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Enhanced survival of shrimp, Penaeus (Marsupenaeus) japonicus from white spot syndrome disease after oral administration of recombinant VP28 expressed in Brevibacillus brevis
Fish & Shellfish Immunology (2008) 25, 315e320
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SHORT COMMUNICATION
Enhanced survival of shrimp, Penaeus (Marsupenaeus) japonicus from white spot syndrome disease after oral administration of recombinant VP28 expressed in Brevibacillus brevis Christopher Marlowe A. Caipang a,1, Noel Verjan b, Ei Lin Ooi b, Hidehiro Kondo a, Ikuo Hirono a, Takashi Aoki a,*, Hiroshi Kiyono b, Yoshikazu Yuki b a Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan b Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 108-8639, Japan
Received 14 February 2008; revised 8 April 2008; accepted 27 April 2008 Available online 2 May 2008
KEYWORDS White spot syndrome virus; WSSV; Shrimp Penaeus japonicus; Recombinant VP28 protein; Oral administration; Immunostimulant
Abstract White spot syndrome virus (WSSV) disease is a major threat to shrimp culture worldwide. Here, we assessed the efficacy of the oral administration of purified recombinant VP28, an envelope protein of WSSV, expressed in a Gram-positive bacterium, Brevibacillus brevis, in providing protection in shrimp, Penaeus japonicus, upon challenge with WSSV. Juvenile shrimp (2e3 g in body weight) fed with pellets containing purified recombinant VP28 (50 mg/shrimp) for 2 weeks showed significantly higher survival rates than control groups when challenged with the virus at 3 days after the last day of feeding. However, when shrimp were challenged 2 weeks after the last day of feeding, survival rates decreased (33.4% and 24.93%, respectively). Survival rate was dose-dependent, increasing from 60.7 to 80.3% as the dose increased from 1 to 50 mg/shrimp. At a dose of 50 mg/shrimp, the recombinant protein provided protection as soon as 1 day after feeding (72.5% survival). Similar results were obtained with larger-sized shrimp. These results show that recombinant VP28 expressed in a Gram-positive bacterium is a potential oral vaccine against WSSV. ª 2008 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ81 3 5463 0556; fax: þ81 3 5463 0690. E-mail address: [email protected] (T. Aoki). 1 Present address: Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the Philippines in the Visayas, Miag-ao 5023, Iloilo, Philippines. 1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.04.012
316 White spot syndrome virus (WSSV) is the causative agent of a serious disease in penaeid shrimp culture worldwide. It affects a diverse range of crustacean species [1,2] and can cause 100% mortality in farmed shrimp within a few days resulting in tremendous economic losses in the shrimp culture industry [3]. WSSV possesses varied genome sizes depending on its place of isolation; WSSV-China with w305 kb (AF332093), WSSV-Taiwan with w307 kb (AF440570) and WSSV-Thailand with w93 kb (AF369029). WSSV-Thailand has been reported to have 184 reading frames [4]. It is an enveloped virus, having an ellipsoid to bacilliform shape with a tail-like appendage at one end [4,5] and is the type species of the genus Whispovirus in the family Nimaviridae [6]. VP-28, one of the five major proteins of WSSV, has been examined and shown to be a potential WSSV vaccine candidate [7e14]. In particular, the feasibility of oral vaccination using the prokaryotic system to express and purify recombinant VP28 has been tested because VP28 protein is not glycosylated [15] and the system is commercially established. Significant protection against WSSV has been achieved using Gram-negative bacteria (Escherichia coli)expressed VP28 [8,9,11]. However, the use of Gram-positive bacteria appears to offer a better alternative due to their ability to secrete functional