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Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination
Vaccine 19 (2001) 4011– 4019 www.elsevier.com/locate/vaccine
Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination Scott E. LaPatra a,*, Serge Corbeil b,1, Gerald R. Jones a, William D. Shewmaker a, Niels Lorenzen c, Eric D. Anderson d, Gael Kurath a a Research Di6ision, Clear Springs Foods Inc., PO Box 712, Buhl, ID 83316, USA USGS Western Fisheries Research Center, 6505 NE 65th Street, Seattle, WA 98115, USA c Danish Veterinary Laboratory, Hango6ej 2, 8200 Arhus N, Denmark d Department of Biochemistry, Microbiology, and Molecular Biology, Uni6ersity of Maine, Orono, ME 04469, USA b
Received 12 October 2000; received in revised form 6 March 2001; accepted 14 March 2001
Abstract A DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was shown to provide significant protection as soon as 4 d after intramuscular vaccination in 2 g rainbow trout (Oncorhynchus mykiss) held at 15°C. Nearly complete protection was also observed at later time points (7, 14, and 28 d) using a standardized waterborne challenge model. In a test of the specificity of this early protection, immunization of rainbow trout with a DNA vaccine against another fish rhabdovirus, viral hemorrhagic septicemia virus, provided a significant level of cross-protection against IHNV challenge for a transient period of time, whereas a rabies virus DNA vaccine was not protective. This indication of distinct early and late protective mechanisms was not dependent on DNA vaccine doses from 0.1 to 2.5 mg. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: DNA vaccine kinetics; Fish DNA vaccine specificity; Infectious hematopoietic necrosis virus (IHNV); Viral hemorrhagic septicemia virus (VHSV)
1. Introduction Infectious hematopoietic necrosis (IHN) is the most important rhabdoviral disease of salmon and trout in North America. Annual losses estimated at several millions of dollars occur among commercially-reared rainbow trout (Oncorhynchus mykiss) and IHN also affects both wild and cultured Pacific salmon, steelhead (O. mykiss), and Atlantic salmon (Salmo salar) throughout the Pacific Northwest. Depending upon the species of fish, strain of virus, and environmental conditions, epidemics of IHN may result in losses of greater than 90%. The history of IHNV, its chemical, physical, * Corresponding author. Tel.: + 1-208-5433465; fax: +1-2085434146. E-mail address: [email protected] (S.E. LaPatra). 1 Present address: CSIRO, Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, Vic., 3220, Australia.
and serological characteristics, and factors affecting virulence of the virus for salmon and trout have been comprehensively reviewed [1,2]. Presently, avoidance of exposure by destruction of infected stocks and the use of virus-free water supplies and certified eggs are the only control measures for IHN. Vaccination has been attempted for more than two decades, but no licensed efficacious products are yet available [3]. For fish, the first application of DNA vaccine technology was reported by Anderson et al. [4] who used a plasmid containing the glycoprotein (G) gene of IHNV to stimulate a protective immune response in rainbow trout fry. Further work in our laboratories has shown that a DNA vaccine against IHNV reproducibly provides significant protection in rainbow trout against either waterborne or injection challenges, in fish that range in size from 2 to 160 g [5–7]. Significant high levels of protection against
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IHNV were also observed in vaccine efficacy studies in another economically important species, Atlantic salmon [8]. A DNA vaccine containing the G gene of another rhabdoviral pathogen of rainbow trout, viral hemorrhagic septicemia virus (VHSV), has also been shown to provide significant protection when administered alone [9,10] or in combination with a DNA vaccine against IHNV [11]. The potency of DNA vaccination has been experimentally demonstrated in various host-pathogen systems including large mammals and model hosts such as mice. In some cases, significant antibody levels were obtained as soon as 7 d post-immunization [12]. In BALB/c mice challenged with herpes simplex virus, significant protection was reported as early as 14 d after primary vaccination [13]. To our knowledge protection against viral challenge has not been reported at time points less than 14 d post-vaccination in any mammalian host. Here, we report that vaccination of rainbow trout with a DNA vaccine results in immunity at 4 d at 15°C and provides significant protection against a lethal virus challenge. To further explore this observation the specificity of protection was examined and found to be low for a transient period of time.
strain DH5a and plasmid DNA was purified by the alkaline lysis protocol of Saporito-Irwin et al.[18]. Expression of the proteins from the IHNV and VHSV DNA vaccines in fish cells was confirmed by transfection of fish cells as described in the references above. Expression of the rabies virus G protein in fish cells was confirmed by transfection of EPC cells at both 15 and 28°C (data not shown). Detection of the expressed rabies virus G protein was by immunoflourescence using the rabies G protein monoclonal 514, provided by Dr. D. Lodmell. For DNA vaccination, specific-pathogen-free rainbow trout (mean weight, 2 g; Clear Springs Foods, Inc., Buhl, Idaho) were anaesthetized by immersion in 100 mg/ml of tricaine methane sulfonate (MS-222; Argent Chemical Laboratories, Redmond, WA) and injected intramuscularly at the base of the dorsal fin with 1 mg of the specified DNA vaccine in 50 ml of phosphate buffered saline (PBS). For the DNA vaccine dose experiment 0.1, 1.0 and 2.5 mg were injected in 25 ml. Control fish were injected with the plasmid vector alone, PBS alone, or left unhandled as specified in the text. Treatment groups were placed in 19 l aquaria receiving UV-treated, single-pass spring water (15°C), and fed a dry pelleted diet ad libitum.
2. Materials and methods
2.3. Hematopoietic necrosis 6irus challenge of rainbow trout
2.1. Virus propagation and titration The IHNV challenge virus strain 220– 90 was propagated in the epithelioma papulosum cyprini (EPC) cell line [14] as described by LaPatra et al [15]. Virus challenge dose and virus titers in infected fry were determined by plaque assay on confluent EPC cell monolayers [16].
2.2. DNA 6accination of rainbow trout The pIHNw-G, pIHNw-N, and pLuc DNA vaccines, containing the IHNV G gene, IHNV N gene, and the luciferase gene, respectively, have been described in Corbeil et al. [5] and Corbeil et al [6]. The VHSV G gene DNA vaccine, pCMV-vhsG, is described in Lorenzen et al. [9]. The fish vaccines were constructed using the vector plasmids pcDNA3 or pCDNA3.1 for the VHSV G gene and IHNV G gene, respectively. The DNA vaccine construct containing the G gene of the CVS strain of rabies virus, pCMV4CVSG [17], was generously provided by Dr. D. Lodmell (Rocky Mountain Laboratories, NIAID, Hamilton, MT). This vaccine was constructed using the vector plasmid pCMV-4. The VHSV and rabies virus vaccines were denoted as pVHS-G and pRV-G, respectively, in the studies reported herein. All vaccine plasmids and the vector plasmid pCDNA3.1 were amplified in Escherichia coli
Challenges of rainbow trout were performed on duplicate 25 fish groups that were waterborne exposed to 104 or 105 plaque forming units (PFU)/ml of IHNV strain 220–90 for 60 min with aeration in a volume of water that was 10× the total weight of the fish (g). Mock infected control groups were treated identically but were exposed to cell culture media (MEM) only. Experimental groups were held separately in 19 l aquaria receiving UV-treated, single-pass spring water (15°C), fed a dry pelleted diet ad libitum, and monitored for 21 d for mortality. Cumulative percent mortality data was used in the Fisher exact test to test for differences between replicates within treatment and day. When there was no difference between replicates they were pooled and the Chi-square test was used to identify differences among treatments.
