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The Dose-Dependent Effect on Protection andHumoral Response to a DNA Vaccine against InfectiousHematopoietic Necrosis (IHN) Virus in SubyearlingRainbow TroutScott E. LaPatra a , Serge Corbeil b , Gerald R. Jones c , William D. Shewmaker c & GaelKurath da Clear Springs Foods, Inc., Research Division, Post Office Box 712, Buhl, Idaho, 83316, USAb Pathobiology Department, University of Washington, Box 357238, Seattle, Washington,98195-7238, USAc Clear Springs Foods, Inc., Research Division, Post Office Box 712Buhl, Idaho, 83316, USAd Western Fisheries Research Center, 6505 Northeast 65th Street, Seattle, Washington,98115-5016, USAPublished online: 09 Jan 2011.

To cite this article: Scott E. LaPatra , Serge Corbeil , Gerald R. Jones , William D. Shewmaker & Gael Kurath (2000): TheDose-Dependent Effect on Protection and Humoral Response to a DNA Vaccine against Infectious Hematopoietic Necrosis (IHN)Virus in Subyearling Rainbow Trout, Journal of Aquatic Animal Health, 12:3, 181-188

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Journal of Aquatic Animal Health 12:181–188, 2000q Copyright by the American Fisheries Society 2000

FEATUREThe Dose-Dependent Effect on Protection andHumoral Response to a DNA Vaccine against

Infectious Hematopoietic Necrosis (IHN) Virus inSubyearling Rainbow Trout

SCOTT E. LAPATRA*Clear Springs Foods, Inc., Research Division,Post Office Box 712, Buhl, Idaho 83316, USA

SERGE CORBEIL1

Pathobiology Department, University of Washington,Box 357238, Seattle, Washington 98195-7238, USA

GERALD R. JONES AND WILLIAM D. SHEWMAKER

Clear Springs Foods, Inc., Research Division,Post Office Box 712, Buhl, Idaho 83316, USA

GAEL KURATH

Western Fisheries Research Center,6505 Northeast 65th Street, Seattle, Washington 98115-5016, USA

Abstract.—A dose–response study that used the DNA vaccine pIHNw-G against infectious he-matopoietic necrosis virus (IHNV) showed that complete and highly significant (P , 0.001)protection against a virus injection challenge can be attained in subyearling rainbow trout On-corhynchus mykiss (145–160 g, 8- to 10-months-old) 6 weeks after a single intramuscular injectionwith doses as low as 1 mg. Complete protection was also reproducibly demonstrated at highervaccine doses; however, no protection was observed with a 0.1-mg dose. Virus-neutralizing an-tibody titers were detected in fish that had been vaccinated with different doses of the DNA vaccineand then sham-infected; there appeared to be a dose-dependent effect, with higher titers obtainedwith higher doses of vaccine. The DNA-vaccinated animals that survived virus challenge hadsignificantly (P , 0.05) higher neutralizing antibody titers than sham-infected, DNA-vaccinatedcontrol fish. Additionally, the titers detected in the IHN survivors exhibited a significant (P ,0.05) dose-dependent effect, with the highest titers being present in fish that received the highestvaccine doses.

Infectious hematopoietic necrosis (IHN) is themost economically important viral disease of sal-monids in North America. Originally endemic tothe western coast of North America, the causativerhabdovirus (IHNV) has been detected in Asia andEurope and has become widely established amongpopulations of rainbow trout Oncorhynchus my-kiss. The history of IHNV, its chemical, physical,and serological characteristics, and factors affect-ing the virulence of the virus to salmonids havebeen comprehensively reviewed (LaPatra 1998;

* Corresponding author: scottl@clearsprings.com1 Present address: CSIRO Australian Animal Health

Laboratory, Post Office Bag 24, Geelong, Victoria 3220,Australia.

Received May 2, 2000; accepted May 9, 2000

Wolf 1988). Formerly classified in the Lyssavirusgenus of the family Rhabdoviridae, IHNV is nowthe type species of the newly recognized aquaticrhabdovirus genus Novirhabdovirus (Walker et al.,in press).

