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. 2000 Aug;74(15):7048–7054. doi: 10.1128/jvi.74.15.7048-7054.2000

DNA Vaccines Encoding Viral Glycoproteins Induce Nonspecific Immunity and Mx Protein Synthesis in Fish†

Carol H Kim 1,‡, Marc C Johnson 1, John D Drennan 1, Benjamin E Simon 1, Estela Thomann 1, Jo-Ann C Leong 1,*
PMCID: PMC112222  PMID: 10888644

Abstract

Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.

Antiviral DNA vaccines carrying a gene for a major antigenic viral protein have received considerable attention as a new approach to vaccine development, especially when traditional vaccines have failed. They offer the advantage of mimicking a viral infection, resulting in host production of a single viral protein that is correctly folded and modified, and eliciting both cellular and humoral immune responses (9, 48). DNA vaccines have been developed for a wide variety of viruses, including influenza virus (14, 46), human immunodeficiency virus (7, 15, 42), rabies virus (38), hepatitis B virus (10), rubella virus (41), and foot-and-mouth disease virus (19). Genetic vaccines have also been developed for several other pathogens, including Mycoplasma pulmonis (29), Mycobacterium tuberculosis (34), Plasmodium yoelii (17), and Schistosoma japonicum (49).

For fish viruses, DNA vaccines have been developed for infectious hematopoietic necrosis virus (IHNV) (2, 33) and viral hemorrhagic septicemia virus (6), both rhabdoviruses belonging to the Novirhabdovirus genus. Laboratory trials with fish indicate that these vaccines are considerably more effective in protecting fish from lethal challenge with homologous virus than either the traditional killed vaccine or the subunit vaccine we had developed previously (32). However, the basis for protection by the DNA vaccine had not been determined.

As part of a controlled study to demonstrate the specificity of the immune response to the IHNV vaccine, we developed DNA vaccines for two other serologically distant fish rhabdoviruses, spring viremia of carp virus (SVCV) and snakehead rhabdovirus (SHRV) (26). Surprisingly, both SVCV and SHRV vaccines induced protective immunity to lethal challenge with IHNV. Because SHRV and SVCV are exotic pathogens in the United States, it was not possible to conduct the reverse experiment with IHNV vaccination and subsequent challenge with SVCV or SHRV. Nevertheless, these observations prompted an investigation into the possible reasons why the glycoprotein (G) expression of either SVCV or SHRV would induce protection against lethal challenge from an unrelated virus. We show here that DNA vaccination with a G gene induces a potent interferon (IFN) response in fish and propose that this initial IFN induction is the basis for the heterologous protection.

MATERIALS AND METHODS

Virus propagation.

The Rangen isolate (RA) or the 220-90 isolate of IHNV was used at a multiplicity of infection of 0.01 to infect susceptible chinook salmon embryo (CHSE-214) cell monolayers. The infected cells were incubated at 16°C in complete medium containing Eagle's minimum essential medium supplemented with 5% fetal bovine serum, 1,000 IU of penicillin/ml, 1 mg of streptomycin per ml, and 2.5 μg of amphotericin B per ml and buffered to pH 7.5 with 7.5% sodium bicarbonate. The medium was harvested when viral cytopathic effects were apparent (usually within 48 to 72 h). Cellular debris was removed by low-speed centrifugation, and the resulting clarified medium was subsequently used to infect rainbow trout.

Plasmid constructions.

Plasmid vectors encoding the G gene sequences of IHNV, SHRV, and SVCV were constructed. All of these G gene sequences were previously cloned in our laboratory (24, 25, 28). The G genes of IHNV, SHRV, and SVCV were cloned into the plasmid vector, pcDNA3 (Clontech, Palo Alto, Calif.), which contains a cytomegalovirus promoter upstream from the inserted G genes. DNA sequencing of the first 400 nucleotides of each insert verified the nucleotide sequence and orientation of each plasmid constructed in this study. In vitro translation of the resulting plasmids, pcDNA3-IHNV-G, pcDNA3-SHRV-G, and pcDNA3-SVCV-G, was carried out to verify that each plasmid had been constructed correctly and that G production in eukaryotic cells was possible with each plasmid.

