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Appendix
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Workshop Report
-Preface
-Final Report

Infectious diseases are a major constraint to the future development of aquaculture in the United States (Joint Subcommittee on Aquaculture, 1993). This statement is based on annual surveys conducted by the U.S. Trout Farmers Board of Directors. They found that annual losses in trout production due to disease rose from 52% in 1991 to 84% in 1998. The World Bank estimates that diseases cost the aquaculture industry $3.02 billion annually (Intrafish, 2000). The aquaculture industry must bring its disease problems under control if it is going to provide 10-30% of the world’s fish production by 2010. For many diseases, traditional vaccines and control methods do not work and we need to consider other strategies for disease management including vaccines, diagnostic tools, and disease resistant fish produced with biotechnological tools. DNA vaccines for fish have been developed for several viral pathogens and are being considered for parasitic and bacterial pathogens as well. These vaccines have been very successful in laboratory trials in fish; however, public acceptance of these vaccines will depend upon their perceived safety, not only for consumers, but for the fish as well. Vectors that have no antibiotic resistance and use fish specific promoters instead of human viral promoters have been designed and are being tested in fish. These fish-specific vectors work very well in rainbow trout fry and induce good protection against infectious hematopoietic necrosis virus.

Key Words: DNA vaccine, Genetic Vaccination, Infectious Hematopoietic Necrosis Virus, Aquaculture Biotechnology

Introduction

Fish are susceptible to a wide variety of pathogens whose impacts are especially severe under the intensive rearing conditions of most fish farms. These pathogens can be viruses, bacteria, fungi, and parasites. Strategies employing antibiotics and chemotherapeutic treatments can be used to control bacterial and parasitic diseases but these products have been restricted by government-licensing agencies. Antibiotics accumulate in the flesh of the animals and their widespread use often leads to the selection of antibiotic-resistant strains. Thus, antibiotics are only a short-term remedy (Munn et al., 1994). For viral infections with no commercially available treatments or inexpensive chemotherapeutants, outbreaks are controlled by the destruction of all fish in the rearing facility. This is followed by closure of the facility and extensive decontamination, a costly and labor-intensive procedure. Prophylactic measures such as vaccination and disease diagnosis are desperately needed for the prevention of disease outbreaks in aquaculture. Although there are a few bacterial fish vaccines in the market, aquaculture needs many more vaccines if the industry is going to continue to grow. Many vaccines have been tested under laboratory conditions, but they were not commercially viable because of prohibitive production costs, inconvenient administration requirements, moderate protection, and/or biosafety concerns (Leong et al. 1997, 1998; Winton 1997).

Today, formalin-killed vaccines are commercially available for five bacterial fish pathogens: Yersinia ruckeri, Vibrio anguillarum, Vibrio ordalii, Vibrio salmonicida and Aeromonas salmonicida (Evelyn.,1997). Fish vaccine companies also produce autochthonous killed vaccines for the viral diseases, infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV), and a recombinant DNA based vaccine for IPNV is marketed in Norway by Norbio (Christie, 1997). Attempts to produce formalin-killed vaccines for other bacterial and viral pathogens have not worked well for a number of reasons. For example, some bacteria, like Renibacterium salmoninarum, grow very slowly in vitro and vaccines that have been developed induce only moderate protection. Furthermore, the vaccine must be administered by injection. Viral vaccines require very large antigenic doses and production of enough antigen in tissue culture is too expensive. The subunit viral vaccines that have been developed have produced good antibody responses. However, if the fish immune system is similar to the mammalian system, protection against viral infections requires a strong cell mediated immune response as well. We do not have a clear understanding of the cellular immune response in fish and until we do, development of effective vaccines for many pathogens may be limited.

Viral Vaccines for Fish

Viral vaccines have been in development for over 20 years. Yet, with the exception of the recombinant DNA produced subunit vaccine for IPNV sold by Norbio, there are no commercially available fish viral vaccines. There are many reasons why no fish viral vaccines have been licensed in the United States. First, the licensing process is lengthy and expensive and second, the efficacy of these vaccines has been inconsistent. Also, the safety of attenuated vaccines has been questioned. The requirement for protein based viral vaccines to be administered by injection has also been a drawback. Attempts to vaccinate fish by immersion in suspensions of killed virus have produced unsatisfactory results. For example, inconsistent protection against IPNV was observed by Bootland et al.1990; no protection against viral hemorrhagic septicemia virus (VHSV) was observed by de Kinkelin, 1988; and feeble protection against IHNV was observed by Nishimura et al., 1985, when fish were vaccinated by immersion. Viruses whose virulence has been attenuated by serial passage in cell culture have been developed for IHNV, IPNV, spring viremia of carp virus (SVCV), and VHSV. Vaccination by injection with attenuated virus was shown to be effective against IPNV (McAllister 1984; Bootland et al.,1986; Hill et al.,1980), VHSV (de Kinkelin et al.,1980; Jorgensen 1992) and IHNV (Leong et al., 1988). However, further development of these vaccines has been abandoned for various safety considerations including residual virulence in the target species, virulence for other fish species, persistence in the treated fish and the fear that the virus might revert to virulence. In the case of the IHNV vaccine, although the virus was attenuated for chinook and kokanee salmon, there was significant residual virulence for rainbow trout (Leong et al.,1988). Fijan et al. (1988) attenuated SVCV by multiple passages in cells culture. This vaccine gave good protection but the vaccinated fish became asymptomatic carriers of the virus.