Vaccination has been attempted as a strategy toprevent IHN disease for more than two decades,but no efficacious vaccines are yet available (Win-ton 1997). For fish, the first application of DNAvaccine technology was reported by Anderson etal. (1996), who used a plasmid containing the gly-coprotein (G) gene of the RB1 strain of IHNV (Hsuet al. 1986) to stimulate a protective immune re-sponse in small rainbow trout (mean weight 1 g).Fish injected with 10 mg of the G-encoding plas-mid pCMV4-G exhibited glycoprotein-specificand virus-neutralizing antibody responses. The

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182 LAPATRA ET AL.

vaccinated fish were also protected from subse-quent challenge. Additional studies with this vac-cine construct were recently reported in anothereconomically important species, Atlantic salmonSalmo salar, by Traxler et al. (1999). Presmoltsand smolts (mean weight 57–73 g) were injectedintramuscularly with 25 mg of pCMV4-G, and 8weeks postvaccination, the fish were challengedby immersion and cohabitation procedures andfound to be significantly protected. Fish injectedwith the DNA vaccine exhibited low levels of neu-tralizing antibodies before being challenged; how-ever, neutralizing titers increased in all groups af-ter exposure to the virus.

Further work in our laboratories has resulted ina series of DNA vaccines containing genes fromthe WRAC strain of IHNV (Corbeil et al. 1999).The protective immunogenicity of the WRAC Ggene along with the nucleoprotein (N), phospho-protein (P), matrix protein (M), and nonvirion pro-tein (NV) genes were assessed in rainbow troutfry. The results indicated that neither the internalstructural proteins (N, P, and M) nor the NV pro-tein of IHNV induced protective immunity or neu-tralizing antibodies in rainbow trout (mean weight2 g). However, the G-encoding plasmid pIHNw-Gdid confer nearly complete and significant (P ,0.01) protection in fry up to 80 d postvaccination.In agreement with earlier studies (Corbeil et al.1999), this study showed low titers and preva-lences of antibody-positive fish before challenge.In a minimal-dose study in rainbow trout (meanweight 0.8–1.8 g), doses of this vaccine as low as1–10 ng have been shown to provide significant(P , 0.05) protection, and the optimal dose of 100ng protected fish against a panel of geographicallydiverse strains of IHNV (Corbeil et al. 2000). Anadditional study of delivery routes for the pIHNw-G vaccine showed that intramuscular injection andintradermal gene-gun vaccination are both highlyefficacious methods of delivery, while intraperi-toneal injection provided partial protection in 1-grainbow trout (Corbeil et al. 2000). As in previousstudies, significant (P , 0.01) protection againstIHNV was observed in fish injected with pIHNw-G in groups that had low titers and prevalences ofantibody-positive fish.

The objective of this study was to evaluate thedose–response characteristics of the pIHNw-Gvaccine in subyearling rainbow trout (110–120 g,8–10-months-old) 6 weeks after a single intra-muscular injection. To satisfy this objective, anIHNV injection challenge model was developed.The humoral response in sham-infected and IHN

survivors that were vaccinated with different dosesof pIHNw-G was also evaluated.

Methods

Virus and cell culture.—An epithelioma papu-losum cyprini (EPC) cell line (Fijan et al. 1983)and a cell line from embryonic chinook salmonOncorhynchus tshawytscha (CHSE-214; Lannan etal. 1984) were used for the propagation and iso-lation of IHNV. Cells were maintained at 15–258Cin minimum essential medium (MEM; Gibco BRL,Grand Island, New York) supplemented with 10%fetal bovine serum (Hyclone, Logan, Utah) and 2mM L-glutamine (Gibco BRL). The WRAC strainof IHNV, also designated 039-82, and isolate220-90 (LaPatra et al. 1994) were used to producestock virus. Virus used in the fish injection chal-lenge or isolated from dead fish was quantified byplaque assay procedures previously described(LaPatra et al. 1989).