extracellular proteins directly into the culture medium, lack of pathogenicity and the absence of lipopolysaccharides (endotoxins) from the cell wall [16], which are normally present in Gram-negative bacteria. In the present study, we expressed VP28 in a Gram-positive bacterium, Brevibacillus brevis, purified its recombinant protein, incorporated in the diet and fed to shrimp, Penaeus (Marsupenaeus) japonicus. The onset and duration of protection after oral administration of the recombinant protein in shrimp were investigated by live experimental challenge of the virus at different time points post-feeding. WSSV-free P. japonicus (approximately 2e3 g in body weight) were procured from a commercial shrimp hatchery in Okinawa, Japan. They were reared in tanks with sand beds supplied with a sand-filtered recirculating artificial seawater system (25e28 ppt) at 23e25 C. Prior to each experiment, shrimp were stocked in 60-l aquaria, each fitted with an individual filter system, heater and continuous aeration. All experiments were conducted using artificial seawater (25e28 ppt) at 23e25 C. On the other hand, the virus suspension used in the study was obtained from moribund P. japonicus infected with WSSV. Briefly, muscle tissues were aseptically removed from the cuticle, mixed with equal part phosphate-buffered saline (PBS), homogenized with a tissue grinder and centrifuged at 1500 g at 4 C for 10 min. The supernatant was removed, aliquoted in 1 ml of stock and stored at 80 C until use. For the challenge experiment, ten-fold serial dilutions of the filtrate were prepared using PBS and the WSSV stock was titered by in vivo experiments following the procedures described by van Hulten et al. [17] with some modifications. Briefly, 25 ml of the different virus dilutions were intramuscularly injected in shrimp of approximately 2e3 g in body weight. Mortality was recorded twice a day and dead shrimp were tested for the presence of WSSV either by PCR or a commercial detection kit (Shrimple, EnBioTec Lab. Co.
C.M.A. Caipang et al. Ltd., Tokyo). The timeemortality relationship obtained was used to determine the desired challenge pressure (approximately 50%) in the succeeding infection experiments. To produce, purify and analyze recombinant VP28, B. brevis carrying the recombinant plasmid pNOH3-Vp28 was grown for 3 days at 30 C in S2U media as described previously [18]. The culture was centrifuged and the cell pellet dissolved in PBS and sonicated. The homogeneous solution was centrifuged and the supernatant containing recombinant VP28 was concentrated five-fold using an ultrafiltration device (Amicon, Beverly, MA) through a 10,000 molecular weight cut-off filter. The concentrated solution was mixed with an equal volume of phosphate buffer (NaH2PO4 0.1 M, 300 mM NaCl, 20 mM imidazole, pH 8.2) and loaded into a packed column containing 40 ml of NiNTA agarose beads (Amersham Biosciences). Extensive washing was performed with phosphate buffer containing 20 mM imidazole and eluted with buffers containing 60 and 100 mM imidazole. Elution of the recombinant VP28 was completed with phosphate buffer containing 250 mM imidazole. The protein concentration in the eluted fractions was estimated spectrophotometrically at 280 nm wavelength and fractions containing the purified protein were pooled and concentrated using the Amicon ultrafiltration device (Millipore, Billerica, MA, USA). The concentrated recombinant VP28 was applied to a Sephadex G-100 (Amersham Biosciences AB) column (2 95 cm) equilibrated with PBS, pH 7.4. Protein concentration was determined and stored at 80 C until use. The purified protein was further analyzed by SDS-PAGE and Western blot. Briefly, B. brevis carrying recombinant plasmid or the purified recombinant VP28 were separated in 5e20% precast e-PAGEL (ATTO Co., Tokyo, Japan) under reducing conditions and stained with