3. Results
3.1. Onset of early protection Rainbow trout fry (mean weight, 2 g), in groups of approximately 400 fish each, were injected intramuscularly with 1 mg of the IHNV DNA vaccine (pIHNw-G), or treated as various controls. In the first experiment duplicate groups of 25 fish were removed at 1, 7, 14, 21,
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and 28 d post-vaccination and challenged by immersion in water containing IHNV at a concentration of 104 PFU/ml. Mock-infected fish from each treatment group were handled similarly, but not exposed to virus and were included for each treatment and time interval tested. All groups were monitored for mortalities daily for 21 d and final cumulative percent mortality data is shown in Fig. 1A. All treatment groups that were challenged 1 d after vaccination had high mortality ranging from 62–86%. In fish challenged at 7 d post-vaccination the pIHNw-G groups showed 2% average cumulative mortality compared with 50–58% mortality in the various control groups. Similar significant (P B 0.05) protection in the pIHNw-G vaccinated groups was observed in fish challenged at 14, 21, and 28 d post-immunization. In a second experiment, time points shorter than one week were tested (Fig. 1B). Rainbow trout fry were
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vaccinated with the same treatments and controls as above and challenged at 1, 2, 4, and 7 d post-vaccination. A higher IHNV challenge concentration of 105 PFU/ml was used to provide higher levels of mortality in the control treatments, as a more stringent test of the protection observed. No protection was observed at 1 or 2 d post-vaccination, but by 4 d fish immunized with pIHNw-G were significantly (PB0.05) protected relative to all controls. By 7 d protection was even stronger, with an average mortality of 20% in pIHNw-G vaccinates compared to 85–94% in control groups. In this experiment average cumulative mortalities in all groups were higher than in the first experiment due to the higher dose of challenge virus, but protection in the pIHNw-G vaccinated groups was clearly significant at 4 and 7 d post-vaccination. There were no mortalities in the sham infected control fish. No statistically significant differences in mortality were detected among any
Fig. 1. Cumulative % mortality in rainbow trout O. mykiss (mean weight, 2 g) after lethal virus challenge at different time intervals after injection intramuscularly at the base of the dorsal fin with 1 mg of DNA. The DNA vaccines contained either the infectious (IHNV) G gene (pIHNw-G), IHNV N gene (pIHNw-N), or the firefly luciferase gene (pLuc); two additional groups were either injected with phosphate buffered saline (buffer) or were not injected (unhandled). Duplicate groups of 25 fish were challenged by waterborne exposure to 104 IHNV PFU/ml at 1, 7, 14, 21 and 28 d (A) or 105 IHNV PFU/ml at 1, 2, 4, and 7 d (B) post-vaccination. Asterisks * indicate a significant (P B0.05) difference from controls.
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Fig. 2. Cumulative % mortality in rainbow trout (mean weight, 2 g) after lethal virus challenge at different time intervals after injection with DNA vaccines that contained a different rhabdoviral glycoprotein (G) gene or the plasmid pCDNA 3.1 (vector). DNA vaccines contained the G gene of three antigenically distinct rhabdoviruses including IHNV (pIHNw-G), viral hemorrhagic septicemia virus (pVHS-G, also called pCMV-vhsG [9]), and rabies virus (pRV-G, also called pCMV4CVSG [17]). Duplicate groups of 25 fish were challenged by waterborne exposure to 104 IHNV PFU/ml at 4, 7, 14, and 28 d (A) or 1, 2, 4, and 7 d (B) post-vaccination. Asterisks indicate a significant (PB 0.05) difference from controls.
of the replicates within any of the treatment groups in either study, demonstrating the high degree of reproducibility of this challenge model (data not shown).
3.2. Specificity of protection The specificity of this early immunity was investigated using DNA vaccines containing the G genes of two other rhabdovirus species. The vaccine denoted as pVHS-G protects trout against a major European pathogen, VHSV [9]. VHSV is in the same genus as IHNV, but experiments with trout sera show no serological cross-neutralization activity between these viruses [19]. A DNA vaccine to the CVS strain of rabies virus, denoted here as pRV-G, was included as a vaccine for a mammalian rhabdovirus from a different genus [17]. Rainbow trout fry were injected intramuscularly with 1 mg of pIHNw-G, pVHS-G, pRV-G, or pCDNA3.1
vector alone, or left unhandled. At 4, 7, 14, and 28 d post-vaccination duplicate groups of 25 fish were removed from each treatment and challenged by immersion in 104 PFU/ml of IHNV. Mortalities were monitored daily and the results indicated that significant cross-protection against IHNV challenge could be obtained with the VHSV G gene vaccine for a transient time period (Fig. 2A). Groups of fish vaccinated with the IHNV DNA vaccine showed significant protection (PB 0.05) at all time points, with average cumulative mortalities ranging 6–18% in comparison to 54–90% in control groups injected with vector alone or left unhandled. Fish vaccinated with the VHSV DNA vaccine showed significant protection (PB 0.05) ranging 6–20% CPM at 4, 7, and 14 d post-vaccination, but at 28 d the average mortality of 69% was as high as in the unhandled control groups. This loss of cross-protection by the VHSV vaccine at later time points was observed in two independent experiments (data not shown). Fish vacci-
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nated with the rabies DNA vaccine had mortalities comparable to negative control fish at all time points. A specificity experiment with shorter time points was carried out to assess the timing of the onset of crossprotection (Fig. 2B). Treatment and control groups were prepared as above and challenged at 1, 2, 4, and 7 d post-vaccination using an IHNV immersion challenge dose of 104 PFU/ml. There was no significant protection in any groups when challenged 1 d post-vaccination. At 2 d the pIHNw-G vaccinated groups showed significant protection relative to all other groups, and by 4 and 7 d both the IHNV and VHSV vaccine treatment groups were significantly protected (PB 0.05), with 0–10% mortality compared to 34– 63% in the control groups. As in the first specificity experiment, the rabies vaccine showed no protection at any time point tested.
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3.3. Effect of DNA 6accine dose The possible effect of vaccine dose on short and long duration immunity was investigated using IHNV challenges at 4 and 28 d post-vaccination to represent early and late protection, respectively. The IHNV, VHSV, and rabies DNA vaccines and the pCDNA3.1 vector were each administered to groups of rainbow trout fry at doses of 0.1, 1.0, and 2.5 mg per fish. At 4 and 28 d post-vaccination duplicate groups of 25 fish from each treatment were challenged with IHNV and the cumulative mortalities that occurred over 21 d are shown in Fig. 3. At the 4 d time point the IHNV and VHSV vaccine groups at all doses had overall lower mortalities (4–18%) than the rabies vaccine (20–38%) and vector control groups (20–45%). At 28 d post-vaccination only the IHNV vaccine groups were significantly protected at all doses relative to controls. Although the
Fig. 3. Cumulative % mortality in rainbow trout (mean weight, 2 g) after lethal virus challenge at different time intervals after injection with 0.1, 1.0 or 2.5 mg doses of DNA vaccines that contained a different rhabdoviral glycoprotein (G) gene or the plasmid pCDNA 3.1 (vector) as described in Fig. 2. Duplicate groups of 25-fish were challenged by waterborne exposure to 104 IHNV PFU/ml at 4 d (A) or 28 d (B) post-vaccination. Asterisks indicate a significant (PB 0.05) difference from controls.