DNA plasmid constructs.—The DNA prepara-tions contained either the IHNV G gene or the fireflyluciferase gene, each of which had been cloned intothe pCDNA3.1 vector plasmid (Invitrogen, Carls-bad, California) downstream of the cytomegalovi-rus immediate-early enhancer–promoter sequence(Corbeil et al. 1999). The vaccine construct con-taining the G gene of the WRAC strain of IHNVwas designated pIHNw-G. The negative controlDNA construct containing the firefly luciferase genewas designated pCDNA-Luc. Vaccine plasmidswere prepared by means of an alkaline lysis pro-tocol as previously described (Corbeil et al. 2000).

Development of an IHNV injection challenge.—Subyearling rainbow trout (Clear Springs strain)with a mean weight of about 100 g were used toevaluate the virulence of the WRAC strain ofIHNV and isolate 220-90 in an injection challengeprocedure. Groups of 20 fish were anesthetized ina tricaine methanesulfonate solution (100 mg/L)and injected intraperitoneally with 0.1 mL ofMEM that contained approximately 104 or 106

plaque-forming units (PFU) of each isolate. Eachgroup was held in a separate 144-L fiberglassaquarium receiving single-pass, ultraviolet-light-treated spring water at a constant temperature of158C. Fish were monitored for 14 d, and cumu-lative percent mortality was assessed for each iso-late at each challenge concentration.

Rainbow trout vaccination and challenge.—Twostudies were conducted with subyearling specific-pathogen-free rainbow trout (Clear Springs strain)to determine the dose–response of the pIHNw-Gvaccine. In the first study, groups of 25 fish with

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183IHN VIRUS DNA VACCINE

a mean weight of about 120 g were anesthetizedin a tricaine methanesulfonate solution (100 mg/L) and injected immediately posterior to the dorsalfin with 100 mL phosphate buffered saline (PBS)containing 25, 10, or 1 mg of the pIHNw-G con-struct. Additionally, two 13-fish groups were eitherinjected intramuscularly in the same location with100 mL of PBS containing 25 mg of pCDNA-Lucor 100 mL of PBS. In the second study, the sameexperimental design was used except that the meanweight of the fish was about 110 g at vaccinationand the pIHNw-G doses tested were 10.0, 1.0 and0.1 mg. Two 13-fish groups were also included thatwere either injected with 10 mg pCDNA-Luc orPBS only. These fish were differentially fin clippedand combined. Each group was held in a separate144-L fiberglass aquarium receiving single-pass,ultraviolet-light-treated spring water at a constanttemperature of 158C and fed a standard ration(Clear Springs Foods, Inc., Buhl, Idaho) daily. Fishwere held 42 d before challenge. On the day ofthe IHNV challenge, five fish were removed fromeach of the groups vaccinated with pIHNw-G, dif-ferentially fin clipped, intraperitoneally injectedwith 0.1 mL MEM and pooled in one aquarium.Three fish from each of the other groups (pCDNA-Luc and PBS) were handled similarly and also heldin the same aquarium with the pIHNw-G vacci-nated fish injected with MEM. The remaining fishin each treatment group, which included 20 fish ateach dose of pIHNw-G and 10 fish for both thepCDNA-Luc and PBS, were then anesthetized andinjected intraperitoneally with 0.1 mL MEM con-taining approximately 105 to 106 PFU of IHNVchallenge isolate 220-90. All fish were monitoredfor 28 d and fed ad libitum daily. Kidney andspleen specimens from each fish that died weretested by plaque assay, and IHNV concentrationswere determined. Cumulative percent mortality ofthe IHNV challenged fish was analyzed by chi-square analysis to determine if statistically signif-icant differences existed among groups vaccinatedwith different doses of the pIHNw-G or injectedwith pCDNA-Luc or PBS.