The plasmids, pcDNA3, pcDNA3-IHNV-G, pcDNA3-SHRV-G, and pcDNA3-SVCV-G, were each used to transform competent Escherichia coli JM109, and these transformed organisms were grown in the presence of ampicillin. Large-scale preparations of each plasmid were made with the Qiagen (Valencia, Calif.) plasmid purification kit, according to the manufacturer's protocol. The concentration of purified plasmids was determined by optical density at 260 nm.

Plasmid injection of rainbow trout.

The rainbow trout strain used in the studies presented here was a cross between the Klamath strain (Klamath Hatchery, Chiloquin, Oreg.) and the Cape Cod strain (Roaring River Hatchery, Scio, Oreg.). Eggs from both strains were obtained from their corresponding hatcheries and then fertilized at the Oregon State University Salmon Disease Laboratory, Corvallis. Fry were held in 100-liter tanks until the fish mean weight was 0.5 g at 2 months posthatch. Fish were then randomly assigned to one of five groups of fish. Each group of 200 fish received one of the vaccines or phosphate-buffered saline (PBS).

One of the plasmid vaccines, pcDNA3-IHNV-G, pcDNA3-SHRV-G, pcDNA3-SVCV-G, or pcDNA3, was used to vaccinate the fish by intramuscular injection. PBS-injected fish were used as challenge controls. Plasmid DNA concentrations were adjusted to 10 μg/25 μl by dilution in PBS, pH 7.4, and the DNA concentration was confirmed by spectrophotometry. The fish (mean weight, 0.56 g) were anesthetized in 0.1% tricane methanesulfonate (MS-222; Finquel), injected intramuscularly with 25 μl of the corresponding plasmid or PBS, and placed in 25-liter tanks with 50 fish per tank. A total of 20 tanks were used: 4 for pcDNA3-, 4 for pcDNA3-IHNV-G-, 4 for pcDNA3-SHRV-G-, 4 for pcDNA3-SVCV-G-, and 4 for PBS-injected fish. Tank locations were randomized to avoid possible effects of tank position.

IHNV challenge of rainbow trout.

At 30 days postvaccination (dpv), fish (mean weight, 1.33 g) were challenged by immersion with the IHNV-RA strain. Fish were challenged with the viral doses 103 or 105 PFU/ml. Two tanks (50 fish per tank) of each group were exposed to each IHNV-RA dose in 2 liters of water at 15°C for 5 h. The fish were monitored for clinical signs of disease, such as a distended abdomen, petechial hemorrhaging, and a whirling form of swimming. Postchallenge fish mortality in each tank was recorded, and dead fish were removed daily for 30 days. Plaque assays were performed to confirm that the fish were infected with IHNV (data not shown). The relative percent survival (RPS) was calculated for each vaccination and challenge. RPS was calculated by the following formula, as described by Johnson et al. (23): RPS = [1 − (% mortality of vaccinated fish/% mortality of control fish)] × 100, where pcDNA3-injected fish were used as the control fish for these calculations.

In a parallel experiment, fish (mean weight at 4 months posthatch, 2.0 g) were challenged by immersion with 105 PFU of IHNV per ml at 70 dpv. In this experiment, IHNV strain 220-90 was used for the challenge, since our experience with IHNV indicated that strain 220-90 was more virulent in larger fish. The conditions of this challenge were identical to those described above.

Amino acid sequence analysis.

The amino acid sequences of IHNV, SHRV, and SVCV Gs were imported into a genetic data analysis program (CLUSTAL) and aligned with the sequence editor (Wisconsin Package version 9.1, Genetics Computer Group, Madison, Wis.). Conserved amino acids were identified among these three Gs and used to determine percent amino acid identity.

Preparation of tissues and immunoblot analyses for Mx protein.