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overall level of mortality in the control groups was lower in this experiment than in the previous challenges, it was evident that the levels of immunity elicited by vaccine doses of 0.1 to 2.5 mg were not significantly different.
3.4. Hematopoietic necrosis 6irus challenge of rainbow trout In all experiments a minimum of 20% of the fish that died on any day were processed for virus isolation and quantitation by plaque assay (described above) to confirm infection with IHNV. Among the fish that were challenged and died in the experiments shown in Fig. 1 87% (565/651) were IHNV-positive with a mean concentration of 107.0 PFU/g (range 102.0 to \107.3 PFU/ g). For the experiments in Figs. 2 and 3, 95% (465/489) and 86% (234/271) of the fish that were challenged and died were IHNV-positive, with mean concentrations of 106.8 PFU/g (range 102 to \ 107.3 PFU/g) and 106.7 PFU/g (range 102.3 to \107.3 PFU/g), respectively. There was no detectable virus in any of the small number of mock-infected control fish that died in these experiments.
4. Discussion Immunity to rhabdoviruses is generally believed to be mediated through both humoral and cellular mechanisms [20,21]. Although fish immunology is a young science relative to mammalian immunology, it is known that the humoral and cellular responses of fish share basic features in form and function with those of mammals [22–30]. Aspects of immunity against IHNV in rainbow trout, including nonspecific as well as specific mechanisms, have been reviewed recently [2,31]. It has been demonstrated that neutralizing and protective antibodies can be induced by immunization with the viral G protein [32]. However, it is also known that adaptive acquired immune responses of an aquatic poikilotherm are directly influenced by the temperature of the environment. A humoral immune response is generally not detectable until 3– 4 weeks post-exposure to IHNV in 2 g rainbow trout at 15°C [33]. Similar kinetics of neutralizing antibody production have been reported following DNA vaccination of larger trout with both IHNV and VHSV G gene vaccines [11]. Thus, although neutralizing antibody against IHNV is highly protective [34,11], the potential for this adaptive acquired mechanism to function at the early protection time points described here would seem to be negligible. Our observation of the onset of protection against IHNV 4 d after DNA vaccination is the earliest protection time point thus far reported for DNA vaccines.
Similarly, Lorenzen et al. [35] recently showed protection against VHSV 8 d after vaccination of rainbow trout fry with a DNA vaccine containing the G gene of VHSV, suggesting that early protection may be a common feature of DNA vaccines against fish rhabdoviruses. It is also possible that early protection may occur with DNA vaccination in mammalian hosts, but studies describing the onset of protection are lacking, likely due to difficulties with using the large numbers of animals needed to test multiple time points. Significant immunoprotection at these early time points suggests that the DNA vaccine elicited antigenspecific cellular immunity or non-specific anti-viral factors that were responsible for conferring protection at early stages of viral infection [35–39]. There is little known about the role of cellular immunity in protection against fish rhabdovirus infections due to the present lack of lymphocyte subpopulation markers. However, up-regulation of MHC class II gene expression has been observed in trout 7 d after vaccination with a VHSV G gene DNA vaccine [11]. This suggests that T-cell activation could be involved in the response to a DNA vaccine in fish. The timing allows the possibility of involvement in the early protection mechanism, but no shorter time periods were tested and no conclusions can therefore be drawn without further investigation. Among non-specific immune functions it is well established that fish cells can secrete type I interferon molecules in response to virus infection [40]. Several reports indicate that interferon induction in trout can occur within 48 h after virus infection [41– 43] or exposure to poly I:C [44]. In response to DNA vaccination, Boudinot et al. [11] observed the interferon inducible Mx gene mRNA within 7 d, which was the earliest time point tested. This suggests that an interferon related non-specific mechanism(s) may be active through the time period defined here as early protection. The nature of the component(s) involved in eliciting the early protection mechanism is of great interest. With the VHSV G gene DNA vaccine, the G gene mRNA and G protein were both detected as early as 7 d post-vaccination, the earliest time point tested [11]. Studies with luciferase marker gene constructs have shown that foreign genes in DNA vaccines are expressed within one day after intramuscular injection into trout [45,46]. Therefore it is likely that the IHNV G protein is actively expressed before the onset of the early protection mechanism at 4 d post-vaccination. It has been reported that vaccination of rainbow trout with an attenuated strain of IHNV was not protective against challenge at 1 d post-vaccination, but did produce significant protection as early as 7 d post-vaccination [47]. Similarly, we have observed protection against IHNV challenge as early as 4 d after intramuscular vaccination with an attenuated IHNV vaccine strain (LaPatra et al., unpublished). This suggests that
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the early protection described in this report may not be unique to DNA vaccination, but is a feature of the natural response to endogenous expression of the viral G protein by somatic cells in fish. In our study of the specificity of protection, the transient nature of the cross-protection provided by the VHSV vaccine indicated a difference between the mechanism of protection acting at early and later time points post-vaccination. Therefore, we hypothesize the existence of at least two temporally distinct protective mechanisms that are initiated by DNA vaccination of rainbow trout. At time points beginning 4 d after vaccination there is an early protective mechanism(s) characterized by rapid onset and low specificity. The consistent protection after 4 d and the cross-protection results suggest that this mechanism functions for at least 14 d. By 3–4 weeks post-vaccination protection is likely mediated at least to a large extent by more specific adaptive immune response components presumably including both antibodies and cellular factors. It has been shown that the neutralizing antibody response to either virus infection or DNA vaccination in trout is highly specific, with no cross-neutralizing activity between sera from fish infected with IHNV and VHSV [19] or injected with IHNV and VHSV G gene DNA vaccines [11]. It is possible that in our experiment the loss of the cross-protection against IHNV challenge at the 28 d time point reflects the down-regulation of the non-specific early protection mechanism when the more specific humoral response becomes active. The failure of the rabies virus DNA vaccine to stimulate a protective response suggests that unique feature(s) of the host and/or the poikilothermic rhabdovirus G proteins may be responsible for the early and semi-specific protection observed. Although the IHNV and VHSV G proteins have only approximately 38% amino acid identity (54% similarity), phylogenetic analyses clearly place IHNV and VHSV together in the novirhabdovirus genus, separate from the lyssavirus genus which contains rabies virus [48,49]. In addition, the folded tertiary structures of the IHNV and VHSV G proteins are predicted to be more similar to each other than they are to rabies G protein [50,51], and although there is no cross-neutralizing activity, there is some cross-reaction between IHNV and VHSV G proteins in western blot analyses [52]. Whether the inactivity of the rabies virus vaccine was due to the G gene being phylogenetically distant, or to other factors such as an adverse effect of the low temperature of fish hosts on the rabies G protein tertiary structure, is not known. However, the expression of the rabies G protein from the pRV-G DNA vaccine transfected into fish cells at both 15 and 28°C was confirmed by immunoflourescence with a monoclonal antibody. It is also possible that features such as different numbers of CpG motifs, or the pCMV4 vector backbone in the