Coomassie brilliant blue R-250. The separated proteins in a second gel were electro-blotted on an AE6667 (ATTO Co., Tokyo, Japan) membrane, and then incubated with anti-his tag monoclonal antibody (Amersham Biosciences) or a rabbit polyclonal antibody anti-VP28 of shrimp white spot syndrome virus (Genesis Biotech Inc.). The second antibody was rabbit anti-mouse IgG or goat anti-rabbit IgG conjugated with alkaline phosphatase (Promega, Madison, WI, USA. and the last interaction revealed with the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP-NBT) alkaline phosphatase substrate (Sigma-Aldrich, St. Louis, MO, USA). The WSSV ORF encoding VP28 fused with a His6 tag was overexpressed in B. brevis. The band corresponding to the His6 tag fusion protein was observed at the expected molecular weight of approximately 23 kDa (Fig. 1a). The purified recombinant viral protein was confirmed to be Vp28 by Western analysis with anti-WSSV polyclonal antiserum (Fig. 1b). We then used this recombinant VP28 for the feeding experiments. Feed pellets were first produced by mixing finely ground commercial shrimp feed with the purified recombinant VP28 dissolved in PBS. The mixture was placed in a 50 ml syringe and slowly extruded to form feed pellets. The extruded moist pellets were placed in a dark room at 4 C and allowed to dry. The dried pellets were mixed with 1% fish oil (w/v) [12] to prevent dispersion of the feed particles in the water. Similar preparations using supernatant of
Survival of Penaeus (Marsupenaeus) japonicus from WSSV
Figure 1 (A) SDS-PAGE and (B) Western blot analysis of recombinant VP28 (rVP28) expressed in Brevibacillus brevis. M, protein standard; lane 1, B. brevis carrying recombinant plasmid pNOH3-VP28 and expressing rVP28 (indicate with an arrowhead); lane 2, purified rVP28. Purified rVP28 (enclosed in box) was detected using rabbit polyclonal antibody anti-VP28 of WSSV.
sonicated B. brevis dissolved in PBS and PBS only were used as negative controls in the experiment. The amount of recombinant protein in the diet was calculated based on
Table 1
317 the average body weight of the shrimp, dose, feeding rate (5%) and duration of feeding. Four feeding experiments were conducted (Table 1). The duration of protection by the recombinant VP28 in juvenile shrimp was determined in the first and second experimental runs. In the third experimental run, the effective dose of the recombinant VP28 to protect the shrimp against WSSV disease and the onset of protection were determined. Finally, on the fourth experimental run, the efficiency of using recombinant VP28 in protecting commercial size shrimp against WSSV after oral administration was determined. There were six groups in the first experimental run. One group was fed with shrimp pellets containing 50 mg/shrimp of purified recombinant VP28 for 2 weeks, then challenged with the virus at 3 days post-feeding. Another group was fed with a similar preparation of the recombinant VP28 for 2 weeks, followed by a 2-week feeding of non-treated shrimp pellets. Two groups fed with shrimp pellets containing supernatant of sonicated B. brevis (1 mg ml1) for 2 weeks followed by virus challenge at 3 and 14 days postfeeding, respectively. Another two groups fed with normal shrimp pellets served as controls. In the second experimental run, three groups (Treatment 1, 2 and 3) were fed with shrimp pellets containing purified recombinant VP28 (50 mg/shrimp) for 2 weeks followed by feeding with non-treated shrimp pellets for 3, 7 and 14 days, respectively. This was immediately followed by virus challenge. Another three groups were fed following the preceding feeding scheme using the preparation containing supernatant of sonicated B. brevis (1 mg ml1) and a group fed with non-treated shrimp pellets were used as controls.
Set-up of the vaccination experiments
Experimental run
Treatment no.