Analysis of the humoral response.—Four weekspostchallenge with IHNV (10 weeks postvacci-nation), fish were anesthetized as described pre-viously and bled by caudal puncture, and serumwas collected from IHN survivors. Sham-infectedcontrol fish were also tested. The presence and titerof neutralizing antibodies were determined bymeans of a plaque reduction method (LaPatra etal. 1993). Twofold serial dilutions of each serumsample in Hanks’ balanced salt solution were test-

ed for neutralizing activity by reacting with ap-proximately 80–100 PFU of IHNV isolate 220-90for 1 h at 178C. Complement in unheated serumcollected from specific-pathogen-free rainbowtrout was added to test wells and allowed to in-cubate an additional 1 h at 178C. The antibodytiter was reported as the reciprocal of the highestserum dilution that resulted in a 50% reduction inthe average number of plaques compared with thenegative controls. Neutralization titers detected inDNA-vaccinated animals that were sham-infectedwere compared with titers detected in IHN sur-vivors by means of chi-square analysis. Addition-ally, the titers detected in DNA-vaccinated IHNsurvivors were compared by vaccine dose to de-termine if significant differences existed betweengroups.

Results

Development of an IHNV Injection Challenge

Subyearling rainbow trout (mean weight 100 g)exhibited a mortality pattern that appeared to bedependent on IHNV isolate and virus challengeconcentration. Fish injected with 104 PFU of theWRAC strain of IHNV had no mortality, whereasfish injected with 106 PFU had 5% (1 of 20) mor-tality. Fish injected with 104 or 106 PFU of isolate220-90 had cumulative mortalities of 25% and95%, respectively. Injection challenge with isolate220-90 at a concentration of 106 PFU was selectedfor use in the vaccine dose–response evaluations.

DNA Vaccine Dose–Response

The average weight of the subyearling rainbowtrout 42 d after vaccination was about 160 g in thefirst dose–response study and 145 g in the second.In the first study, complete and highly significant(P , 0.001) protection was attained after a singleintramuscular injection with a dose as low as 1 mgof vaccine (Table 1). Complete protection was alsodemonstrated at higher vaccine concentrations. Inthe second study, one fish that was injected with10 mg of pIHNw-G died before virus challenge,but no virus was detected. Complete and signifi-cant (P , 0.001) protection was also observed inthe second study with the 10.0-mg and 1.0-mgpIHNw-G doses; however, no protective effect wasobserved at the 0.1-mg dose. The injected virusconcentration was 106.40 and 105.06 for the first andsecond challenges, respectively. Overall, 100%(35 of 35) of the fish that died in the challengeswere positive for IHNV, with a mean concentrationof 107.09 PFU/g of kidney and spleen (range: 104.95

to 107.30 PFU/g).

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184 LAPATRA ET AL.

TABLE 1.—Cumulative percent mortality of rainbowtrout injected with different doses of a DNA vaccine con-taining an infectious hematopoietic necrosis (IHN) virusglycoprotein gene (pIHNw-G) compared with that of twocontrol groups, one injected with a firefly luciferase gene(pCDNA-Luc) and one injected with phosphate bufferedsaline (PBS). The mean weights of the fish challenged inthe two experiments were 160 g and 145 g, respectively.Six weeks postvaccination, the fish were injection chal-lenged with the IHN virus and monitored for 28 d. Thenumbers in parentheses are the number of fish that diedrelative to the total number of fish challenged. Differencesin cumulative percent mortality between the vaccine andcontrol groups were tested by means of chi-square analy-sis. Asterisks indicate significant (P , 0.001) differences;ND 5 not done.

Percent mortality ofPercent mortality at vaccine dose control group

25 mg 10 mg 1.0 mg 0.1 mg pCDNA-Luc PBS

Experiment 10*

(0/20)0*

(0/20)0*

(0/20)ND 90

(9/10)90

(9/10)

Experiment 2

ND 0*(0/19)a

0*(0/20)

40(8/20)

40(4/10)

50(5/10)

a One fish died prior to challenge.