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pRV-G plasmid influenced the results, but in previous studies an IHNV G gene DNA vaccine constructed with the pCMV4 vector elicited the same strong protection as the IHNV DNA vaccine used here [4]. The specificity experiments described here are particularly relevant in comparison to an interesting study recently reported by Kim et al. [39]. In this work rainbow trout fry that had received DNA vaccines containing the G genes of three different fish rhabdovirus species, IHNV, snakehead rhabdovirus (SHRV), and spring viremia of carp virus (SVCV), were all protected against IHNV challenge 30 d post-vaccination. By 70 d post-vaccination the cross-protection by the SHRV and SVCV vaccines was no longer evident, and only the IHNV DNA vaccine provided protection. These results are very similar to ours with the exception that the timing of the two phases referred to as ‘‘early’’ and ‘‘late’’ protection is dramatically different, such that the 28 d time point that we consider late protection is equivalent to the early protection time point for Kim et al. [39]. The major difference between the two studies is that the heterologous DNA vaccines contained G genes of different rhabdoviruses, although other differences, such as rainbow trout and challenge virus strain differences may also affect the kinetics of cross-protection. The study by Kim et al. [39] also used a larger vaccine dose of 10 ug DNA per fish which may prolong the non-specific response. Our experiments here suggest that vaccine doses of 0.1 to 2.5 mg did not have a significant effect on the cross-protection phenomenon. However, additional studies that use higher doses of the vaccines are required before any conclusion can be made on the dose response relationship. Despite differences in timing, the combined results of these two studies show evidence of cross-protection against IHNV by the G genes of three different heterologous fish rhabdoviruses from two different genera (VHSV and SHRV are in the novirhabdovirus genus while SVCV is a vesiculovirus). The lack of cross-protection by the rabies virus DNA vaccine suggests that the cross-protection may be limited to fish rhabdoviruses. In both our study and that of Kim et al. [39] it was not possible to test specificity by challenging IHNV-G gene DNA vaccinated fry with different viruses due to restrictions regarding in vivo infection experiments with viruses exotic to North America. Therefore, the experiments involved vaccinating rainbow trout fry with various DNA vaccines and then testing for protection against in vivo challenge with IHNV. The reciprocal studies have recently been carried out in Denmark with rainbow trout fry that were vaccinated with the same DNA vaccines against VHSV and IHNV used in this report, and then challenged at early and late time points with VHSV. Very similar early and cross-protection results were obtained (Niels Lorenzen, Danish
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Veterinary Laboratory, Aarhus, Denmark, personal communication), confirming the reciprocal ability of IHNV and VHSV G genes to cross-protect against heterologous fish rhabdovirus challenge at early time points. Advantages of DNA vaccines over other types of vaccines have been well described [53]. Our results illustrate the potency of a DNA vaccine against IHNV in rainbow trout and the early protection that is observed in these animals. The feature of early protection after DNA vaccination could be exploited as an effective management tool in a variety of domesticated animal industries including aquaculture. Additionally, these results substantiate that rainbow trout provide an excellent model for understanding host-pathogen interactions and the effect of vaccines in a controlled laboratory setting. Thus, DNA vaccines against IHNV have great potential both as a practical biologic for protection of fish and as an important tool for investigation of the teleost immune system.
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Acknowledgements
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The authors thank Don Lodmell for the generous gift of the rabies DNA vaccine and the rabies G protein monoclonal antibody. We thank Dorothy Chase and Katja Einer-Jensen for performing the transfection and immunofluoresence tests to confirm expression of the rabies DNA vaccine in fish cells. We also thank Rhonda LaPatra for help with creating the figures, and Jim Winton for valuable review of the manuscript. This work was supported in part by USDA/NRICGP award number 97-35204-4735.
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antiserum raised against recombinant rainbow trout (Onchorynchus mykiss) MHC class II beta-chain (MhcOnmy-DAB). Fish Shellfish Immunol 1998;8:231 –43. Shum PP, Azumi K, Zhang S, Kehrer SR, Raison RL, Detrich W, et al. Unexpected B2-microglobulin sequence diversity in individual rainbow trout. Proc Natl Acad Sci 1996;93:2779 – 84. Nakanishi T, Ototake M. The graft-versus-host reaction (GVHR) in the ginbuna crucian carp, Carassius auratus langsdorfii. Dev Comp Immunol 1999;23:15 – 26. Jaso-Friedmann L, Evans DL. Mechanisms of cellular cytotoxic innate resistance in tilapia (Oreochromis nilotica). Dev Comp Immunol 1999;23:27 –35. Vallejo AN, Miller NW, Clem LW. Antigen processing and presentation in teleost immune responses. Annual Rev Fish Dis 1992;2:73– 89. Hansen JD. Characterization of rainbow trout terminal deoxynucleotidyl transferase structure and expression. TdT and RAG1 co-expression define the trout primary lymphoid tissues. Immunogenetics 1997;46:367 –75. Hansen JD, Strassburger P, Du Pasquier L. Conservation of a master hematopoietic switch gene during vertebrate evolution: isolation and characterization of Ikaros from teleost and amphibian species. Eur J Immunol 1997;46:367 –75. Lorensen N, LaPatra SE. Immunity to rhabdoviruses in rainbow trout: the antibody response. Fish Shellfish Immunol 1999;9:345 – 60. Engelking HM, Leong JC. The glycoprotein of infectious hematopoietic necrosis virus elicits neutralizing antibody and protective responses. Virus Res 1989;13:213 –30. LaPatra SE, Turner T, Lauda KA, Jones GR, Walker S. Characterization of the humoral response of rainbow trout to infectious hematopoietic necrosis virus. J Aquat Anim Health 1993;5:165 – 71. LaPatra SE, Lauda KA, Jones GR, Walker S, Shewmaker WD. Development of passive immunotherapy for control of infectious hematopoietic necrosis. Dis Aquat Org 1994;20:1 –6. Lorenzen E, Einer-Jensen K, Martinussen T, LaPatra SE, Lorenzen N. DNA vaccination of rainbow trout against VHS virus: a dose-response and time course study. J Aquat Anim Health 2000;12:167 – 80. Bahloul C, Jacob Y, Tordo N, Perrin P. DNA based immunization for exploring the enlargement of immunological cross-reactivity against lyssaviruses. Vaccine 1997;16:417 –25. Krieg AM, Yi AK, Schorr J, Davis HL. The role of CpG dinucleotides in DNA vaccines. Trends Microbiol 1998;6:23 – 7. Whitton JL, Rodriquez F, Zhang J, Hassett DE. DNA immunization: mechanistic studies. Vaccine 1999;17:1612 –9. Kim CH, Johnson MC, Drennan JD, Simon BE, Thomann E, Leong JC. DNA vaccines encoding viral glycoproteins induce