Feeding scheme and virus challenge
No. of shrimp
1a
1 2 3 4 5 6
2 weeks feeding 50 mg/shrimp, challenge 3 days post-feeding 2 weeks feeding, challenge 2 weeks post-feeding Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 1 Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 2 Commercial shrimp feed Commercial shrimp feed
18 15 21 16 30 10
2
1 2 3 4 5 6 7
2 weeks feeding 50 mg/shrimp, challenge 3 days post-feeding 2 weeks feeding, challenge 1 week post-feeding 2 weeks feeding, challenge 2 weeks post-feeding Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 1 Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 2 Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 3 Commercial shrimp feed
30 21 33 30 27 27 32
3
1 2 3 4 5
2 weeks feeding 50 mg/shrimp, challenge 3 days post-feeding 2 weeks 10 mg/shrimp, challenge 3 days post-feeding 2 weeks 1 mg/shrimp, challenge 3 days post-feeding 1 day 50 mg/shrimp, challenge 1 day post-feeding Commercial shrimp feed
30 28 27 30 33
4
1 2 3
3 days feeding 50 mg/shrimp, challenge 1 day post-feeding 1 week feeding 50 mg/shrimp, challenge 1 day post-feeding Commercial shrimp feed
14 16 16
a
In Experiment 1, Treatment 5 served as the control for Treatments 1 and 3, while Treatment 6 was the control for Treatments 2 and 4.
318
Figure 2 Timeemortality relationship of experiment 1. Juvenile shrimp were fed recombinant VP28 for 2 weeks followed by viral challenge at (A) 3 days and (B) 2 weeks post-feeding. Cumulative mortality rates of shrimp from the experimental groups, as indicated in Table 1 are plotted against time (15day period) after challenge.
Five experimental groups were used in the third experimental run. Three groups (Treatment 1, 2 and 3) were fed with shrimp pellets containing recombinant VP28 at a dose of 1, 10 and 50 mg/shrimp, respectively. The feeding scheme lasted for 2 weeks followed by feeding with nontreated shrimp pellets for 3 days before virus challenge. A group (Treatment 4) fed with shrimp pellets containing purified recombinant VP28 at a dose of 50 mg/shrimp for 1 day followed by virus challenge was included to determine whether the onset of protection is observed immediately after oral administration of the purified recombinant protein. Shrimp fed with the non-treated shrimp pellets were used in the control group (Treatment 5). To determine the efficiency of using the purified recombinant VP28 in protecting bigger-size shrimp against WSSV, commercial size shrimp (approximately 12e15 g body weight) were used in the fourth experimental run. Treatment 1 and 2 were composed of shrimp fed with shrimp pellets containing purified recombinant VP28 at a dose of 50 mg/shrimp for 3 and 7 days, respectively. The shrimp were challenged with the virus 1 day after the last feeding of the purified recombinant VP28-treated pellets. Treatment 3, which is composed of shrimp fed with the non-treated shrimp pellets, was used as a control. Adjustments were made at the start of the feeding scheme such that virus challenge would commence at the same time. The challenge tests were conducted by
C.M.A. Caipang et al. intramuscularly injecting individual shrimps in the experimental groups with 50 ml of the WSSV dilution (105 dilution of the virus stock solution) at a challenge pressure of at least 50%. Dead shrimp were collected twice a day to prevent cannibalism and mortality was monitored over a 15-day period. Statistical analysis was done using c2 test (SYSAT ver. 8, Chicago, Il.) at the 0.05 level of significance. The protection against WSSV disease after oral administration of the purified recombinant VP28 was assessed by calculating the relative percent survival [19]. The timeemortality relationships in the first experiment are shown in Fig. 2. In the first run (Fig. 2a), the groups fed with commercial shrimp pellets and those containing supernatant of sonicated B. brevis for 2 weeks followed by virus challenge at 3 days after last feeding, had cumulative mortalities of 76.7% and 90.5%, respectively. In contrast to the group fed with shrimp pellets containing purified recombinant VP28 (second run), a significantly lower cumulative mortality of 5.5% was observed (relative survival of 92.8%, Table 2). However, the relative survival decreased to 33.4% when the shrimp were challenged with the virus 14 days post-feeding, regardless of the treatment (Table 2, Fig. 2b). The relative survival values in the groups fed with sonicated B. brevis, and commercial feed only were 12.5% and 0%, respectively (Table 2). In the second experiment (Fig. 3), the group fed with purified recombinant protein for 2 weeks followed by WSSV
Table 2 Relative survival of shrimp in the different experimental runs Experimental run
Treatment no.
