Vaccination trials with Penaeus japonicus to induce resistance to white spot syndrome virus

Aquaculture 229 (2004) 25 – 35 www.elsevier.com/locate/aqua-online

Vaccination trials with Penaeus japonicus to induce resistance to white spot syndrome virus Atsushi Namikoshi a, Jin Lu Wu a, Takayoshi Yamashita a, Toyohiko Nishizawa b, Toyohiro Nishioka c, Misao Arimoto c, Kiyokuni Muroga a,* a

Laboratory of Fish Pathology, Graduate School of Biosphere Sciences, Hiroshima University, Higashihiroshima 739-8528, Japan b Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan c Kamiura Station of Japan Sea-Farming Association, Kamiura, Oita 879-2602, Japan Received 1 March 2003; received in revised form 23 April 2003; accepted 27 April 2003

Abstract Crustaceans do not possess an adaptive immune response with immunoglobulins; however, recently, ‘‘quasi-immune response’’ has been reported by which kuruma shrimp (Penaeus japonicus) surviving from natural or experimental white spot syndrome virus (WSSV) infections possess a resistance against challenge with WSSV. In this study, efficacy of vaccines made of inactivated WSSV with or without immunostimulants (h-1,3-glucan or killed Vibrio penaeicida) and of recombinant proteins of WSSV (rVP26, rVP28) were tested by intramuscular vaccination followed by intramuscular challenge of kuruma shrimp with WSSV. The shrimp vaccinated with formalininactivated WSSV showed a resistance to the virus on 10th day post-vaccination (dpv) but not on 30th dpv. Heat-inactivated WSSV did not induce a resistance in the shrimp even on 10th dpv. Additional injections with glucan or V. penaeicida enhanced the efficacy of formalin-inactivated WSSV vaccine; however, the relative percent survival (RPS) values did not exceed 60% even when shrimp were vaccinated three times. On the other hand, two injections with rVP26 or rVP28 induced a higher resistance, with RPS values 60% and 95%, respectively, in the shrimp on 30th dpv. These results indicate the possibility of vaccination of kuruma shrimp with recombinant proteins against WSSV. D 2004 Elsevier Science B.V. All rights reserved. Keywords: White spot syndrome virus; Vaccination; Penaeus japonicus; Quasi-immune response; Recombinant vaccine

* Corresponding author. Present address: Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. Tel.: +81-22-717-8724; fax: +81-22-717-8727. E-mail address: [email protected] (K. Muroga). 0044-8486/$ - see front matter D 2004 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00363-6

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1. Introduction Since 1993, the Asian shrimp industry has been hampered by the appearance of white spot syndrome (WSS), which has now been spread to the Americas (Lightner, 1996; Jory and Dixon, 1999). Various studies have been made on the causative virus of WSS (WSSV) including molecular biological analyses (van Hulten et al., 2001a), and WSSV has now been placed in the new genus of the new family: Genus Whispovirus, Family Nimaviridae (http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm). The infection does not occur only in shrimp but also in other marine and freshwater crustaceans, including crabs and crayfish (Lo et al., 1996; Maeda et al., 1998). In shrimp farming system, horizontal transmission of the virus occurs via oral ingestion and the waterborne route (Corsin et al., 2001), and cannibalism has been demonstrated to be important in transmission of the virus in experimental conditions (Soto and Lotz, 2001; Wu et al., 2001). In hatcheries, vertical transmission has been identified as the primary route and a control method by segregation of infected spawners or contaminated eggs has been established in kuruma shrimp (Penaeus japonicus) (Mushiake et al., 1999). Strategies for prophylaxis and control of WSS theoretically include improvement of environmental conditions, stocking of specific pathogen free (SPF) or specific pathogen resistant (SPR) postlarvae, and enhancement of disease resistance by using vaccines and immunostimulants. Protective effects of various immunostimulants have been reported against WSSV infection, for example, oral administration of peptidoglycan or lipopolysaccharide in P. japonicus (Itami et al., 1998; Takahashi et al., 2000), and glucan in Penaeus monodon (Song et al., 1997; Chang et al., 1999). However, crustaceans do not possess an adaptive immune response with immunoglobulins (So¨derha¨ll and Thornqvist, 1997; Lee and So¨derha¨ll, 2002), thus few trials have been made to vaccinate them. Recently an interesting phenomenon, called quasi-immune response, has been reported against WSSV in P. japonicus (Venegas et al., 2000). The resistance against WSSV appeared 3 weeks after the virus exposure and was maintained for about 1 month, which was endorsed by the appearance of virus neutralizing factor(s) in the hemolymph of ‘‘immune’’ kuruma shrimp (Wu et al., 2002a). Our study was carried out to explore the possibility of protecting kuruma shrimp from WSSV infection by vaccination with inactivated WSSV or recombinant proteins of WSSV. As the term ‘‘quasi-immune response’’ is used, the nature of the resistance induced in the shrimp by virus infection or injections with inactivated virus or its recombinant proteins is unknown; nevertheless, the terms ‘‘vaccination’’ and ‘‘vaccines’’ are used in the text by way of comprehensible explanations for the experiments.