Humoral Response Analysis

Virus-neutralizing antibody titers were not de-tected in sham-infected fish that had been injectedwith PBS or pCDNA-Luc. However, sham-infect-ed fish that had been vaccinated with different dos-es of pIHNw-G had such titers, and there appearedto be a dose-dependent effect, with higher titersobtained with increased vaccine dose (Table 2).Challenge survivors were also tested for neutral-izing titers, and pIHNw-G vaccinated animals hadsignificantly (P , 0.05) higher titers than sham-infected, pIHNw-G vaccinated control fish thatwere injected with MEM. Additionally, the titersdetected in the DNA-vaccinated IHN survivors ex-hibited a significant (P , 0.05) dose-dependenteffect in the first study, with the highest neutral-ization titers being present in fish that received thehighest dose of the pIHNw-G vaccine. A similartrend was observed in the second study (Table 2).Fish injected with pCDNA-Luc or PBS that wereexposed to the virus and survived exhibited a rangeof neutralization titers. In the first challenge, onesurviving fish injected with pCDNA-Luc had aneutralization titer of 160 or higher and one in-jected with PBS had a titer of 20. In the secondchallenge, six pCDNA-Luc and five PBS injectedfish survived; 83% (five of six) of the former and

60% (three of five) of the latter had IHNV neu-tralization titers of 160 or higher. The other fishhad neutralization titers of either 40 (1 fish) or 80(2 fish).

Discussion

Rainbow trout provide an excellent model forunderstanding host–pathogen interactions and theeffect of DNA vaccines because these fish have awell-developed immune system that can provideinformation on immunological parameters that areimportant in understanding the mechanisms of pro-tection. Small rainbow trout, typically 1–2 g insize, that are susceptible to waterborne infectionshave been shown to be significantly protectedagainst IHNV with DNA vaccines (Anderson etal. 1996; Corbeil et al. 1999; Corbeil et al. 2000).However, scientific investigations into the mech-anisms of DNA vaccine protection that use a small-fish model system are limited. Tissue and serumsamples generally have to be pooled for immu-nological and biological analyses, which decreasesthe total number of data points obtained and canmask individual responses by pooling the sera ortissues of responders with those of nonresponders.Because a high degree of fish-to-fish variation hasbeen well documented in immunological studiesof fish, pooling samples can lead to inaccurate sta-tistical evaluation of the data. The use of a larger-fish model for basic research investigations canfacilitate immunological and biological assays onindividual fish.

In recent studies with a DNA vaccine in Atlanticsalmon Salmo salar, it was shown that fish thatranged in weight from 57 to 73 g were susceptibleto IHNV waterborne and cohabitation challengesand that significant protection could be attainedwith a 25-mg dose of vaccine. Observations in-volving experimental waterborne infections withIHNV in rainbow trout indicate that fish larger than20 g generally exhibit nonlethal, localized infec-tions in the skin and gills (our unpublished data);however, this can be influenced by the rainbowtrout stock, fish age, and strain of IHNV (LaPatra1998). Previous studies have shown that the in-tegument appears to function as an innate defensemechanism against IHN in larger fish. If the in-tegument is bypassed by direct injection of thevirus, the virulence may be increased 100-fold to1,000-fold (our unpublished data). The results ob-tained in this study regarding the development ofan IHNV injection challenge for subyearling rain-bow trout (;100 g) suggest that a virus dose-dependent mortality pattern can be obtained; the

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185IHN VIRUS DNA VACCINE

TABLE 2.—Virus-neutralizing antibody titers, shown as the percent prevalence at each titer level, in fish vaccinatedwith different doses of the DNA vaccine pIHNw-G and subsequently challenged with a live virus or a sham infection.Values in parentheses are numbers of fish. The mean weights of the fish in the two experiments were 160 g and 145g, respectively. Differences in the titers, both between challenge survivors and control groups and between challengesurvivors that received different doses of the vaccine, were tested by means of chi-square analysis. In experiment 1,the titers of the challenge survivors were significantly (P , 0.05) different from those of the sham-infected controls. Inaddition, the titers of the challenge survivors injected with 25 mg pIHNw-G were highly significantly (P , 0.01) differentfrom those of the challenge survivors injected with 1 mg, and the titers of the challenge survivors injected with 10 mgpIHNw-G were significantly (P , 0.05) different from those of the challenge survivors injected with 1 mg. In experiment2, the titers of the challenge survivors were very highly significantly (P , 0.001) different from those of the sham-infected controls.