Survival (%)
Mortalitya
RPS (%)b
1
1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 1 2 3
94.5 60.0 9.5 56.2 23.3 50.0 70.0 57.1 27.3 6.7 14.8 18.5 3.1 83.3 67.9 66.7 76.7 15.2 78.6 81.2 18.2
5.5 40.0 90.5 43.8 76.7 50.0 30.0 42.9 72.7 93.3 85.2 81.5 96.9 16.7 32.1 33.3 23.3 84.8 21.4 18.8 81.2
92.8* 33.4 0 12.5 0 0 69.03* 55.8* 24.9 3.66 12.07 15.9 0 80.3* 62.09* 60.7* 72.5* 0 73.7* 76.9* 0
2
3
4
(1/18) (6/15) (19/21) (7/16) (23/30) (5/10) (9/30) (9/21) (24/33) (28/30) (23/27) (22/27) (31/32) (5/30) (9/28) (9/27) (7/30) (28/33) (3/14) (3/16) (13/16)
An asterisk denotes significantly different (p > 0.05) from the control group in each experimental run. a Values in parentheses indicate the ratio of dead shrimp over the total shrimp in each treatment. b Relative percentage survival (RPS) was calculated based on the formula of Amend, 1981 [19].
Survival of Penaeus (Marsupenaeus) japonicus from WSSV
319
challenge at 3 days after cessation of feeding resulted in a cumulative mortality of 30.0% (relative survival of 69.03%). Cumulative mortality increased when the groups were challenged with the virus at 7 and 14 days after the last day of feeding, with relative survival of 55.8% and 24.9%, respectively. The cumulative mortality in the sonicated B. brevis-fed groups ranged from 81.5% to 93.3%. The positive control (normal fed) group had 96.9% cumulative mortality, while the negative control group showed no mortality. To determine the effective dose that is required for protection against the viral disease, varying amounts (50, 10 and 1 mg/shrimp) of the purified recombinant VP28 were incorporated in the diet and fed to the shrimp for 2 weeks followed by virus challenge 3 days post-feeding. The lowest cumulative mortality (16.6%) was obtained in the group fed with 50 mg/shrimp of the purified recombinant VP28 (relative survival of 80.3%) (Fig. 4). The cumulative mortality in the group fed with 1 mg/shrimp of the purified recombinant VP28 was 33.3%, but its relative survival was 60.7%. Shrimp fed pellets containing 50 mg/shrimp of the purified recombinant VP28 for 1 day and then challenged with the virus 1 day after feeding showed a cumulative mortality of 23.3% (relative survival of 72.5%). The negative control group had a cumulative mortality of 84.8%.
The relative survival of larger shrimp (12e15 g) fed with recombinant VP28 for 3 and 7 days, immediately followed by virus challenge a day post-feeding was 73.6 and 76.9%, respectively (Fig. 5). The negative control group had relative survival of 91.3%. For each shrimp a total of 50 mg over a 2-week period followed by virus challenge at 3 days post-feeding gave the highest relative survival (92.8%). The duration of protection against the disease was 1 week post-feeding with recombinant VP28, if the shrimp were fed for 2 weeks at a dose of 50 mg/shrimp. The minimum dose of recombinant VP28 that is sufficient to provide protection against white spot syndrome is 1 mg/shrimp, if the shrimp are fed for at least 2 weeks. If shrimp are fed with recombinant VP28 at a dose of 50 mg/shrimp, they are effectively protected against the disease even at 1 day post-feeding. These results exhibit a positive correlation between protection and dosage, even if the treatment is administered for a short period of time (1e3 days), and show that the level of protection in shrimp against the disease significantly decreased at 2 weeks post-feeding regardless of the dose and duration of feeding. These results are similar to those of previous studies that showed decreased survival in shrimp at least 3 weeks post-feeding with recombinant VP28 [8,9] Such protection was suggested to be caused by the blocking of host cell receptors for WSSV of envelope proteins preventing viral infection and therefore maintenance of protection for about 2 weeks [20]. If this is the case, then the 50 mg/shrimp VP28 dose used in this study might have been enough to cover all receptors for WSSV, such that short-term treatment can already confer protection. Such a mechanism is supported by the finding that a low VP28 dose (1 mg/shrimp) can also induce protection if administered for a longer period (at least 2 weeks). We also actually analyzed whether inactivated bacteria induce a non-specific immune response when sonicated B. bacillus was used as one of the controls. Results showed that shrimp fed with the sonicated bacteria were not protected from subsequent infection. Similar findings were also obtained by Witteveldt and co-workers [8] using E. coli. We believe that feeding shrimp with inactivated bacteria does not trigger an immune response that would protect the shrimp against WSSV.