Fish tissues were prepared for immunoblot analyses to detect Mx protein. Kidney and liver tissues were harvested and then immediately placed in 100 μl of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (0.5 M Tris-Cl [pH 8.8], containing 0.4% SDS, 20% glycerol, 2% β-mercaptoethanol, 0.1% [wt/vol] bromophenol blue). The tissues were homogenized, heated at 95°C for 2 min, and thoroughly mixed. Ten microliters of sample was loaded onto a SDS–12% PAGE acrylamide gel and run at 150 V until the dye front reached the bottom of the gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (New England Nuclear, Wilmington, Del.) with cold Towbin buffer (25 mM Tris-HCl [pH 8.3], 192 mM glycine, 20% methanol) for 1 h at 50 V. The membrane was blocked overnight in 2× TTBS (1× Tris-buffered saline [100 mM Tris-HCl, pH 7.6, 0.9% NaCl], 2% bovine serum albumin, 0.1% Tween 20). Rabbit anti-Mx polyclonal antibody serum was diluted 1:500 in 1× TTBS and incubated for 1 h at room temperature (46). The membrane was washed three times for 10 min each with 1× TTBS. Horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (Kirkegaard & Perry, Inc., Gaithersburg, Md.) was diluted 1:24,000 in 2× TTBS and incubated with the membrane for 1 h. The membrane was washed as described above and subsequently incubated with chemiluminescence substrate (SuperSignal; Pierce, Rockford, Ill.) for 10 min and used to expose X-ray film.

Neutralizing antibody.

At 70 dpv, fish were bled, and serum was collected. The serum samples were tested for the presence of neutralizing antibodies to IHNV, as previously described (13, 30).

RESULTS

G sequence analysis and CpG motif analysis.

An amino acid sequence alignment of the G genes from SVCV, SHRV, and IHNV is shown in Fig. 1. Although approximately 40% of the amino acids are conserved between SHRV and IHNV, only 11% of the amino acids were conserved among all three viral G genes. The largest block of homology comprises 3 consecutive amino acids, and in no place were there more than 4 of 10 conserved amino acids.

FIG. 1.

FIG. 1

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G amino acid sequence analysis. Alignment of the predicted amino acid sequences from SHRV, SVCV, and IHNV. ∗, amino acids conserved among all three genes.

Potent immune stimulation has been reported to result from an unmethylated, six-nucleotide sequence consisting of two purine residues followed by CG and two pyrimidine residues, RRCGYY, called CpG motifs (29). CpG-containing oligonucleotides have been shown to activate antigen-presenting cells and upregulate the expression of certain surface molecules which activate the immune response. These oligonucleotides also induce secretion of such interleukins (ILs) as IL-1, IL-12, IFN-γ, and tumor necrosis factor alpha. More recently, CpG-containing oligonucleotides were shown to be efficient adjuvants for the induction of protective immunity with peptide vaccines in mice (41). It is thought that this effect is a result of binding to the surface of B cells and macrophages. There is some evidence that CpG motif-containing DNA also acts in the same manner in fish (J. Heppell, J. Sanchez-Dardon, A. M. Krieg, and H. L. Davis, Proc. 3rd Int. Symp. Aquat. Anim. Health, abstr. S3-1, p. 101, 1998). Thus, we examined the sequences of the vaccine and control plasmids to determine whether there were any significant differences in the number of CpG motifs. In each of the three G gene sequences, there were only two CpG motifs. The sequence of the parental plasmid, pcDNA3, had 25 copies of the CpG motif.

Effect of DNA immunization against IHNV challenge 30 dpv.

Immunized and control fish were challenged by immersion at 30 dpv with 105 and 103 PFU of IHNV-RA per ml. The fish were monitored daily for signs of disease, and dead individuals were removed on a daily basis. The effect of pcDNA3-IHNV-G, pcDNA3-SHRV-G, or pcDNA3-SVCV-G immunization on cumulative percent mortality (CPM) resulting from a challenge dose of 105 PFU/ml is shown in Fig. 2A. Significant protection was observed with a CPM of only 4% in the pcDNA3-IHNV-G, 1% in the pcDNA3-SHRV-G, and 3% in the pcDNA3-SVCV-G vaccination treatment groups (Fig. 2A). The RPS for these groups of fish was 93% for pcDNA3-IHNV-G, 98% for pcDNA3-SHRV-G, and 95% for pcDNA3-SVCV-G. The control fish that received either PBS or pcDNA3 had CPM rates of 55 and 57%, respectively (Fig. 2A). As expected, there were fewer mortalities in the challenged fish who received 103 PFU/ml but with results similar to those observed in fish receiving 105 PFU/ml, with CPM/RPS ratios (percentages) of 0/100 for pcDNA3-IHNV-G, 1/94 for pcDNA3-SHRV-G, and 2/88 for pcDNA3-SVCV-G (Fig. 2B).