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nonspecific immunity and M × protein synthesis in fish. J Virology 2000;74:7048 – 54. Yano T. The nonspecific immune system: humoral defense. In: Iwama G, Nakanishi T, editors. The Fish Immune System. San Diego: Academic Press, 1996:105 – 57. Dorson M, de Kinkelin P. Mortalite´ et production d’interfe´ ron circulant chez la truite arc-en-ciel apre`s infection expe´ rimentale avec le virus d’Egtved: influence de la tempe´ rature. Ann Rech Veter 1974;5:365 – 72. Renault T, Torchy C, de Kinkelin P. Spectrophotometric method for titration of trout interferon, and its application to rainbow trout fry experimentally infected with viral haemorrhagic septicaemia virus. Dis Aquat Org 1991;10:23 – 9. Trobridge GD, Chiou PP, Kim CH, Leong JC. Induction of the Mx protein of rainbow trout Oncorhynchus mykiss in vitro and in vivo with poly I:C dsRNA and infectious hematopoietic necrosis virus. Dis Aquat Org 1997;30:91 – 8. Eaton WD. Antiviral activity in four species of salmonids following exposure to poly(I) – poly(C). Dis Aquat Org 1990;9:193 – 8. Anderson ED, Mourich DV, Leong JC. Gene expression in rainbow trout (Oncorhynchus mykiss) following intramuscular injection of DNA. Mol Mar Biol Biotechnol 1996;5(2):105 –13. Heppell J, Lorenzen N, Armstrong NK, Wu T, Lorenzen E, Einer-Jensen K, et al. Development of DNA vaccines for fish: vector design, intramuscular injection and antigen processing using viral hemorrhagic genes as a model. Fish Shellfish Immunol 1998;8:271 – 86. Roberti KA, Rohovec JS, Winton JR. Vaccination of rainbow trout against infectious hematopoietic necrosis (IHN) by using attenuated mutants selected by neutralizing monoclonal antibodies. J Aquat Anim Health 1998;10:328 – 37. Morzunov SP, Winton JR, Nichol ST. The complete genome structure and phylogenetic relationship of infectious hematopoietic necrosis virus. Virus Res 1995;38:175 – 92. Bjorklund HV, Higman KH, Kurath G. The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp and hirame rhabdovirus:analysis of relationships with other rhabdoviruses. Virus Res 1996;42:65 – 80. Einer-Jensen K, Krogh TN, Roepstorff P, Lorenzen N. Characterization of the intramolecular disulfide bonds and secondary modifications of the glycoprotein from viral hemorrhagic septicemia virus, a fish rhabdovirus. J Virol 1998;72:10189 –96. Walker PJ, Kongsuwan K. Deduced structural model for animal rhabdovirus glycoproteins. J Gen Virol 1999;80:1211 – 20. Nishizawa T, Yoshimizu M, Winton J, Ahne W, Kimura T. Characterization of structural proteins of hirame rhabdovirus, HRV. Dis Aquat Org 1991;10:167 – 72. Lai WC, Bennett M. DNA vaccines. Crit Rev Immunol 1998;18:449 – 84.
Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination Scott E. LaPatra a,*, Serge Corbeil b,1, Gerald R. Jones a, William D. Shewmaker a, Niels Lorenzen c, Eric D. Anderson d, Gael Kurath a a Research Di6ision, Clear Springs Foods Inc., PO Box 712, Buhl, ID 83316, USA USGS Western Fisheries Research Center, 6505 NE 65th Street, Seattle, WA 98115, USA c Danish Veterinary Laboratory, Hango6ej 2, 8200 Arhus N, Denmark d Department of Biochemistry, Microbiology, and Molecular Biology, Uni6ersity of Maine, Orono, ME 04469, USA b
Received 12 October 2000; received in revised form 6 March 2001; accepted 14 March 2001
Abstract A DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was shown to provide significant protection as soon as 4 d after intramuscular vaccination in 2 g rainbow trout (Oncorhynchus mykiss) held at 15°C. Nearly complete protection was also observed at later time points (7, 14, and 28 d) using a standardized waterborne challenge model. In a test of the specificity of this early protection, immunization of rainbow trout with a DNA vaccine against another fish rhabdovirus, viral hemorrhagic septicemia virus, provided a significant level of cross-protection against IHNV challenge for a transient period of time, whereas a rabies virus DNA vaccine was not protective. This indication of distinct early and late protective mechanisms was not dependent on DNA vaccine doses from 0.1 to 2.5 mg. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: DNA vaccine kinetics; Fish DNA vaccine specificity; Infectious hematopoietic necrosis virus (IHNV); Viral hemorrhagic septicemia virus (VHSV)
1. Introduction Infectious hematopoietic necrosis (IHN) is the most important rhabdoviral disease of salmon and trout in North America. Annual losses estimated at several millions of dollars occur among commercially-reared rainbow trout (Oncorhynchus mykiss) and IHN also affects both wild and cultured Pacific salmon, steelhead (O. mykiss), and Atlantic salmon (Salmo salar) throughout the Pacific Northwest. Depending upon the species of fish, strain of virus, and environmental conditions, epidemics of IHN may result in losses of greater than 90%. The history of IHNV, its chemical, physical, * Corresponding author. Tel.: + 1-208-5433465; fax: +1-2085434146. E-mail address: [email protected] (S.E. LaPatra). 1 Present address: CSIRO, Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, Vic., 3220, Australia.
and serological characteristics, and factors affecting virulence of the virus for salmon and trout have been comprehensively reviewed [1,2]. Presently, avoidance of exposure by destruction of infected stocks and the use of virus-free water supplies and certified eggs are the only control measures for IHN. Vaccination has been attempted for more than two decades, but no licensed efficacious products are yet available [3]. For fish, the first application of DNA vaccine technology was reported by Anderson et al. [4] who used a plasmid containing the glycoprotein (G) gene of IHNV to stimulate a protective immune response in rainbow trout fry. Further work in our laboratories has shown that a DNA vaccine against IHNV reproducibly provides significant protection in rainbow trout against either waterborne or injection challenges, in fish that range in size from 2 to 160 g [5–7]. Significant high levels of protection against
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(01)00113-X
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IHNV were also observed in vaccine efficacy studies in another economically important species, Atlantic salmon [8]. A DNA vaccine containing the G gene of another rhabdoviral pathogen of rainbow trout, viral hemorrhagic septicemia virus (VHSV), has also been shown to provide significant protection when administered alone [9,10] or in combination with a DNA vaccine against IHNV [11]. The potency of DNA vaccination has been experimentally demonstrated in various host-pathogen systems including large mammals and model hosts such as mice. In some cases, significant antibody levels were obtained as soon as 7 d post-immunization [12]. In BALB/c mice challenged with herpes simplex virus, significant protection was reported as early as 14 d after primary vaccination [13]. To our knowledge protection against viral challenge has not been reported at time points less than 14 d post-vaccination in any mammalian host. Here, we report that vaccination of rainbow trout with a DNA vaccine results in immunity at 4 d at 15°C and provides significant protection against a lethal virus challenge. To further explore this observation the specificity of protection was examined and found to be low for a transient period of time.