Figure 4 Timeemortality relationship of experiment 3. Shrimp were fed with varying levels of recombinant VP28 for 2 weeks and challenged with the virus at 3 days post-feeding. Cumulative mortality rates of shrimp from the experimental groups, as indicated in Table 1 are plotted against time (15day period) after challenge.
Figure 5 Timeemortality relationship of experiment 4. Marketable-sized shrimp (12e15 g body weight) were fed with recombinant VP28 at certain duration and challenged with WSSV. Cumulative mortality rates of shrimp from the experimental groups, as indicated in Table 1 are plotted against time (15-day period) after challenge.
Figure 3 Timeemortality relationship of experiment 2. Cumulative mortality rates of shrimp from the experimental groups, as indicated in Table 1 are plotted against time (15day period) after challenge.
320 By using B. brevis, a Gram-positive bacterium, to produce VP28, we were able to generate a higher relative survival than was achieved in previous studies that utilized Escherichia coli, a Gram-negative bacterium [8,9,12]. This is probably because Gram-positive bacteria secrete functional extracellular proteins directly into the culture medium, and are less toxic, i.e., they do not produce lipopolysaccharides (endotoxins) from the cell wall [16]. Taken together, our results show that oral administration of recombinant VP28 expressed in B. brevis was successful in protecting shrimp against WSSV, confirming previous reports of the efficacy of VP28 protein as a protective antigen in shrimp [8,9,21e23]. The exact mechanism that would explain the efficacy of VP28 is still not clear, although it is now known that VP28 is located on the surface of WSSV, which allows the virus particle to bind to shrimp cells and eventually enter the cytoplasm [17,24]. In conclusion, purified recombinant VP28 expressed in a Gram-positive bacterium, B. brevis, clearly protected shrimp against white spot syndrome disease. The use of this bacterial species in the production of recombinant protein eliminates the possibility of endotoxin contamination, which is common in Gram-negative bacteria. Hence, this study is a first step in addressing safety issues in producing vaccines for the shrimp aquaculture industry. Future studies will focus on optimizing the combination of the following parameters: dose of the recombinant protein, size of the shrimp, feeding duration and start of virus challenge, as these will pave the way in the application of this feeding scheme in protecting shrimp against WSSV infections on a large-scale basis.
Acknowledgements This study was supported in part by a grant from the Japan Science and Technology Corporation (JST): Creation and Support Program for Start-ups from Universities.