2. Materials and methods 2.1. Shrimp WSSV-free P. japonicus, produced and reared in the Kamiura Station of the Japan SeaFarming Association (JASFA), were used in this study. They were kept in tanks with sand beds supplied with sand-filtered, ozone-treated and flow-through seawater at 24 F 0.5 jC,

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and fed commercial crumbled feed at 5% of body weight per day before and during the experiment. 2.2. Virus The viral suspension used in this study was prepared from experimentally WSSVinfected P. japonicus as previously reported (Wu et al., 2002b). Briefly, the hemolymph was withdrawn with a 4-fold volume of phosphate-buffered saline (PBS) from moribund P. japonicus, divided into 0.3 ml aliquots, and stored at 80 jC. It was confirmed that the virulence of the virus in the stored hemolymph materials was stable for at least 16 months (Wu et al., 2002b). Before each challenge test, an aliquot was thawed and centrifuged at 1500  g at 4 jC for 10 min. The supernatant fluid was diluted to the required concentrations and injected intramuscularly (IM) into shrimp. 2.3. Inactivated WSSV vaccines For preparation of formalin-inactivated WSSV, formalin (0.5% v/v) was added to the viral suspension prepared as described above, and the virus was inactivated for 10 min at 25 jC. Formalin was removed by two centrifugations at 30,000  g for 1 h at 4 jC. The pellet was resuspended in PBS to furnish the viral concentration of one tenth of the original viral stock solution, and used as a vaccine. For preparation of a heat-inactivated WSSV vaccine, the viral suspension was diluted 10-fold in PBS and inactivated for 10 min at 60 jC. 2.4. Immunostimulants In order to enhance immune response of vaccinated shrimp, formalin-inactivated Vibrio penaeicida (strain KH-1) and h-1,3-glucan from yeast were parenterally administered to the shrimp. Vibrio penaeicida, the major causative agent of vibriosis of kuruma shrimp in Japan (Ishimaru et al., 1995), cultured on Marine Agar 2216 (Difco) at 25 jC for 24 h, was suspended in PBS at 1 mg (wet weight)/ml with 0.5% formalin and incubated at 25 jC for 30 min. The inactivated bacterial suspension was washed three times with PBS by centrifugation at 1500  g for 5 min at 4 jC to remove formalin and its sterility was confirmed by culture. h-1,3-Glucan extracted from yeast by Taitou (Tokyo) was dissolved in PBS prior to injection into shrimp. 2.5. Recombinant protein vaccines For nucleotide sequence analysis, viral DNA was extracted from naturally WSSVinfected kuruma shrimp and submitted to PCR amplification with two different primer sets, VP26F-VP26R and VP28F-VP28R. These primers were designed based on the nucleotide sequences of structural proteins (VP26, VP28) of WSSV in the GenBank (accession number: AF173992 and AF173993) deposited by van Hulten et al. (2000). The first PCR primer set, VP26F (5V-GTAAAGGAAGAACTTCCATC-3V) and VP26R (5VTATATTTGTACAATTCCCACTTTA-3V), was used for the amplification of the 670 base