FIG. 2.

FIG. 2

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CPM for fish exposed to 105 PFU (A) or 103 PFU (B) of IHNV per ml at 30 dpv. The average mortality for the virus is shown on the ordinate and is plotted against the number of days postexposure. The range of mortality did not vary significantly between tanks and thus is not shown.

Effect of DNA immunization against IHNV challenge at 70 dpv.

A parallel group of 25 fish was vaccinated with each G DNA vaccine or with the pcDNA3 control or PBS and then held for 70 days. At 70 dpv, the fish were challenged by immersion with 105 PFU of IHNV per ml (Fig. 3). The fish were observed for 30 days for clinical signs of disease, and dead fish were removed on a daily basis. The challenge data (Fig. 3) show greater differences in protective effects for the three different DNA vaccines at 70 dpv than was observed for fish challenged at 30 dpv (Fig. 2). The groups receiving the heterologous vaccines, pcDNA3-SHRV-G and pcDNA3-SVCV-G, had RPS values of only 26% (68% CPM) and 17% (76% CPM), respectively, while the pcDNA3-IHNV-G vaccine produced an RPS of 87% (12% CPM) (Fig. 3). The pcDNA3 and PBS provided little protection, resulting in CPMs of 91 and 96%, respectively.

FIG. 3.

FIG. 3

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CPM for fish exposed to 105 PFU of IHNV per ml at 70 dpv. The average mortality for the virus is shown on the ordinate and is plotted against the number of days postexposure. The range of mortality did not vary significantly between tanks and thus is not shown.

Mx expression 0 and 7 dpc.

To determine the possible role of IFN induction in protection with these DNA vaccines, immunoblots of the liver and kidney tissues of the vaccinated fish were performed with antiserum against the rainbow trout Mx protein (Fig. 4). IFN has not yet been cloned from any fish species, and there are no available reagents that will detect trout IFN directly; however, the Mx gene of rainbow trout has been cloned and characterized, and antiserum has been raised against it in rabbits (44). As an IFN-inducible protein, Mx antiserum can be used as an indicator of IFN induction (43). Kidney and liver tissues were harvested from fish and homogenized in SDS-PAGE loading buffer. Although total protein amounts were not calculated, the fish were approximately the same size, and the tissues were homogenized in the same amount of buffer. This approach provides an approximation of the relative amounts of Mx protein expressed in the tissues. The results at 0 and 7 day postchallenge (dpc) with 105 PFU of IHNV per ml are shown in Fig. 4. It should be noted that the 0 dpc was 30 dpv. At 0 dpc, Mx was detected in kidney and liver samples from fish that were vaccinated with pcDNA3-IHNV-G. Two of three kidney and liver samples expressed detectable levels of Mx in the pcDNA3-SHRV-G- and pcDNA3-SVCV-G-vaccinated fish. In contrast, none of the pcDNA3- and PBS-injected control fish expressed Mx in either kidney or liver tissue.

FIG. 4.

FIG. 4

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Mx expression at 0 and 7 dpc. Immunoblot analyses of kidney (K) and liver (L) tissues from fish that were vaccinated with IHNV, SHRV, and SVCV G vaccines or with a pcDNA3 or PBS control, and then subsequently challenged with 105 PFU of IHNV per ml are shown. The immunoblots were probed with an Mx antiserum primary antibody and an alkaline phosphatase-conjugated donkey anti-rabbit secondary antibody as described in Materials and Methods.