Subunits vaccines prepared by recombinant DNA technology consist of only a portion of a pathogen that stimulates protective immunity (often a single protein or important antigenic domain) expressed in bacteria, yeast or eukaryotic cells. With IPNV, good protection was obtained following immersion vaccination in a crude bacterial lysate of E.coli expressing the IPNV segment A (Manning and Leong.,1990). Glycoprotein genes of the IHNV or VHSV expressed in E.coli were prepared as a crude lysate and used to vaccinate fish by immersion. The first laboratory trials with these vaccines were very promising. However, subsequent field trials yielded inconsistent results with only moderate levels of protection despite the fact that neutralizing antibody was induced in the vaccinated animals (Gilmore et al.,1988; Lorenzen et al. 1993; Leong et al., 1997). Antibody production was observed in fish vaccinated with recombinant G proteins of both IHNV and VHSV produced in an attenuated strain of Aeromonas salmonicida (Noonan et al.,1995). A portion of the IHNV glycoprotein gene expressed as part of the S-layer protein of Caulobacter crescentus also produced only moderate protection in vaccinated fish (Simon et al., 2001). One reason for the disappointing results may be that bacterial expression systems lack the appropriate chaperones for the correct folding of foreign, eukaryotic proteins. Thus, eukaryotic expression systems have been tested and the reports of baculovirus/insect cell or yeast produced proteins as vaccines have not been encouraging. For instance, the VHSV-G protein expressed in yeast was glycosylated and immunologically reactive with anti-G antibody but did not induce protection against viral challenge (Thiry et al.1990). Protection was observed after intraperitoneal injection into rainbow trout with a baculovirus produced VHSV-G protein, but immunization performed by immersion failed (Lecocq-Xhonneux.,1994).

DNA Vaccines

Genetic immunization or the direct administration of antigen-encoding DNA into animals is one of the latest approaches in vaccine design that offers several advantages. DNA vaccines permit the correct folding of the viral antigen, are safe from reversion problems because the DNA encodes only a single viral protein, and induce both cellular and humoral immunity (Cohen, 1998; Babiuk et al., 1998). From a practical point of view, they are relatively inexpensive and easy to produce. There is no a need to purify the pathogen or immunoprotective antigen and all DNA vaccines require an identical production process. This technology is based on the finding that skeletal muscle cells injected with purified plasmid DNA express the plasmid-encoded proteins (Wolff et al.,1990; Tang et al.,1992). The newly synthesized antigen can then stimulate strong, long-lasting humoral and cell-dependent immune responses without boost, similar to that conferred by live vaccines, but without the risk of inadvertent infection (Davis and McCluskie,1999; Fynan et al.,1993; Rabinovich et al.,1994; Vogel and Sarver, 1995). DNA vaccines have been developed for a wide variety of viruses, including influenza virus (Webster et al.,1994; Fu et al., 1999), rabies virus (Osorio et al,1999), hepatitis B virus (Davis,1999), rubella virus (Pougatcheva et al.,1999), and human immunodeficiency virus (Robinson et al.,1999; Bryder et al.,1999). The first evidence that DNA vaccines work in fish was provided by the development of a DNA vaccine encoding the IHNV glycoprotein under the regulatory control of the cytomegolovirus immediate early promoter/enhancer (CMVIEP). Survival to IHNV challenge was 83-85% for the DNA injected fish (Anderson et al., 1996). The fish in the experimental group also developed G protein-specific and virus neutralizing antibody responses after DNA vaccination. Subsequently, a combined DNA vaccination experiment using VHSV and IHNV glycoprotein genes was shown to induce a protective antibody response (Boudinot et al.,1998).