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region including an open reading frame (ORF) for VP26 between nucleotides (nt) 35-649. The other primer set, VP28F (5V-CAAGCAACGTTCGATAAAGA-3V) and VP28R (5VGAAGGTTAAGTAGGTCAATAA-3V), was for the 754 base region including an ORF for VP28 between nt 33-647. Amplification was performed in a DNA thermal cycler (PC800, Astec, Japan) with the following cycle parameters: initial 72 jC for 10 min and 95 jC for 6 min, followed by 30 cycles of 95 jC for 1 min, 55 jC for 1 min and 72 jC 1 min, and final extension at 72 jC for 5 min. The PCR products were cloned into SrfI site of the plasmid vector pCR-Script SK(+) to transform Escherichia coli (DH5a) according to the manufacturer’s instructions. The nucleotide sequences of cloned products corresponding vp26 and vp28 were determined using a dye terminator cycle sequencing kit (ABI) and analyzed with DNASIS (Hitachi Software Engineering). As a result, the sequences obtained in our study were completely in accord with vp26 and vp28, respectively, reported by van Hulten et al. (2000). The PCR primers, VP26expF (5V-aaagaattcat-ATGGAATTTGGCAACCTAAC-3V) and VP26expR (5V-gctgtcgac-TTACTTCTTCTTGATTTCGTCC-3V) for vp26 gene expression and VP28expF (5V-aaagaattcat-ATGGATCTTTCTTTCACTCTTTC-3V) and VP28expR (5V-gctgtcgac-TTACTCGGTCTCAGTGCC-3V) for vp28 gene expression were, respectively, designed based on the determined nucleotide sequences of vp26 and vp28. VP26expF and VP28expF primers included 11 additional bases of linker sequence as recognition sites for the NdeI and EcoRI while the VP26expR and VP28expR primers have nine additional bases of linker sequence as the SalI recognition site. The vp26 and vp28 genes were amplified from the extracted nucleic acid of WSSV with expression primer sets under the same PCR condition as described above. After digestion with NdeI or EcoRI and SalI, the PCR products were ligated into the expression vector plasmid, pET25b (+) (Novagen) to transform E. coli (BL21). Recombinant VP26 or VP28 (rVP26, rVP28) in the transformed E. coli with the expression vector plasmid was induced by culturing at 37 jC for 3 h in LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 1% NaCl, pH 7.4) containing 50 Ag/ml ampicillin and 1 mM isopropyl-1-1-thio-h-D-galactoside (IPTG). The cells were resuspended in 50 mM Tris – HCl (pH 8.0)– 2 mM EDTA solution containing 100 Ag/ml lysozyme and 0.1% Triton X-100 and incubated at 30 jC for 15 min. The cells were then sonicated until the viscosity of the solution was lost. The induced product from the target gene in the insoluble fraction was washed twice by centrifugation (12,000  g, 15 min) and resuspended in PBS as recombinant protein vaccines. It was shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) that the expressed proteins had molecular weights of approximately 25.5 and 27.8 kDa, respectively (Fig. 1). 2.6. Vaccination 2.6.1. Trial 1. Vaccination with formalin or heat-inactivated WSSV Kuruma shrimp (mean body weight: MBW 1.4 g) were divided into four groups including 50 shrimp each, and one group was IM vaccinated with 25 Al of formalininactivated WSSV per shrimp, the second group was IM vaccinated with heat-inactivated WSSV at the same dose as formalin-inactivated WSSV, and the last two groups served as

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Fig. 1. SDS-PAGE analysis of expressed proteins, rVP26 and rVP28 of WSSV. The proteins in the 15% gel were stained with Coomassie brilliant blue. M: molecular marker, 1: rVP26, 2: rVP28.