Titer

Challenge survivors

Dose 1 Dose 2 Dose 3

Sham-infected controls

Dose 1 Dose 2 Dose 3

Experiment 1a

25mg 10mg 1mg 25mg 10mg 1mg$1,280

640320804020

,20

60 (12)15 (3)5 (1)

15 (3)5 (1)0 (0)0 (0)

35 (7)25 (5)10 (2)10 (2)5 (1)

10 (2)5 (1)

5 (1)5 (1)5 (1)

35 (7)30 (6)15 (3)5 (1)

0 (0)20 (1)20 (1)20 (1)20 (1)20 (1)0 (0)

0 (0)0 (0)

20 (1)20 (1)20 (1)20 (1)20 (1)

0 (0)0 (0)0 (0)0 (0)

20 (1)20 (1)60 (3)

Experiment 2a

10mg 1.0mg 0.1mg 10mg 1.0mg 0.1mg$160

804020

,20

68 (13)16 (3)16 (3)0 (0)0 (0)

50 (10)20 (4)15 (3)10 (2)5 (1)

33 (4)42 (5)17 (2)8 (1)0 (0)

60 (3)20 (1)0 (0)

20 (1)0 (0)

0 (0)20 (1)40 (2)0 (0)

40 (2)

0 (0)0 (0)0 (0)0 (0)

100 (5)

a In experiment 1, N 5 20 for dose groups 1–3 and N 5 5 for control groups. In experiment 2,N 5 19, 20, and 12 for dose groups 1–3, respectively; N 5 5 for control groups.

relative virulence of two particular isolates ap-peared similar to that obtained in immersion chal-lenges of small rainbow trout. In developing theinjection challenge model, the WRAC strain ofIHNV was tested because the pIHNw-G vaccineused in this study is constructed with the G genefrom this strain. However, the WRAC strain wassignificantly less virulent than isolate 220-90, aspreviously reported for waterborne challenges in1–5 g rainbow trout (LaPatra et al. 1994). Thehighly virulent 220-90 strain at a challenge doseof 106 PFU was therefore selected to provide suf-ficient mortality in the injection challenge modeldeveloped here for subyearling rainbow trout. Ad-ditionally, challenge with strain 220-90 confirmedthe results of previous studies that have shown thatthe glycoprotein of one IHNV strain induces pro-tective immunity against heterologous strains (An-derson et al. 1996; Corbeil et al. 1999; Traxler etal. 1999; Corbeil et al. 2000), suggesting that avaccine against a single type of IHNV may beefficacious against all IHNV strains.

In the dose–response study that used the DNAvaccine pIHNw-G, complete and highly significant

protection was attained in subyearling rainbowtrout (145–160 g, 8210-months-old) 6 weeks aftera single intramuscular injection with doses as lowas 1 mg. Complete protection was also reproduc-ibly demonstrated at higher vaccine concentrationsin a standardized injection challenge model; how-ever, no protection was observed with a 0.1-mgdose. In the first challenge, the groups injectedwith pCDNA-Luc and PBS each had 90% cumu-lative mortality, whereas no fish died in the p-IHNw-G vaccinated groups injected with doses of25, 10, and 1 mg. In the second challenge, completeprotection was again observed with the 10-mg and1.0-mg doses. However, mortality in the controlgroups only reached 40%–50%, and the 0.1-mgpIHNw-G group also exhibited a 40% cumulativemortality after injection with IHNV. The differencein the mortality levels between the two challengescould be explained by the 10-fold reduced con-centration of virus injected in the second chal-lenge. However, the results were still very consis-tent and allow definition of the minimal vaccinedose in larger fish.