strain DH5a and plasmid DNA was purified by the alkaline lysis protocol of Saporito-Irwin et al.[18]. Expression of the proteins from the IHNV and VHSV DNA vaccines in fish cells was confirmed by transfection of fish cells as described in the references above. Expression of the rabies virus G protein in fish cells was confirmed by transfection of EPC cells at both 15 and 28°C (data not shown). Detection of the expressed rabies virus G protein was by immunoflourescence using the rabies G protein monoclonal 514, provided by Dr. D. Lodmell. For DNA vaccination, specific-pathogen-free rainbow trout (mean weight, 2 g; Clear Springs Foods, Inc., Buhl, Idaho) were anaesthetized by immersion in 100 mg/ml of tricaine methane sulfonate (MS-222; Argent Chemical Laboratories, Redmond, WA) and injected intramuscularly at the base of the dorsal fin with 1 mg of the specified DNA vaccine in 50 ml of phosphate buffered saline (PBS). For the DNA vaccine dose experiment 0.1, 1.0 and 2.5 mg were injected in 25 ml. Control fish were injected with the plasmid vector alone, PBS alone, or left unhandled as specified in the text. Treatment groups were placed in 19 l aquaria receiving UV-treated, single-pass spring water (15°C), and fed a dry pelleted diet ad libitum.
2. Materials and methods
2.3. Hematopoietic necrosis 6irus challenge of rainbow trout
2.1. Virus propagation and titration The IHNV challenge virus strain 220– 90 was propagated in the epithelioma papulosum cyprini (EPC) cell line [14] as described by LaPatra et al [15]. Virus challenge dose and virus titers in infected fry were determined by plaque assay on confluent EPC cell monolayers [16].
2.2. DNA 6accination of rainbow trout The pIHNw-G, pIHNw-N, and pLuc DNA vaccines, containing the IHNV G gene, IHNV N gene, and the luciferase gene, respectively, have been described in Corbeil et al. [5] and Corbeil et al [6]. The VHSV G gene DNA vaccine, pCMV-vhsG, is described in Lorenzen et al. [9]. The fish vaccines were constructed using the vector plasmids pcDNA3 or pCDNA3.1 for the VHSV G gene and IHNV G gene, respectively. The DNA vaccine construct containing the G gene of the CVS strain of rabies virus, pCMV4CVSG [17], was generously provided by Dr. D. Lodmell (Rocky Mountain Laboratories, NIAID, Hamilton, MT). This vaccine was constructed using the vector plasmid pCMV-4. The VHSV and rabies virus vaccines were denoted as pVHS-G and pRV-G, respectively, in the studies reported herein. All vaccine plasmids and the vector plasmid pCDNA3.1 were amplified in Escherichia coli
Challenges of rainbow trout were performed on duplicate 25 fish groups that were waterborne exposed to 104 or 105 plaque forming units (PFU)/ml of IHNV strain 220–90 for 60 min with aeration in a volume of water that was 10× the total weight of the fish (g). Mock infected control groups were treated identically but were exposed to cell culture media (MEM) only. Experimental groups were held separately in 19 l aquaria receiving UV-treated, single-pass spring water (15°C), fed a dry pelleted diet ad libitum, and monitored for 21 d for mortality. Cumulative percent mortality data was used in the Fisher exact test to test for differences between replicates within treatment and day. When there was no difference between replicates they were pooled and the Chi-square test was used to identify differences among treatments.
3. Results
3.1. Onset of early protection Rainbow trout fry (mean weight, 2 g), in groups of approximately 400 fish each, were injected intramuscularly with 1 mg of the IHNV DNA vaccine (pIHNw-G), or treated as various controls. In the first experiment duplicate groups of 25 fish were removed at 1, 7, 14, 21,
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and 28 d post-vaccination and challenged by immersion in water containing IHNV at a concentration of 104 PFU/ml. Mock-infected fish from each treatment group were handled similarly, but not exposed to virus and were included for each treatment and time interval tested. All groups were monitored for mortalities daily for 21 d and final cumulative percent mortality data is shown in Fig. 1A. All treatment groups that were challenged 1 d after vaccination had high mortality ranging from 62–86%. In fish challenged at 7 d post-vaccination the pIHNw-G groups showed 2% average cumulative mortality compared with 50–58% mortality in the various control groups. Similar significant (P B 0.05) protection in the pIHNw-G vaccinated groups was observed in fish challenged at 14, 21, and 28 d post-immunization. In a second experiment, time points shorter than one week were tested (Fig. 1B). Rainbow trout fry were
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vaccinated with the same treatments and controls as above and challenged at 1, 2, 4, and 7 d post-vaccination. A higher IHNV challenge concentration of 105 PFU/ml was used to provide higher levels of mortality in the control treatments, as a more stringent test of the protection observed. No protection was observed at 1 or 2 d post-vaccination, but by 4 d fish immunized with pIHNw-G were significantly (PB0.05) protected relative to all controls. By 7 d protection was even stronger, with an average mortality of 20% in pIHNw-G vaccinates compared to 85–94% in control groups. In this experiment average cumulative mortalities in all groups were higher than in the first experiment due to the higher dose of challenge virus, but protection in the pIHNw-G vaccinated groups was clearly significant at 4 and 7 d post-vaccination. There were no mortalities in the sham infected control fish. No statistically significant differences in mortality were detected among any
Fig. 1. Cumulative % mortality in rainbow trout O. mykiss (mean weight, 2 g) after lethal virus challenge at different time intervals after injection intramuscularly at the base of the dorsal fin with 1 mg of DNA. The DNA vaccines contained either the infectious (IHNV) G gene (pIHNw-G), IHNV N gene (pIHNw-N), or the firefly luciferase gene (pLuc); two additional groups were either injected with phosphate buffered saline (buffer) or were not injected (unhandled). Duplicate groups of 25 fish were challenged by waterborne exposure to 104 IHNV PFU/ml at 1, 7, 14, 21 and 28 d (A) or 105 IHNV PFU/ml at 1, 2, 4, and 7 d (B) post-vaccination. Asterisks * indicate a significant (P B0.05) difference from controls.
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Fig. 2. Cumulative % mortality in rainbow trout (mean weight, 2 g) after lethal virus challenge at different time intervals after injection with DNA vaccines that contained a different rhabdoviral glycoprotein (G) gene or the plasmid pCDNA 3.1 (vector). DNA vaccines contained the G gene of three antigenically distinct rhabdoviruses including IHNV (pIHNw-G), viral hemorrhagic septicemia virus (pVHS-G, also called pCMV-vhsG [9]), and rabies virus (pRV-G, also called pCMV4CVSG [17]). Duplicate groups of 25 fish were challenged by waterborne exposure to 104 IHNV PFU/ml at 4, 7, 14, and 28 d (A) or 1, 2, 4, and 7 d (B) post-vaccination. Asterisks indicate a significant (PB 0.05) difference from controls.
of the replicates within any of the treatment groups in either study, demonstrating the high degree of reproducibility of this challenge model (data not shown).