A summary of the immunoblot data at 0, 1, 2, 5, and 7 dpc from fish challenged with 103 PFU/ml and 105 PFU/ml is shown in Table 1. The expression of Mx protein in the pcDNA3-IHNV-G-vaccinated fish declined over time so that by 7 dpc, all tissue samples from fish were negative for both virus challenge doses. The pcDNA3-SHRV-G-vaccinated fish also showed a change in Mx protein expression over time. By 7 dpc, only one of the kidney tissue samples was positive for Mx in the 105 PFU/ml-challenged fish, but two kidney samples and two liver samples from the 103 PFU/ml-challenged fish were positive (Table 1). Mx expression in the group vaccinated with pcDNA3-SVCV-G did not change over time, as it did in the other vaccination groups. At 7 dpc, Mx expression was present in two kidney tissues and all three liver tissues from fish in both virus challenge groups.

TABLE 1.

Mx expression in fish vaccinated with IHNV, SHRV, and SVCV G DNA vaccinesa

Treatment Challenge dose
103 PFU/ml at dpc:
105 PFU/ml at dpc:
0
1
2
5
7
1
2
5
7
K L K L K L K L K L K L K L K L K L
IHNV-G 3/3 3/3 3/3 3/3 2/3 3/3 2/3 1/3 0/3 0/3 2/3 3/3 3/3 2/3 0/3 2/3 0/3 0/3
SHRV-G 2/3 2/3 3/3 3/3 2/3 2/3 1/3 2/3 2/3 2/3 3/3 3/3 3/3 3/3 2/3 2/3 1/3 0/3
SVCV-G 2/3 2/3 3/3 2/3 3/3 2/3 2/3 2/3 2/3 3/3 3/3 2/3 3/3 3/3 1/3 3/3 2/3 3/3
pcDNA3 0/3 0/3 0/3 0/3 0/3 0/3 2/3 1/3 3/3 3/3 0/3 0/3 2/3 1/3 2/3 3/3 3/3 3/3
PBS 0/3 0/3 0/3 0/3 1/3 1/3 2/3 2/3 3/3 3/3 0/3 0/3 0/3 0/3 3/3 3/3 2/3 2/3
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a

Mx expression for fish exposed to 103 or 105 PFU of IHNV per ml at 0, 1, 2, 5, and 7 dpc is shown. The ratios denote the number of fish expressing the Mx protein versus the total number of fish examined. K, kidney; L, liver. 

A contrasting profile was observed for fish in the two control groups which received either pcDNA3 or PBS. Fish in both groups started at 0 dpc with no Mx expression in the liver or kidney tissues; however, by 7 dpc, all of the pcDNA3-vaccinated fish receiving both challenge doses and the PBS-injected fish receiving the 103 PFU/ml dose expressed the Mx protein. Mx was detected in two kidney and two liver tissue samples from PBS-vaccinated fish challenged with 105 PFU/ml.

Neutralizing antibody to IHNV.

Fish were bled and tested for neutralizing antibody against IHNV. Neutralizing antibody to IHNV was not detected in any of the fish injected with pcDNA3-IHNV-G, pcDNA3-SHRV-G, pcDNA3-SVCV-G, pcDNA3, or PBS. Only positive-control fish that were challenged with IHNV had detectable levels of IHNV-neutralizing antibody, in serum samples taken from fish 130 dpv. These titers ranged from 20 to 80 and were calculated from the reciprocal of the last dilution of antisera with detectable neutralizing activity for 50 PFU. The antisera from fish immunized with the DNA vaccines, pcDNA3-SHRV-G or pcDNA3-SVCV-G, were not examined for neutralizing activity for their respective viruses.

DISCUSSION

Although it has been clearly demonstrated that the IHNV DNA vaccine provides good protection against challenge in fish (2, 6), the mechanisms which form the foundation of its success remain unclear. DNA vaccines for mammalian viruses induce strong, long-lasting protective antibody and cytolytic T-cell responses (11, 40). The basis for this strong protection is thought to reside in the fact that DNA vaccines produce viral protein within the host cells and this permits presentation of peptides in the context of both major histocompatibility complex class I (MHC-I) and MHC-II molecules. Clearly, protection is assumed to be due to the induction of specific immunity and the production of specific antibody and cytolytic T-cell responses. In this study of fish, we asked whether the protection was, indeed, specific and whether different G-encoding DNA vaccines might induce protective immunity to IHNV.