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C.M.A. Caipang et al. [7] Namikoshi A, Wu JL, Yamashita T, Nishioka T, Arimoto M, Muroga K. Vaccination trials with Penaeus japonicus to induce resistance to white spot syndrome virus. Aquaculture 2003; 229:25e35. [8] Witteveldt J, Cifuentes CC, Vlak JM, van Hulten MCW. Protection of Penaeus monodon against white spot syndrome virus by oral vaccination. J Virol 2004;78:2057e61. [9] Witteveldt J, Vlak JM, van Hulten MCW. Protection of Penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine. Fish Shellfish Immunol 2004;16: 571e9. [10] Du H, Xu Z, Wu X, Li W, Dai W. Increased resistance to white spot syndrome virus in Procambarus clarkii by injection of envelope protein VP28 expressed using recombinant baculovirus. Aquaculture 2006;260:39e43. [11] Jha RK, Xu ZR, Shen J, Bai SJ, Sun JY, Li WF. The efficacy of recombinant vaccines against white spot syndrome virus in Procambarus clarkii. Immunol Lett 2006;105:68e76. [12] Xu Z, Du H, Xu Y, Sun J, Shen J. Crayfish Procambarus clarkii protected against white spot syndrome virus by oral administration of viral proteins expressed in silkworms. Aquaculture 2006;253:179e83. [13] Vaseeharan B, Prem Anand T, Murugan T, Chen JC. Shrimp vaccination trials with the VP292 protein of white spot syndrome virus. Lett Appl Microbiol 2006;43:137e42. [14] Jha RK, Xu ZR, Bai SJ, Sun JY, Li WF, Shen J. Protection of Procambarus clarkii against white spot syndrome virus using recombinant oral vaccine expressed in Pichia pastoris. Fish Shellfish Immunol 2007;22:295e307. [15] Van Hulten MCW, Reijus M, Vermeesch AMG, Zandbergen F, Vlak JM. Identification of VP19 and VP15 of white spot syndrome virus (WSSV) and glycosylation of the WSSV major structural proteins. J Gen Virol 2002;83:257e65. [16] Simonen M, Palva I. Protein secretion in Bacillus species. Microbiol Rev 1993;57:109e37. [17] Van Hulten MCW, Witteveldt J, Snippe M, Vlak JM. White spot syndrome virus envelope protein VP28 in involved in the systemic infection of shrimp. Virology 2001;285:228e33. [18] Yuki Y, Hara-Yakoyama C, Guadiz AA, Udaka S, Kiyono H, Chatterjee S. Production of a recombinant cholera toxin B subunit-insulin B chain peptide hybrid protein by Brevibacillus choshinensis expression system as a nasal vaccine against autoimmune diabetes. Biotechnol Bioeng 2005;92: 803e9. [19] Amend DF. Potency testing of fish vaccines. Dev Biol Standard 1981;49:447e55. [20] Flegel TW. Update on viral accommodation, a model for hostviral interaction in shrimp and other arthropods. Dev Comp Immunol 2007;31:217e31. [21] Witteveldt J, Vlak JM, Van Hulten MCW. Increased tolerance of Litopenaeus vannamei to white spot syndrome virus (WSSV) infection after oral application of the viral envelope protein VP28. Dis Aquat Org 2006;70:167e70. [22] Rout N, Kumar S, Jaganmohan S, Murugan V. DNA vaccines encoding viral envelope proteins confer protective immunity against WSSV in black tiger shrimp. Vaccine 2007; 25:2778e86. [23] Wei KQ, Xu ZR. Effect of white spot syndrome virus envelope protein Vp28 expressed in silkworm (Bombyx mori) pupae on disease resistance in Procambarus clarkii. Acta Biol Exp Sin 2005;38:190e8. [24] Yi G, Wang Z, Qi Y, Yao L, Qian J, Hu L. VP28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells. J Biochem Mol Biol 2004; 37:726e34.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/fsi
SHORT COMMUNICATION
Enhanced survival of shrimp, Penaeus (Marsupenaeus) japonicus from white spot syndrome disease after oral administration of recombinant VP28 expressed in Brevibacillus brevis Christopher Marlowe A. Caipang a,1, Noel Verjan b, Ei Lin Ooi b, Hidehiro Kondo a, Ikuo Hirono a, Takashi Aoki a,*, Hiroshi Kiyono b, Yoshikazu Yuki b a Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan b Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 108-8639, Japan
Received 14 February 2008; revised 8 April 2008; accepted 27 April 2008 Available online 2 May 2008
KEYWORDS White spot syndrome virus; WSSV; Shrimp Penaeus japonicus; Recombinant VP28 protein; Oral administration; Immunostimulant