From an IHN management standpoint, these re-

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sults suggest that this vaccine may be cost-effectivewhere injection vaccination is feasible, such as invaluable stocks of threatened and endangered sal-monids. Corbeil et al. (2000) determined that itwould cost only U.S.$0.01 for reagents to provide asufficient amount of the pIHNw-G plasmid to vac-cinate 50 fry at the optimal dose of 100 ng. Thissuggests that a 1-mg dose of vaccine for a larger fishwould cost about $0.002 cent. A strain of chinooksalmon listed under the Endangered Species Act re-cently suffered losses from IHN at a larger fish size,and using injection vaccination as a preventativestrategy could have been beneficial in this case (S.Onjukka, Oregon Department of Fish and Wildlife,personal communication). The results of this studyalso support the conclusions of Traxler et al. (1999),who showed that an injectable DNA vaccine may beuseful for sea-pen-farmed Atlantic salmon, whereIHN was reported to have caused losses of 1–3 kgfish (Armstrong et al. 1993).

Immunity to rhabdoviruses is generally believedto be mediated through both humoral and cellularmechanisms. Particularly with the rabies virus,neutralizing antibodies are thought to play an im-portant role in preventing the spread of virus par-ticles throughout the body and thus in stemmingthe subsequent progress of the disease (Celis1990). The ability of antibodies to interact withfree virus particles, and thereby to prevent the in-fection from reaching nerve cells, may thus be acritical aspect of this particular neurotropic virus;by contrast, cytotoxic cells interacting with in-fected cells alone are not able to protect miceagainst disease (Lafon 1994). Although fish im-munology is a young science, it is known that thehumoral immune response of fish shares basic fea-tures of form and function with that of mammals.These include the immunoglobulin structure of theprimary tetrameric isotype in fish, cellular com-ponents for B-lymphocyte activation, neutraliza-tion, complement fixation, opsonization, and an-amnestic responses (Kaattari and Piganelli 1996).

Aspects of immunity to IHNV in rainbow trout,including nonspecific as well as specific mecha-nisms, have been reviewed recently (LaPatra 1998;Lorenzen and LaPatra 1999). It has been clearlydemonstrated that the presence of a neutralizingantibody significantly reduces the risk of a lethalIHNV infection. However, a humoral immune re-sponse is generally not detectable until a minimumof 2 weeks after exposure to IHNV in 16-g rainbowtrout at 158C (LaPatra et al. 1993). This suggeststhat for a naive animal, humoral immunity is nota primary defense mechanism but would be im-

portant in preventing reinfection and providinglong-term protection.

In this work, the assay for neutralizing antibodytiters used the same IHNV strain (220-90) that wasemployed in the virus challenge protocol. Thisstrain is heterologous relative to the DNA vaccinepIHNw-G, which contained the G gene of theWRAC strain of IHNV. In analysis of humoralresponse, it is known that the strain of virus usedas a test antigen can affect the sensitivity and endpoint titer determinations. However, only one se-rotype of IHNV exists (Engelking et al. 1991), andprevious studies have shown that IHNV isolate220-90 performs as well as or better than otherisolates in the IHNV plaque reduction test (LaPatra1996). Previous studies have also shown that an-tibodies produced against one strain will neutralizeheterologous isolates in vitro and provide nearlycomplete protection in vivo after passive transfer(LaPatra et al. 1994). Thus, the plaque reductionprotocol, which was designed to correlate with thechallenge protocol, should be a reliable indicatorof the humoral response against IHNV in general.Although, as noted earlier, both the WRAC and220-90 IHNV strains were isolated from the samegeographic location, they differ dramatically intheir virulence for rainbow trout. Sequence com-parison indicates that these two strains differ by3.6% (11 of 303 nucleotides) in the mid-G generegion (which is known to vary among IHNV iso-lates), resulting in four amino acid differences be-ing predicted within this region (Troyer et al., inpress; see Genbank L40883 and AF237983 for themid-G sequences of the WRAC and 220-90 strains,respectively).