controls being injected with diluted naive shrimp hemolymph similarly treated with formalin or heat. On 10th day post-vaccination (dpv), each of the groups was divided into two subgroups. First subgroups were challenged by IM injection with 25 Al of a 10 4 dilution of the virus preparation, and the remaining subgroups were challenged on 30th dpv. 2.6.2. Trial 2. Vaccination with inactivated WSSV and immunostimulants Kuruma shrimp (MBW 3.4 g) were divided into four groups (each group n = 25), and each of them was vaccinated by IM injection with formalin-inactivated WSSV at the same dose as before, formalin-inactivated WSSV + glucan, formalin-inactivated WSSV + V. penaeicida, or PBS. The injected amount of immunostimulants was 50 Ag/g shrimp for h-1,3-glucan, and 0.15 mg/g shrimp for formalin-inactivated V. penaeicida. Each group was challenged by IM injection with 25 Al of a 10 4 dilution of the virus preparation on the 30th dpv. 2.6.3. Trial 3. Three vaccinations with inactivated WSSV and inactivated V. penaeicida Kuruma shrimp (MBW 1.0 g) were divided into three groups (each group n = 25), each of which was vaccinated three times at 10-day intervals with formalin-inactivated WSSV, inactivated WSSV with V. penaeicida, or PBS by the same method as of the trial 2, except that the dose of V. penaeicida was 0.1 mg/g shrimp. Each group was challenged by the same method as before on the 30th day post-third vaccination (50th day post-initial vaccination). 2.6.4. Trial 4. Vaccination with recombinant proteins Kuruma shrimp (MBW 1.1 g) were divided into three groups (each group n = 50), and each shrimp of these groups was IM vaccinated with 20 Al of rVP26, rVP28 or E. coli proteins (control). The dose of recombinant proteins was determined as 0.1 mg/g shrimp, because 80% of the shrimp injected with rVP26 at 1 mg/g shrimp died but no shrimp injected at 0.1 mg/g died in a preliminary test. On the 20th dpv, each group was divided into two subgroups. The first subgroups were vaccinated again by the same method and

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challenged by the same method as before on the 10th day post-second vaccination (30th day post-initial vaccination), and the remaining subgroups without a booster shot were challenged on the 30th dpv. 2.7. Statistical analysis The mortalities of the tested and control groups were compared statistically using the chi-square test (v2) at a significance level of 5%. The relative percent survival (RPS) values were calculated according to Amend (1981).

3. Results 3.1. Trial 1. Vaccination with inactivated WSSV No mortality was recorded in vaccinated or mock-injected shrimp before the challenge tests. The first mortality was observed on the 3rd or 4th day post-challenge (dpc) in all challenged groups in either experiment made on the 10th or 30th dpv. The challenge test made on the 10th dpv resulted in significantly lower mortalities ( P < 0.05) in the formalininactivated WSSV vaccinated group (40%) compared with its control group (88%), and RPS was calculated at 50%. The cumulative mortalities in the heat-inactivated WSSV vaccinated group and its control were 68% and 80%, respectively, and the calculated RPS was 15%. Following the challenge test made on the 30 dpv, the cumulative mortalities in the formalin vaccine group, its control group, heated vaccine group, and its control group were 72%, 76%, 80% and 60%, respectively; no significant differences in mortalities were found between each vaccinated and control groups (Table 1).

Table 1 Resistance against experimental WSSV infection in kuruma shrimp vaccinated with formalin or heat-inactivated WSSV Time of challenge 10 dpv Formalin-inactivated WSSV Control Heat-inactivated WSSV Control 30 dpv Formalin-inactivated WSSV Control Heat-inactivated WSSV Control

Dead/tested

Mortality (%)

RPS (%)

p-value

10/25 22/25 17/25 20/25

40 88 68 80

50 – 15 –

0.0012*

18/25 19/25 20/25 15/25

72 76 80 60

5 – 33 –

1

0.5190

–

Control group shrimp were injected with naive shrimp hemolymph treated with formalin or heat. Cumulative mortalities, relative percent survival (RPS) values and P-values (v2 test) were determined at the termination (10th day) of challenge test made at 10th and 30th days post-vaccination (dpv). Significant difference (5% level) compared with the corresponding unvaccinated group is indicated by *.