The study was prompted by the work of Ito and colleagues, who showed that the binding of viral Gs to mammalian spleen cells induced these cells to produce IFN-α/β (20, 22). Ito and his colleagues also showed that mice given an intravenous injection of isolated viral Gs of purified Sendai virus produced circulating levels of IFN (21). We asked whether it was possible that DNA vaccines encoding a viral G worked because of the induction of nonspecific protective immunity, i.e., IFN. We had shown previously that injection of a plasmid expressing the IHNV G provides strong protection to subsequent challenge with IHNV (2), but we did not know whether this protection was a result of an antigen-specific response. Although the first appearance of anti-G antibody was at 8 weeks postvaccination, we routinely observed protection against virus challenge as early as 3 to 4 weeks. Also, virus-neutralizing antibody at very low titers only appeared in some fish at 6 weeks postvaccination. All of these observations led us to ask whether the success of the IHNV DNA vaccine in fish was actually due to a specific immune response.

The ideal experiment to answer the question of specificity would be to vaccinate fish with a DNA vaccine for the IHNV G, then challenge the fish with either IHNV or another immunologically unrelated viral pathogen, and compare levels of protection. Unfortunately, this is not possible because the fish rhabdoviruses that were used, SHRV and SVCV, are both considered exotic viruses in the United States, and their growth in fish is forbidden by state regulatory agencies. In addition, SHRV and SVCV infect warm-water (20 to 28°C) fishes, and there was a strong likelihood that these viruses would not be pathogenic in rainbow trout at 10 to 15°C. Thus, we chose to run the experiment by vaccinating fish with DNA vaccines prepared from the G genes of IHNV, SHRV, and SVCV and challenging the vaccinated fish with IHNV. IHNV and SHRV both belong to the genus Novirhabdovirus but are unrelated serologically (26). SVCV is most closely related to rhabdoviruses in the genus Vesiculovirus and is also unrelated serologically to either IHNV or SHRV (5).

We found that fish vaccinated with DNA encoding either IHNV, SVCV, or SHRV Gs were protected against IHNV challenge at 30 dpv. The induction of an IFN-α/β response in the vaccinated animals was confirmed by immunoblots which detected the increased production of the IFN-inducible Mx protein. It was only in fish that were vaccinated and not challenged until 70 dpv that differences in protection were observed among the different vaccines. In this case, the pcDNA3-IHNV-G vaccine protected the fish against virus challenge, and the heterologous vaccines were much less protective.

One interpretation of these results is that vaccination with a plasmid encoding a viral G resulted in the production of IFN-α which provided an early and effective protection against virus infection. Over time, the nonspecific antiviral state waned, either as a result of decreasing glycoprotein production or as the result of some other downregulation event. As this antiviral state decreased, the long-term specific protection provided by the DNA vaccine became the important player in host immunity. This later DNA vaccine-induced protection in fish appears to be independent of neutralizing antibodies and may be due to a cellular immune response.

Evidence that a nonspecific immune response could protect fish against the lethal effects of IHNV challenge was first observed by Eaton (12), who showed that injection with the synthetic double-stranded RNA, polyI-C, significantly reduced mortality in fish challenged with IHNV. In addition, it has been shown that fish exposed to heterologous, unrelated viruses were also protected against IHNV (31). This protection was presumed to result from nonspecific defense induced by the original virus infection. In a recently completed study, the IFN-inducible protein, Mx, was detected in the liver and kidney tissues of fish injected with the IHNV-G DNA vaccine but not in fish injected with a formalin-killed or subunit IHNV vaccine or vaccine controls (C. H. Kim, E. Thomann, H. C. Carlson, and I. C. Leong, unpublished data). These data indicate that IFN may play a key role in the protection produced by the DNA vaccines.