3.2. Specificity of protection The specificity of this early immunity was investigated using DNA vaccines containing the G genes of two other rhabdovirus species. The vaccine denoted as pVHS-G protects trout against a major European pathogen, VHSV [9]. VHSV is in the same genus as IHNV, but experiments with trout sera show no serological cross-neutralization activity between these viruses [19]. A DNA vaccine to the CVS strain of rabies virus, denoted here as pRV-G, was included as a vaccine for a mammalian rhabdovirus from a different genus [17]. Rainbow trout fry were injected intramuscularly with 1 mg of pIHNw-G, pVHS-G, pRV-G, or pCDNA3.1
vector alone, or left unhandled. At 4, 7, 14, and 28 d post-vaccination duplicate groups of 25 fish were removed from each treatment and challenged by immersion in 104 PFU/ml of IHNV. Mortalities were monitored daily and the results indicated that significant cross-protection against IHNV challenge could be obtained with the VHSV G gene vaccine for a transient time period (Fig. 2A). Groups of fish vaccinated with the IHNV DNA vaccine showed significant protection (PB 0.05) at all time points, with average cumulative mortalities ranging 6–18% in comparison to 54–90% in control groups injected with vector alone or left unhandled. Fish vaccinated with the VHSV DNA vaccine showed significant protection (PB 0.05) ranging 6–20% CPM at 4, 7, and 14 d post-vaccination, but at 28 d the average mortality of 69% was as high as in the unhandled control groups. This loss of cross-protection by the VHSV vaccine at later time points was observed in two independent experiments (data not shown). Fish vacci-
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nated with the rabies DNA vaccine had mortalities comparable to negative control fish at all time points. A specificity experiment with shorter time points was carried out to assess the timing of the onset of crossprotection (Fig. 2B). Treatment and control groups were prepared as above and challenged at 1, 2, 4, and 7 d post-vaccination using an IHNV immersion challenge dose of 104 PFU/ml. There was no significant protection in any groups when challenged 1 d post-vaccination. At 2 d the pIHNw-G vaccinated groups showed significant protection relative to all other groups, and by 4 and 7 d both the IHNV and VHSV vaccine treatment groups were significantly protected (PB 0.05), with 0–10% mortality compared to 34– 63% in the control groups. As in the first specificity experiment, the rabies vaccine showed no protection at any time point tested.
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3.3. Effect of DNA 6accine dose The possible effect of vaccine dose on short and long duration immunity was investigated using IHNV challenges at 4 and 28 d post-vaccination to represent early and late protection, respectively. The IHNV, VHSV, and rabies DNA vaccines and the pCDNA3.1 vector were each administered to groups of rainbow trout fry at doses of 0.1, 1.0, and 2.5 mg per fish. At 4 and 28 d post-vaccination duplicate groups of 25 fish from each treatment were challenged with IHNV and the cumulative mortalities that occurred over 21 d are shown in Fig. 3. At the 4 d time point the IHNV and VHSV vaccine groups at all doses had overall lower mortalities (4–18%) than the rabies vaccine (20–38%) and vector control groups (20–45%). At 28 d post-vaccination only the IHNV vaccine groups were significantly protected at all doses relative to controls. Although the
Fig. 3. Cumulative % mortality in rainbow trout (mean weight, 2 g) after lethal virus challenge at different time intervals after injection with 0.1, 1.0 or 2.5 mg doses of DNA vaccines that contained a different rhabdoviral glycoprotein (G) gene or the plasmid pCDNA 3.1 (vector) as described in Fig. 2. Duplicate groups of 25-fish were challenged by waterborne exposure to 104 IHNV PFU/ml at 4 d (A) or 28 d (B) post-vaccination. Asterisks indicate a significant (PB 0.05) difference from controls.
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overall level of mortality in the control groups was lower in this experiment than in the previous challenges, it was evident that the levels of immunity elicited by vaccine doses of 0.1 to 2.5 mg were not significantly different.
3.4. Hematopoietic necrosis 6irus challenge of rainbow trout In all experiments a minimum of 20% of the fish that died on any day were processed for virus isolation and quantitation by plaque assay (described above) to confirm infection with IHNV. Among the fish that were challenged and died in the experiments shown in Fig. 1 87% (565/651) were IHNV-positive with a mean concentration of 107.0 PFU/g (range 102.0 to \107.3 PFU/ g). For the experiments in Figs. 2 and 3, 95% (465/489) and 86% (234/271) of the fish that were challenged and died were IHNV-positive, with mean concentrations of 106.8 PFU/g (range 102 to \ 107.3 PFU/g) and 106.7 PFU/g (range 102.3 to \107.3 PFU/g), respectively. There was no detectable virus in any of the small number of mock-infected control fish that died in these experiments.
4. Discussion Immunity to rhabdoviruses is generally believed to be mediated through both humoral and cellular mechanisms [20,21]. Although fish immunology is a young science relative to mammalian immunology, it is known that the humoral and cellular responses of fish share basic features in form and function with those of mammals [22–30]. Aspects of immunity against IHNV in rainbow trout, including nonspecific as well as specific mechanisms, have been reviewed recently [2,31]. It has been demonstrated that neutralizing and protective antibodies can be induced by immunization with the viral G protein [32]. However, it is also known that adaptive acquired immune responses of an aquatic poikilotherm are directly influenced by the temperature of the environment. A humoral immune response is generally not detectable until 3– 4 weeks post-exposure to IHNV in 2 g rainbow trout at 15°C [33]. Similar kinetics of neutralizing antibody production have been reported following DNA vaccination of larger trout with both IHNV and VHSV G gene vaccines [11]. Thus, although neutralizing antibody against IHNV is highly protective [34,11], the potential for this adaptive acquired mechanism to function at the early protection time points described here would seem to be negligible. Our observation of the onset of protection against IHNV 4 d after DNA vaccination is the earliest protection time point thus far reported for DNA vaccines.
