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Recombinant vaccines against infectious pancreatic necrosis (IPN) in salmonid fish


Our five-partner consortium (FAIR CT-95-0353) is concerned with developing recombinant DNA vaccines for Infectious Pancreatic Necrosis (IPN). IPN is now recognised as the most serious problem in salmonid farming in Norway and affects both juvenile and post-smolt fish. Recent estimates of losses due to IPN disease are circa 54 Million ECU per annum. A major part of our research initiative is the development of nucleic acid vaccines against IPN. To date, this has focused on the use of naked DNA constructs and packaged RNA viral particles harbouring the IPN VP2 gene. The VP2 gene product is known to be a protective antigen and the world's first recombinant subunit fish vaccine based on this protein purified from Escherichia coli has recently entered the market place. However, there is considerable evidence that conformational epitopes are important in protection and thus, a bacterial product is unlikely to adopt the correctly folded structure. Nucleic acid vaccination circumvents this problem as the protein is made and folded in its natural host cellular environment. DNA vaccination is now emerging as an exciting technology in fish vaccine research. Several groups have shown that the Cytomegalovirus (CMV) immediate early promoter works very efficiently when injected into fish muscle. As in other biological systems, injected DNA persists in the muscle tissue for a considerable time where it can stimulate extended antigen synthesis. It appears to exist as an episomal plasmid and does not integrate in the host genome. We have made recombinant plasmids harbouring the VP2 gene under the control of the CMV promoter and have tested their ability to express VP2 in cell culture. These plasmids have also been injected into fish muscle and expression and immunological analyses are on going. We are also investigating the ability of an Alpha virus system based on a Semliki Forest Virus vector to deliver recombinant RNA. This system allows one to package RNA encoding VP2 and infect fish cells with suicide virus particles. Recombinant VP2 particles have been produced and are currently being tested in fish. The merits of DNA vs RNA delivery will be presented. In addition, the prospects for the use of genetic immunisation to combat fish disease and the likely public perception are discussed.
DNA encoding the VP2 gene was generated using standard Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) techniques. This fragment was then cloned into the naked DNA vector pCDNA3 (Invitrogen) and the SFV expression vector pSFV1 (6, Figure 1). Clones were isolated with the VP2 gene in the desired orientation and expression was confirmed using a combination of indirect immunofluorescence (using a rabbit polyclonal antibody to the purified virus) of transfected baby hamster kidney (BHK) cell monolayers (data now shown) and immunoprecipitation of 35S-labelled polypeptides (Figure 2). The reactivity of the clones was also investigated using a panel of monclonal antibodies to VP2, some of which were conformationally dependent (2). The results of the latter experiments were somewhat variable although in general, the viral encoded proteins generally reacted better than the plasmid-encoded DNA products. Expression was also confirmed in fish cell culture, although in the case of the SFV-encoded protein this was found to be temperature dependent, with little or no expression detected at growth temperatures below 20°C.

FIGURE 1. Replicons of the SFV expression system. The structure of the wild type SFV genome is shown at the top. nsP1-4 are the four-replicase proteins, which are made as a polyprotein, which self-cleaves to the individual polypeptides, which form the replication, complex. C is the capsid protein gene, p62, 6K and E1 are transmembrane proteins. The right-handed arrow denotes the promoter, which is recognised by the viral replicase for transcription of the subgenomic RNA encoding the structural proteins or the heterologous sequences. MCS indicates a multiple cloning site. The helper encodes for all structural proteins of SFV but has nearly the complete replicase region deleted, including the packaging signal. When co-transfected with the basic vector it results in the production of packaged suicide viral particles harbouring the RNA encoded by the SFV vector. These particles can then be used to infect cells in culture or to vaccinate animals.

(For Figure 1 contact the Coordinator)

FIGURE 2. Immuno-precipitation of 35S-labelled SFV-encoded proteins. BHK cells were transfected with various SFV recombinant RNA molecules and labelled with 35S Methionine 14 hours after transfection. Cell lysates were then immuno-precipitated with rabbit polyclonal antiserum raised against IPNV and fractionated on SDS-PAGE. Lane 1, SFV construct encoding LacZ protein, lanes 2 and 3, SFV constructs encoding IPNV VP2, lane 4, untransfected cell control. A 58 kDa VP2 protein is selectively precipitated in lanes 2 and 3.

(For Figure 2 contact the Coordinator)


The VP2 gene has been expressed in a naked DNA vector and a viral vector based on the Semliki Forest Virus replicon. Both constructs promoted the expression of a VP2 protein, which was recognised by polyclonal rabbit antiserum and in some cases by a panel of monoclonal antibodies to VP2. The SFV-encoded protein was the same size as the mature protein on the native virus. Expression has also been confirmed in fish cell culture for both constructs. However, expression from the SFV vector was temperature dependent with little or no expression detected below 20°C. This suggests that the SFV replicon does not function well in cell culture unless the growth temperature is sufficiently high. Experiments are underway to investigate if SFV particles can function in fish tissue at the normal (lower) temperatures at which salmonids grow. The results of these experiments will determine the feasibility of the SFV approach in cold water species.