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Fig. 2. Cumulative mortalities of kuruma shrimp vaccinated with formalin-inactivated WSSV combined with h1,3-glucan or formalin-inactivated V. penaeicida, and challenged with WSSV on 30th day post-vaccination.

3.2. Trial 2. Vaccination with inactivated WSSV and immunostimulants The cumulative mortalities on the 14th dpc in the groups vaccinated with inactivated WSSV alone, inactivated WSSV + glucan, inactivated WSSV + V. penaeicida, and PBS were 60%, 44%, 40% and 64%, respectively. Calculated RPS values were 6%, 31% and 38% for the inactivated WSSV, inactivated WSSV + glucan, and inactivated WSSV + V. penaeicida groups, respectively. No significant difference in cumulative mortalities was observed between any of the vaccination groups and the control group (Fig. 2).

Fig. 3. Cumulative mortalities of kuruma shrimp vaccinated three times at 10-day intervals with inactivated WSSV and V. penaeicida and challenged with WSSV 30 days after the third vaccination.

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Table 2 Resistance against experimental WSSV infection in kuruma shrimp vaccinated once or twice (at 20th day postinitial vaccination) with recombinant WSSV structural proteins rVP26 or rVP28 Vaccination Vaccination once rVP26 rVP28 Control Vaccination twice rVP26 rVP28 Control

Dead/tested

Mortality(%)

RPS (%)

P-value

10/25 19/25 23/25

40 76 92

57 17

0.0001* 0.1228

8/25 1/25 20/25

32 4 80

60 95

0.0006* 0.0000*

Control group shrimp were injected with E. coli proteins. Cumulative mortalities, relative percent survival (RPS) values and P-values (v2 test) were determined at the termination (10th day) of challenge test made on 30th day post-initial vaccination. Significant difference (5% level) compared with the corresponding unvaccinated group is indicated by *.

3.3. Trial 3. Three vaccinations with inactivated WSSV and V. penaeicida The challenge test with WSSV on the 30th day post-third vaccination resulted in significantly lower mortalities ( P < 0.05) in the group vaccinated with WSSV + V. penaeicida (40%) compared with the control group (72%), and the RPS was 44%. But the mortality in the group vaccinated with WSSV alone, even though vaccinated three times, was not different (80%) from that of the control group (72%) (Fig. 3). 3.4. Trial 4. Vaccination with recombinant proteins The cumulative mortalities during the 10 days following challenge with WSSV on the 30th dpv in the groups vaccinated once with rVP26, rVP28 and E. coli proteins (control) were 40%, 76% and 92%, respectively. A significant difference in cumulative mortalities was observed ( P < 0.05) between rVP26-vaccinated group and the control group, but not between rVP28-vaccinated group and the control. RPS values for the rVP26 and rVP28 groups were 57% and 17%, respectively. The cumulative mortalities following challenge in the groups vaccinated two times with rVP26, rVP28 and E. coli proteins were 32%, 4% and 80%, respectively, and the cumulative mortalities for both vaccinated groups were significantly lower ( P < 0.05) than that for the control group. RPS values of rVP26 and rVP28 groups were 60% and 95%, respectively (Table 2).

4. Discussion Four different trials based on different immunogens were run to examine the possibility of immunization of kuruma shrimp against WSSV. In trial 1, vaccination with formalininactivated WSSV enhanced the resistance of shrimp against WSSV (Table 1), but the resistance did not persist for 20 days. On the other hand, heat-inactivated WSSV did not evoke measurable resistance even temporarily against the virus in shrimp. Thus, we