In mammals, IFN released from virus-infected cells binds to specific receptors on the plasma membrane in an autocrine as well as paracrine fashion, upregulating the expression of over 20 IFN-regulated proteins, including the Mx protein, that can directly or indirectly interfere with viral replication. Mx proteins are IFN-inducible proteins that are synthesized in response to virus infection and polyI-C treatment. This protein appears to be present in all vertebrate species and has been found in humans (1, 18), rats (35, 36), sheep (8), pigs (37), ducks (3), chickens (4), perch (43), and trout (44). Although double-stranded RNA is the best-characterized inducer of IFN, it has been proposed that expression of viral Gs also induces IFN via lectin-like activity similar to concanavalin A and phytohemagglutinin, which induce IFN in lymphoid cells (20). This response was only seen when the viral G was expressed on the surface of a host cell and was not seen in response to extracellular G. To date, IFN has not been cloned from a fish, but the detection of IFN-like activity has been well documented (16). Although it has not been possible to detect IFN directly in fish, it is possible to detect IFN induction indirectly by assessing the production of IFN-inducible proteins, e.g., Mx. Antiserum had been generated to an E. coli-expressed region of the rainbow trout Mx protein, permitting the detection of Mx by immunohistochemistry and immunoblot assays (46).

In a recent paper by Boudinot et al. (6), adult fish (150 to 200 g) were injected with a pcDNA3 plasmid that contained either the IHNV-G genes, the VHSV-G gene, or both G genes on the same plasmid. Substantial levels of specific neutralizing antibody were detected in these DNA-vaccinated fish. In addition, elevated levels of Mx and MHC-II transcripts were detected by reverse transcription PCR, indicating a nonspecific response mediated by IFN as well as a cellular immune response. The researchers were able to detect neutralizing antibodies in vaccinated fish; however, the fish used in that study were adult rainbow trout that would be more immunocompetent than the juvenile fish used in our experiments. Despite the variation in the immunocompetence of fish based on antigen, size, age, and species of fish, it has been shown that adult fish are more resistant to pathogen infection than their juvenile counterparts (47). It should also be noted that although neutralizing antibodies were detected, Boudinot et al. (6) were unable to correlate antibody production with protection against subsequent challenge, since the fish were too large to be killed by virus infection.

It could also be the case that the production of the Mx protein itself results in the nonspecific protection observed with the pcDNA3-SHRV-G- and pcDNA3-SVCV-G-injected fish at 30 dpv. In cell culture experiments, however, the overexpression of the Mx protein did not inhibit replication of IHNV (45). The role of Mx in fish at the systemic level has not been examined. It may be that Mx does play a more important role in antiviral immunity in the whole animal, rather than simply being an indicator of IFN expression.

It is tempting to conclude that Mx expression may be used as an indicator of vaccine efficacy and long-term protection. There was an increase in Mx protein expression in fish receiving any of the three DNA vaccines. This was not observed in the control fish vaccinated with pcDNA3. When the fish were challenged with live virus, there was a sharp decrease in the production of Mx protein in the pcDNA3-IHNV-G-vaccinated fish and, to a lesser degree, in the pcDNA3-SHRV-G-vaccinated fish. The virus challenge did not reduce Mx expression in the pcDNA3-SVCV-G-vaccinated fish. It appeared that decreased Mx production upon virus challenge was correlated with the specificity of the vaccine G. One explanation might be the removal of the G-expressing cells by specific immunity upon virus challenge. Further studies examining the persistence of plasmid DNA in vaccinated fish after virus challenge may provide some explanation for the decrease in Mx production. Previous studies have shown that plasmid DNA persisted in the fish for as long as 160 dpv (2); however, examining the fish for DNA after virus challenge has never been attempted.

ACKNOWLEDGMENTS

This research was supported by the United States Department of Agriculture (USDA) National Research Initiative Competitive Grants Program (grants 9603020 and CO140A), a USDA grant to the Western Regional Aquaculture Consortium (grant 683021), and the National Oceanic and Atmospheric Administration, Office of Sea Grants (grant NA89AA-D-SG108).

The authors gratefully acknowledge the help of S. LaPatra (Clear Springs Foods, Buhl, Idaho) for the virus neutralization assays.

Footnotes

†

Oregon State University Agriculture Experiment Station Technical paper 11,686.

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