Comparative Efficacy of different IHNV vaccines

The comparative efficacies of different viral vaccine formulations have been examined for only a few viruses. The IHNV vaccines have been examined in greater detail because the assay for vaccine efficacy is well established. Rainbow trout fry at 1 g are vaccinated and then held at 12-15 C in well water at a flow rate of 0.5 gal/min. After 30 days, the fish are challenged with 3 different doses of lethal IHNV. The doses are selected to span an expected LD50 for the virus and the fish. Mortalities in the unvaccinated controls usually begin to appear at 10 days post-infection and continue until day 24. The challenge trial is terminated at day 30 post-infection. In Table 1, data for the cumulative mortalities for the different vaccines: killed, recombinant protein produced in E. coli, and DNA vaccine are shown with each respective control. In this study, the DNA vaccine was much more effective than the killed or E.coli produced vaccine.

Table 1: Comparative Efficacy of IHNV vaccines

Vaccine Type % Mortality

Adjuvant alone 67%

Killed IHNV-formalin 52%

pATH 3 vector alone 61%

pXL3 subunit vaccine 49%

pCMV luciferase 58%

pCMV-G vaccine 2%

Recently, a collaborative and preliminary study of the relative efficacy of several different IHNV vaccines was conducted. The work was funded by the Western Regional Aquaculture Center to Sandra Ristow at Washington State University, Jo-Ann Leong and Carol Kim at Oregon State University, Ron Hedrick and Mark Adkinson at the University of California at Davis, Scott LaPatra at Clear Springs Trout and Jim Winton at the University of Washington in Seattle (Table 2). The study conducted at the Salmon Disease Laboratory at Oregon State University examined the efficacy of the different vaccines. Again, the results show clearly the DNA vaccine for IHNV is much more effective than any of the protein based vaccines (Table 3). What was also surprising was the finding that the -propiolactone inactivated IHNV was very effective. Unfortunately, we were unable to run the control, -propiolactone alone experimental group.

The ordinate is shown in Per Cent Cumulative Mortality and the abscissa denotes the challenge dose of virus, 10+5 PFU/ml and 10+3 PFU/ml

Future of fish vaccines

With the exception of an E.coli produced vaccine against IPNV in Norway, no approved vaccines against virus are available (Christie, 1997). The requirements for low cost/dose, ease of application and safety have restricted their commercial development. The ideal viral vaccine for aquaculture must be effective in preventing death, safe, inexpensive to produce, provide immunity of long duration (i.e., T- and B-cell type immunity), and be easily administered. In addition, vaccines must prevent the formation of virus persistence. This is especially true for IHNV, which persists in survivors in presence of high antibody levels (Leong et al.,1997). Since resolution of virus persistence is correlated with cell-mediated immunity, vaccines that include stimulators of this kind of immunity such as MHC-1 or cytokines must be developed for fish.

DNA vaccines are very effective in inducing protective humoral and cellular immune response in mammals (Ulmer et al.,1997; Wang et al.,1995; Xiang et al.,1995). However, their use in animals destined for human consumption is restricted by a number of safety considerations that must be solved, such as the source of DNA encoding the antigen and the regulatory elements controlling antigen expression. With a DNA vaccine, there is the additional requirement that the product be considered safe in the eyes of the consumer. The safety regulations for fish DNA vaccines are in the process of being drafted by the USDA.

Delivery methods are also important determinants for the potential application of this technology in commercial fish farms. Although injection is considered to be the most efficacious method of fish vaccination, alternative methods of administration will have to be developed for small fish and low value species, for which intramuscular injection is not practical or cost effective (Piganelli et al.,1994; Wong et al.,1992). Oral or immersion vaccination would be more appropriate in this case. Specialized approaches such as delivery of antigen with chimaeric viral vectors like polio or adenovirus, encapsulated vaccines for oral ingestion and bacterial carriers such as salmonella or shigella, have been used to induce protective immunity in mammals (Mathiowitz et al.,1997; Kreuter, 1996; Piganelli et al.,1994). Some of these strategies for delivery of DNA vaccines should be examined for fish vaccines.

In summary, we recommend the following for fish vaccines:

Short Term Recommendations (Research):

1. Design of DNA vaccine vectors that are biologically safe
2. Design efficient delivery systems for vaccines
3. Develop immunological markers for vaccination efficacy
4. Genomic and Proteomic analysis of key aquaculture pathogens
5. Development of Guidelines for the licensing of DNA vaccines for fish.

Mid and LongTerm Recommendations (Research):

1. Characterize the immune system of different fish and shellfish species
1. cellular immune response assays need to be developed
2. syngeneic lines of rainbow trout, catfish, and tilapia must be developed
3. examine role of innate immunity in protection
2. Characterize the immune response genes in key aquaculture species
3. Several laboratories are beginning to analyze the immune response in fish and shellfish by identifying genes that are expressed upon infection, vaccination, etc. There should be a coordinated effort to characterize these genes displayed in subtraction hybridization libraries.

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