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concluded that the substance(s) triggering ‘‘quasi-immune response’’ is heat-labile. Accordingly to enhance protection of shrimp against WSSV, trial 2 was performed using formalin-inactivated WSSV combined with immunostimulants, i.e. h-1,3-glucan or formalin-inactivated V. penaeicida. Glucan or inactivated Vibrio have already been reported to enhance the resistance not only against Vibrio infections (Sung et al., 1994; Teunissen et al., 1998; Sritunyalucksana et al., 1999), but also against WSSV infections in shrimp. As expected, the protection of shrimp was enhanced by this dual administration, because the shrimp showed resistance (RPS values were 36 and 31%) even on the 30th dpv. However, efficacy as represented by these RPS values was not satisfactory. The trial 3 where shrimp were vaccinated three times with inactivated WSSV and the bacterium showed that the multiple vaccinations could improve protection (Fig. 3), because the formalin-inactivated WSSV + V. penaeicida group gave significantly lower mortality ( P < 0.05) than the control group. The RPS value (44%) of this multiple vaccinations, however, did not meet the criterion (RPS: 60%) for effective fish vaccines (Amend, 1981). As the shrimp vaccinated three times with inactivated virus alone did not show any enhanced resistance against WSSV (Fig. 3), it is likely that the enhanced resistance in the shrimp vaccinated with the virus and bacteria may have been induced by the bacteria, and thus is not specific. In addition, the three vaccinations at 10-day intervals gave significant stress to shrimp because molting occurred in many shrimp after each vaccination and the growth of these shrimp was reduced. Based on these results we concluded that vaccination with inactivated WSSV is not promising. Wu et al. (2002a) reported that resistance against WSSV in P. japonicus surviving from experimental WSS appeared 3 weeks after exposure to the virus and persisted for another month. The resistance induced by formalin-inactivated WSSV commenced earlier and persisted for a shorter period compared with that induced by the live virus. This difference seems to be caused by the differences in initial quantity of the virus and duration of the existence of the virus in shrimp, i.e. the shrimp in the Wu et al. (2002a) experiment were injected with a low dose of the live virus, while the shrimp of the present study were injected with a larger amount of inactivated WSSV. Vaccines based on recombinant proteins have already been developed for some fish viruses (Leong and Fryer, 1993; Lorenzen and Olesen, 1997; Hu´sgard et al., 2001). A recombinant protein vaccine has the advantage that animals can be vaccinated with a large amount of specific antigens. This vaccine is convenient especially for shrimp viruses including WSSV because they cannot be cultured due to the lack of susceptible cell lines. Therefore, we produced recombinant WSSV structural proteins related to nucleocapsid (rVP26) or envelope (rVP28) reported by van Hulten et al. (2000), and vaccinated shrimp with these recombinant proteins. Results of trial 4 showed highly significant resistance in the shrimp vaccinated with rVP26 once (RPS: 57%) against WSSV by the 30th day post-initial vaccination (Table 2), but not in the shrimp vaccinated with rVP28. On the other hand, two vaccinations of shrimp with rVP26 or rVP28 at a 20day interval resulted in significantly lower mortalities than those occurred among the control animals (RPS values were 60% and 95%, respectively). van Hulten et al. (2001b) reported that the rabbit antiserum against rVP28 was able to neutralize WSSV, and VP28 is likely to play a key role in the initial steps of the systemic WSSV infection in shrimp. In our separate experiment, the capacity of both anti-rVP26 and anti-rVP28 rabbit sera to

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neutralize WSSV were confirmed in a bioassay (data not shown), and recently, VP26 has been shown to be an envelope protein (Zhang et al., 2002). Our results indicate that both VP26 and VP28 are ‘‘protective antigens’’ which can evoke protection of shrimp by vaccination. Keith et al. (1992) reported that one decapod crustacean the American lobster Homarus americanus vaccinated with inactivated Aerococcus viridans var. homari developed resistance against the bacterium on the 6th dpv and the resistance persisted for 93 days. Our study seems to be the first successful vaccination in shrimp against the viral pathogen WSSV, although it is arguable as to whether usage of the term ‘‘vaccination’’ is relevant or not. These results should be of assistance in elucidating the mechanism of ‘‘quasi-immune response’’ or induced resistance in shrimp.

Acknowledgements This work has been supported by the grants in aid of JASFA and the Japan Fisheries Resource Conservation Association.

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