Analysis of virus-neutralizing antibody titers inthis study indicated that titers could be detectedin fish that had been vaccinated with different dos-es of the pIHNw-G vaccine and then sham-in-fected, and there appeared to be a dose-dependenteffect, with higher titers being obtained with in-creased vaccine dose. Challenge survivors exhib-ited a significant dose-dependent effect, with thehighest neutralization titers being present in fishthat received the increased pIHNw-G dose. Chal-lenge survivors had significantly higher titers thanpIHNw-G vaccinated, sham-infected control fish.Similar results have been observed in other DNAvaccine model systems, including vaccines againstother rhabdoviruses such as rabies (Bahloul et al.1998). In these studies, it was shown that the DNAvaccine primed an antibody response that was doseand booster dependent. If a similar mechanism oc-curs with pIHNw-G vaccinated fish, it will be in-

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teresting to determine the rate at which serocon-version occurs following virus exposure. Sham-infected fish vaccinated with 1 mg of pIHNw-Gexhibited low seroconversion, but virus-chal-lenged fish that had the same vaccine dose showedsignificant immunoprotection. This could indicatethat the pIHNw-G vaccine elicited antigen-specificcellular immunity or nonspecific antiviral factorsthat were responsible for conferring protection(Boudinot et al. 1998; Kreig et al. 1998; Whittonet al. 1999). Sham-infected fish vaccinated at the0.1-mg dose exhibited no detectable seroconver-sion from vaccination, and those that were vac-cinated at this dose and challenged were not pro-tected against IHN. Sham-infected fish injectedwith PBS and pCDNA-Luc also had no detectableantibody titers, and additional fish from these treat-ments that were injection challenged with IHNVexhibited mortality patterns similar to the 0.1-mgpIHNw-G vaccinated groups. However, 83% (5 of6) of the surviving fish injected with pCDNA-Lucand 60% (3 of 5) of those injected with PBS hadneutralizing antibody titers of 160 or more, com-pared with only 33% (4 of 12) in the 0.1-mg p-IHNw-G survivors. This suggests that antibodypriming is not the primary defense mechanism orthat the 0.1-mg vaccine dose is below the thresholdneeded to elicit this response. However, additionalstudies will be required to substantiate these hy-potheses.

In this study, we were able to determine that thelower dose limit of pIHNw-G vaccine efficacy forrainbow trout weighing approximately 100 g liesbetween 0.1 and 1.0 mg. This is analogous to theresults obtained in 1–2 g rainbow trout, where thelower limit of pIHNw-G efficacy was shown to bebetween 0.001 and 0.01 mg (Corbeil et al. 2000).The ratio of minimal vaccine dose to fish bodyweight is clearly demonstrated by the results ofthese two studies. A 100-fold increase in fishweight increased the minimal vaccine dose re-quired for significant protection by essentially thesame factor. A similar protective-dose:host-weightratio has been reported for several viral DNA vac-cines in mammalian hosts (reviewed in Corbeil etal. 2000), suggesting that the ratio of protectiveDNA-vaccine-dose:host-weight may be relativelyconstant across diverse virus–host systems.

Although significant protection can be obtainedwith doses as low as 1 mg of pIHNw-G in sub-yearling rainbow trout, higher vaccine doses maybe beneficial because they induce higher antibodytiters for long-duration protection against reinfec-tion with IHNV. Our results further illustrate the

potency of a DNA vaccine against IHNV in rain-bow trout and substantiate that rainbow trout pro-vide an excellent model for understanding host–pathogen interactions and the effect of DNA vac-cines. Additionally, a reproducible injection-viruschallenge procedure that uses a large-fish modelwill be valuable in enhancing our understandingof the mechanism of protection and in improvingvaccine efficacy. DNA vaccines against IHNVhave great potential, both as a practical biologicalfor protecting fish and as an important tool forinvestigating the teleost immune system.

Acknowledgments

We thank Scott Williams for the statistical anal-ysis and Elaine Thompson for editorial assistance(both from Clear Springs Foods, Inc.). This workwas supported in part by NRICPG/USDA award97–35204–4753.

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