We have recently initiated a vaccine trial in Atlantic salmon pre-smolts to test the efficacy of the naked DNA and SFV vaccine prototypes. The vaccines were administered by intramuscular and intraperitoneal injection, respectively. Samples have been taken for immuno-histochemical analysis to detect expression of VP2. Serum antibody and T-cell proliferative responses are also being monitored. The ability of these vaccines to stimulate protective immunity following challenge will also be intestigated.

It remains to be seen how the general public and licensing authorities will react to the concept of introducing recombinant DNA or RNA into fish tissue destined for human consumption. Naked DNA would appear to hold the most promise as a vaccine delivery system. Concern has been expressed that integration of foreign DNA into the host genome could lead to tumorigenic events in the vaccinated animal. However, this has not been detected in fish or in other vaccinated species to date. We believe that nucleic acid poses no threat to the consumer, even if it persists in fish tissue until the time of harvest. Thus, we see no reason why this technology cannot be applied in the construction of safe efficacious vaccines for the aquaculture industry.


This project is funded by the EU FAIR programme (CT-95-0353, 1996-1998).


1. Anderson, E.D. Mourich, D.V. Fahrenkrug, S.C. LaPatra, S. and Leong, J.-A.C.
1996. Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious
hematopoietic necrosis virus. Molec. Marine Biol. Biotech. 5, 105-113.
2. Christie, K.E. 1997. Immunization with viral antigens. Infectious Pancreatic Necrosis. In: Fish Vaccinology, eds. R. Gudding, A. Lillehaug, P. Midtlyng, F. Brown. Developments in Biological Standardization, vol. 90, pp. 191-199. Karger, Basel.
3. Donnelly, J.J. Ulmer, J.B. and Liu, M.A. 1996. DNA vaccines. Life Sci. 60, 163-172.
4. Heppell, J., Lorenzen, N., Lorenzen, E., Jensen, K.E. Wu, T. and Davis, H.L. 1998. Development of DNA vaccines for fish: Vector design, antigen expression and demonstration of efficacy using viral haemorrhagic septicaemia virus as model. Fish Shellfish Immunol. (in press).
5. Lorenzen, N., Lorenzen, E., Einer-Jensen, K., Heppell, J., Wu, T. and Davis, H.L. 1998. Protective immunity to VHS in rainbow trout (Oncorhynchus mykiss, Walbaum) following DNA vaccination. Fish Shellfish Immunol. (in press).
6. Liljestrom, P., and H. Garoff. 1991. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Bio/Technology 9, 1356-1361.
7. Midtlyng, P.J. 1997. Vaccinated Fish Welfare: Protection Versus Side-Effects. In: Fish Vaccinology, eds. R. Gudding, A. Lillehaug, P. Midtlyng, F. Brown. Developments in Biological Standardization, vol. 90, pp. 371-379. Karger, Basel.

Infectious disease poses the biggest single threat to aquaculture. The introduction of a new generation of both oil- and non-oil adjuvants has greatly improved the efficacy of bacterial vaccines and has resulted in an impressive reduction in mortalities, especially against furunculosis. These adjuvants are not without side effects, however, which can include visceral adhesions, pathology and growth checks in vaccinated fish (7). Current research is aimed at reducing this toxicity. Viral diseases remain an even greater problem for the industry. Recently, the world's first recombinant subunit fish vaccine was launched in Norway against Infectious Pancreatic Necrosis (IPN) (2) and a competitor whole virus vaccine has also now entered the marketplace. IPN is the only disease for which a commercial vaccine exists, although others are being developed. The most exciting development in vaccine research in recent years has been the emergence of the technique known as genetic immunisation (also known as naked DNA vaccination). This is based on the discovery in 1990 that naked plasmid DNA could be taken up by muscle (or, as subsequently shown, skin cells) and expressed in the host cell to produce the protein product encoded by that DNA (reviewed in 3). This approach has been investigated in a wide variety of animal models and, where tested, has generally resulted in protection from challenge against the homologous disease agent. Typically, protective antigen(s) are placed under the control of a strong eucaryotic promoter such as the Cytomegalovirus (CMV) immediate-early promoter, which drives expression of the desired gene product(s). This technology has been tested in fish by a number of investigators with encouraging results. Recently, two groups have reported that vaccination of fish with CMV plasmids expressing protective antigens from Infectious Haematopoietic Necrosis Virus (IHNV) and Viral Haemorrhagic Septicaemia Virus (VHSV) resulted in high-level protection against viral challenge (1, 4, 5). Thus, there is little doubt that this technology represents the most promising approach to date in fish viral vaccine design.
We are investigating the ability of naked DNA plasmids under the control of the CMV promoter to deliver the IPNV protective antigen, VP2 to fish muscle. cDNA encoding the VP2 gene has been isolated from a virulent Norwegian field isolate and cloned in a naked DNA expression vector. In parallel, we are also investigating the ability of a novel Alphavirus delivery system, based on a suicide Semliki Forest Virus (SFV) vector, to deliver packed recombinant RNA molecules encoding VP2 to fish tissue.

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Enterprise Ireland - Trading as Bioresearch Ireland
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