identify
the B-cell epitopes.
 A region with high sequence variability among
several strains of a pathogen is also chosen as a
candidate for synthetic peptide vaccine.
Epitope mapping and use of peptide vaccines
against fish pathogens are still in its infancy.
Some of the B-cell epitopes have been identified
on some fish viral proteins such as IHNV
glycoprotein.
Synthetic peptide vaccines emulsified with
Freunds complete adjuvant has induced poor
neutralizing antibodies than that of the native
virus fish sera, which indicates that peptides alone
are less immunogenic than the native protein.
 c) Genetically modified live vaccines
Pathogens with defined genetic manipulations or
microbial vectors carrying the gene coding for
immunogenic protein can be used as live vaccines.
Live vaccines replicate inside the recipient host
resembling the natural infection and thus induce
strong immunity.
This kind of vaccine is reported to be highly
immunogenic than the non-replicating vaccine
products.
Selection of a stable non-pathogenic mutant usable as
live vaccine is a complex process .
It involves tedious procedure of growing viruses in
different culture conditions or introducing targeted
mutations, followed by in vivo and in vitro assays.
 Some important methods of selection of
attenuated mutants are, adaptation to
heterologous cell line, adaptation to elevated
temperature and selection of neutralizing
monoclonal antibody escape mutatnts.
The rationale behind selection of strains
adjusted to such extreme conditions is that
these strains are believed to be altered
genetically hence resulting alteration of their
virulence.
Nucleotide sequence analysis of such strains
can confirm the position of mutation.
 Further, in vivo and in vitro analysis can reveal their
phenotypic variation hence aiding in selection of such
strains as candidates for live vaccine.
Defined genetic alterations resulting in mutants with
desired phenotype can be achieved using site directed
mutagenesis technique also.
Live vaccines have been used against some of the fish
bacterial pathogens such as A. salmonicida and A.
hydrophila.
Several techniques such as homologous
recombination, chemical mutagenesis and transposon
mutagenesis are used to produce mutant bacteria
those are avirulant and capable of being used as live
vaccines.
 d) DNA vaccines
DNA vaccines consist of a suspension of bacterial plasmids
carrying the gene coding for the immunogenic protein
under the control of eukaryotic promoter.
DNA vaccines include
an origin of replication suitable for producing high yields of
plasmid in E. coli,
an antibiotic-resistant gene to confer antibiotic-selected
growth in E. coli,
a strong enhancer/promoter and
an mRNA transcript termination/polyadenylation sequence
for directing expression in mammalian cells.
The plasmids hence constructed are grown in E. coli,
purified and suspended in saline and introduced into the
host either by intramuscular injection or using a gene gun.
 DNA vaccines have been used in fishes with very
encouraging results.
Strong expression of reporter genes in muscle cells
following intramuscular injection of plasmid constructs
carrying gene of interest and reporter gene have been
reported.
When plasmids carrying luciferase gene under the control
of cytomegalovirus immediate early gene promoter is
injected to rainbow trout at a dose of 50g of DNA,
maximum activity is seen at 5 to 7 day post-injection and
the activity of luciferase remains for 115 days.
Combined injection of plasmids carrying VHSV and IHNV
glycoprotein genes shows plasmid DNA to remain in the
muscle cells up to 45 days.
 DNA immunization induced specific as well as non-specific
immune response in the recipient host.
High level of protection in clinical animal model has been
observed due to the generation of specific antibodies and
priming of T-cell responses.
Significant protection of rainbow trout is observed against IHNV
challenged following the injection of construct encoding the
IHNV G protein.
Apart from introducing a part of the genome of pathogen coding
for immunogenic protein, it is possible to introduce a gene
coding for an antibody that can target and destroy the pathogen.
DNA vaccines overcome almost all the drawbacks of all other
form of vaccines.
Advantage of DNA vaccines
Ability to induce production of native form of protein with
appropriate post-translational modifications. This has been shown in
the case of DNA immunization of rainbow trout.
Able to induce long lasting immune response and are economical and
safe.
Practical application of DNA vaccine in fish does not seem to be
encouraging because most of the important fish pathogens,
especially the viruses those affect fish at a very young age.
This makes it difficult for one to administer vaccine to small fish
through injection route, which is so far the only method of
introducing the DNA vaccines.
However, the injection method is useful for immunizing broodstocks
of fairly large fish so as to ensure that immunity is passively
transferred from mother to offspring as this being demonstrated in
controlling Ich.
It is difficult to use DNA vaccines for individual fish on a large scale in
intensive aquaculture unless one can introduce DNA vaccine to fish
orally or through gill filaments via aquatic medium.
 Vaccine delivery system
It can be administered by injection, by immersion
or by spraying directly onto the fish according to
what suits an individual farms preference.
Small fish (1.5 to 5 gms) by direct immersion in
diluted vaccine (1:10) for 30 secs.
Larger fish (70-100 gms) sprayed with vaccine or
immersion for 3-5 secs.
Stress should be avoided at the time of handling.
Maintain the vaccine solution at the same
temperature on the holding tanks, oxygenating the
vaccine solution during the vaccination procedure,
etc.
 Oral vaccination of fish using Artemia as the
vaccine delivery system can also done.
When vaccine is given through oral route there is
possibility of Ag being degraded by the digestive
enzymes in the stomach.
New approach involves first feeding the vaccine (
a killed bacterial suspension) to the Artemia, and
then feeding the Artemia as the first live food to
the fry of the species of interest.
It is thought that the vaccine becomes
incorporated into the lipids of the Artemia and
this protects it from the digestive degradation of
the fish.
 Immunity in vaccinated animals tends to change
with time following vaccination.
Booster vaccination can be given.
Duration of protection depends upon the
method of vaccination, the size of the fish, their
health status at the time of vaccination and the
antigen used to vaccinate them.
Vibrogen -2 vaccine is produced by Aquatic
Health Limited, Greece.
The AHL, Canada has developed another vaccine
called Lipogen Triple bacterin ( a combimnation
furunculosis + vibriosis + hitra bacterin) to
protect against furunculosis.
 Conclusion
Recent advances in the field of molecular biology have
profoundly affected the development of fish vaccines.
Antigens obtained from many fish pathogens with the
potential of being used for vaccine have been
identified.
Genetically engineered vaccines have been increasingly
employed against many of the fish pathogens.
DNA vaccine, have been shown to be highly efficient
against some fish viral diseases.
Field scale efficacy study of some of these vaccines is
being undertaken.
 Although great efforts have been carried out on the
development of efficient vaccines against various fish
pathogens, the delivery systems of the vaccine to fish is
equally important.
One has to make sure that the vaccine can be
effectively delivered and it is suitable for intensive
aquaculture in terms of cost effectiveness.
So far, there is only one recombinant protein vaccine
for commercial use in aquaculture, which is against the
IPNV of trout.
It is expected that some more genetically modified
vaccines may be commercialized in the near future.
Thank you