Similarly, Lorenzen et al. [35] recently showed protection against VHSV 8 d after vaccination of rainbow trout fry with a DNA vaccine containing the G gene of VHSV, suggesting that early protection may be a common feature of DNA vaccines against fish rhabdoviruses. It is also possible that early protection may occur with DNA vaccination in mammalian hosts, but studies describing the onset of protection are lacking, likely due to difficulties with using the large numbers of animals needed to test multiple time points. Significant immunoprotection at these early time points suggests that the DNA vaccine elicited antigenspecific cellular immunity or non-specific anti-viral factors that were responsible for conferring protection at early stages of viral infection [35–39]. There is little known about the role of cellular immunity in protection against fish rhabdovirus infections due to the present lack of lymphocyte subpopulation markers. However, up-regulation of MHC class II gene expression has been observed in trout 7 d after vaccination with a VHSV G gene DNA vaccine [11]. This suggests that T-cell activation could be involved in the response to a DNA vaccine in fish. The timing allows the possibility of involvement in the early protection mechanism, but no shorter time periods were tested and no conclusions can therefore be drawn without further investigation. Among non-specific immune functions it is well established that fish cells can secrete type I interferon molecules in response to virus infection [40]. Several reports indicate that interferon induction in trout can occur within 48 h after virus infection [41– 43] or exposure to poly I:C [44]. In response to DNA vaccination, Boudinot et al. [11] observed the interferon inducible Mx gene mRNA within 7 d, which was the earliest time point tested. This suggests that an interferon related non-specific mechanism(s) may be active through the time period defined here as early protection. The nature of the component(s) involved in eliciting the early protection mechanism is of great interest. With the VHSV G gene DNA vaccine, the G gene mRNA and G protein were both detected as early as 7 d post-vaccination, the earliest time point tested [11]. Studies with luciferase marker gene constructs have shown that foreign genes in DNA vaccines are expressed within one day after intramuscular injection into trout [45,46]. Therefore it is likely that the IHNV G protein is actively expressed before the onset of the early protection mechanism at 4 d post-vaccination. It has been reported that vaccination of rainbow trout with an attenuated strain of IHNV was not protective against challenge at 1 d post-vaccination, but did produce significant protection as early as 7 d post-vaccination [47]. Similarly, we have observed protection against IHNV challenge as early as 4 d after intramuscular vaccination with an attenuated IHNV vaccine strain (LaPatra et al., unpublished). This suggests that
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the early protection described in this report may not be unique to DNA vaccination, but is a feature of the natural response to endogenous expression of the viral G protein by somatic cells in fish. In our study of the specificity of protection, the transient nature of the cross-protection provided by the VHSV vaccine indicated a difference between the mechanism of protection acting at early and later time points post-vaccination. Therefore, we hypothesize the existence of at least two temporally distinct protective mechanisms that are initiated by DNA vaccination of rainbow trout. At time points beginning 4 d after vaccination there is an early protective mechanism(s) characterized by rapid onset and low specificity. The consistent protection after 4 d and the cross-protection results suggest that this mechanism functions for at least 14 d. By 3–4 weeks post-vaccination protection is likely mediated at least to a large extent by more specific adaptive immune response components presumably including both antibodies and cellular factors. It has been shown that the neutralizing antibody response to either virus infection or DNA vaccination in trout is highly specific, with no cross-neutralizing activity between sera from fish infected with IHNV and VHSV [19] or injected with IHNV and VHSV G gene DNA vaccines [11]. It is possible that in our experiment the loss of the cross-protection against IHNV challenge at the 28 d time point reflects the down-regulation of the non-specific early protection mechanism when the more specific humoral response becomes active. The failure of the rabies virus DNA vaccine to stimulate a protective response suggests that unique feature(s) of the host and/or the poikilothermic rhabdovirus G proteins may be responsible for the early and semi-specific protection observed. Although the IHNV and VHSV G proteins have only approximately 38% amino acid identity (54% similarity), phylogenetic analyses clearly place IHNV and VHSV together in the novirhabdovirus genus, separate from the lyssavirus genus which contains rabies virus [48,49]. In addition, the folded tertiary structures of the IHNV and VHSV G proteins are predicted to be more similar to each other than they are to rabies G protein [50,51], and although there is no cross-neutralizing activity, there is some cross-reaction between IHNV and VHSV G proteins in western blot analyses [52]. Whether the inactivity of the rabies virus vaccine was due to the G gene being phylogenetically distant, or to other factors such as an adverse effect of the low temperature of fish hosts on the rabies G protein tertiary structure, is not known. However, the expression of the rabies G protein from the pRV-G DNA vaccine transfected into fish cells at both 15 and 28°C was confirmed by immunoflourescence with a monoclonal antibody. It is also possible that features such as different numbers of CpG motifs, or the pCMV4 vector backbone in the
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pRV-G plasmid influenced the results, but in previous studies an IHNV G gene DNA vaccine constructed with the pCMV4 vector elicited the same strong protection as the IHNV DNA vaccine used here [4]. The specificity experiments described here are particularly relevant in comparison to an interesting study recently reported by Kim et al. [39]. In this work rainbow trout fry that had received DNA vaccines containing the G genes of three different fish rhabdovirus species, IHNV, snakehead rhabdovirus (SHRV), and spring viremia of carp virus (SVCV), were all protected against IHNV challenge 30 d post-vaccination. By 70 d post-vaccination the cross-protection by the SHRV and SVCV vaccines was no longer evident, and only the IHNV DNA vaccine provided protection. These results are very similar to ours with the exception that the timing of the two phases referred to as ‘‘early’’ and ‘‘late’’ protection is dramatically different, such that the 28 d time point that we consider late protection is equivalent to the early protection time point for Kim et al. [39]. The major difference between the two studies is that the heterologous DNA vaccines contained G genes of different rhabdoviruses, although other differences, such as rainbow trout and challenge virus strain differences may also affect the kinetics of cross-protection. The study by Kim et al. [39] also used a larger vaccine dose of 10 ug DNA per fish which may prolong the non-specific response. Our experiments here suggest that vaccine doses of 0.1 to 2.5 mg did not have a significant effect on the cross-protection phenomenon. However, additional studies that use higher doses of the vaccines are required before any conclusion can be made on the dose response relationship. Despite differences in timing, the combined results of these two studies show evidence of cross-protection against IHNV by the G genes of three different heterologous fish rhabdoviruses from two different genera (VHSV and SHRV are in the novirhabdovirus genus while SVCV is a vesiculovirus). The lack of cross-protection by the rabies virus DNA vaccine suggests that the cross-protection may be limited to fish rhabdoviruses. In both our study and that of Kim et al. [39] it was not possible to test specificity by challenging IHNV-G gene DNA vaccinated fry with different viruses due to restrictions regarding in vivo infection experiments with viruses exotic to North America. Therefore, the experiments involved vaccinating rainbow trout fry with various DNA vaccines and then testing for protection against in vivo challenge with IHNV. The reciprocal studies have recently been carried out in Denmark with rainbow trout fry that were vaccinated with the same DNA vaccines against VHSV and IHNV used in this report, and then challenged at early and late time points with VHSV. Very similar early and cross-protection results were obtained (Niels Lorenzen, Danish
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Veterinary Laboratory, Aarhus, Denmark, personal communication), confirming the reciprocal ability of IHNV and VHSV G genes to cross-protect against heterologous fish rhabdovirus challenge at early time points. Advantages of DNA vaccines over other types of vaccines have been well described [53]. Our results illustrate the potency of a DNA vaccine against IHNV in rainbow trout and the early protection that is observed in these animals. The feature of early protection after DNA vaccination could be exploited as an effective management tool in a variety of domesticated animal industries including aquaculture. Additionally, these results substantiate that rainbow trout provide an excellent model for understanding host-pathogen interactions and the effect of vaccines in a controlled laboratory setting. Thus, DNA vaccines against IHNV have great potential both as a practical biologic for protection of fish and as an important tool for investigation of the teleost immune system.
[12]
Acknowledgements
[13]
The authors thank Don Lodmell for the generous gift of the rabies DNA vaccine and the rabies G protein monoclonal antibody. We thank Dorothy Chase and Katja Einer-Jensen for performing the transfection and immunofluoresence tests to confirm expression of the rabies DNA vaccine in fish cells. We also thank Rhonda LaPatra for help with creating the figures, and Jim Winton for valuable review of the manuscript. This work was supported in part by USDA/NRICGP award number 97-35204-4735.
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