Abstract White spot syndrome virus (WSSV) disease is a major threat to shrimp culture worldwide. Here, we assessed the efficacy of the oral administration of purified recombinant VP28, an envelope protein of WSSV, expressed in a Gram-positive bacterium, Brevibacillus brevis, in providing protection in shrimp, Penaeus japonicus, upon challenge with WSSV. Juvenile shrimp (2e3 g in body weight) fed with pellets containing purified recombinant VP28 (50 mg/shrimp) for 2 weeks showed significantly higher survival rates than control groups when challenged with the virus at 3 days after the last day of feeding. However, when shrimp were challenged 2 weeks after the last day of feeding, survival rates decreased (33.4% and 24.93%, respectively). Survival rate was dose-dependent, increasing from 60.7 to 80.3% as the dose increased from 1 to 50 mg/shrimp. At a dose of 50 mg/shrimp, the recombinant protein provided protection as soon as 1 day after feeding (72.5% survival). Similar results were obtained with larger-sized shrimp. These results show that recombinant VP28 expressed in a Gram-positive bacterium is a potential oral vaccine against WSSV. ª 2008 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ81 3 5463 0556; fax: þ81 3 5463 0690. E-mail address: [email protected] (T. Aoki). 1 Present address: Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the Philippines in the Visayas, Miag-ao 5023, Iloilo, Philippines. 1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.04.012
316 White spot syndrome virus (WSSV) is the causative agent of a serious disease in penaeid shrimp culture worldwide. It affects a diverse range of crustacean species [1,2] and can cause 100% mortality in farmed shrimp within a few days resulting in tremendous economic losses in the shrimp culture industry [3]. WSSV possesses varied genome sizes depending on its place of isolation; WSSV-China with w305 kb (AF332093), WSSV-Taiwan with w307 kb (AF440570) and WSSV-Thailand with w93 kb (AF369029). WSSV-Thailand has been reported to have 184 reading frames [4]. It is an enveloped virus, having an ellipsoid to bacilliform shape with a tail-like appendage at one end [4,5] and is the type species of the genus Whispovirus in the family Nimaviridae [6]. VP-28, one of the five major proteins of WSSV, has been examined and shown to be a potential WSSV vaccine candidate [7e14]. In particular, the feasibility of oral vaccination using the prokaryotic system to express and purify recombinant VP28 has been tested because VP28 protein is not glycosylated [15] and the system is commercially established. Significant protection against WSSV has been achieved using Gram-negative bacteria (Escherichia coli)expressed VP28 [8,9,11]. However, the use of Gram-positive bacteria appears to offer a better alternative due to their ability to secrete functional extracellular proteins directly into the culture medium, lack of pathogenicity and the absence of lipopolysaccharides (endotoxins) from the cell wall [16], which are normally present in Gram-negative bacteria. In the present study, we expressed VP28 in a Gram-positive bacterium, Brevibacillus brevis, purified its recombinant protein, incorporated in the diet and fed to shrimp, Penaeus (Marsupenaeus) japonicus. The onset and duration of protection after oral administration of the recombinant protein in shrimp were investigated by live experimental challenge of the virus at different time points post-feeding. WSSV-free P. japonicus (approximately 2e3 g in body weight) were procured from a commercial shrimp hatchery in Okinawa, Japan. They were reared in tanks with sand beds supplied with a sand-filtered recirculating artificial seawater system (25e28 ppt) at 23e25 C. Prior to each experiment, shrimp were stocked in 60-l aquaria, each fitted with an individual filter system, heater and continuous aeration. All experiments were conducted using artificial seawater (25e28 ppt) at 23e25 C. On the other hand, the virus suspension used in the study was obtained from moribund P. japonicus infected with WSSV. Briefly, muscle tissues were aseptically removed from the cuticle, mixed with equal part phosphate-buffered saline (PBS), homogenized with a tissue grinder and centrifuged at 1500 g at 4 C for 10 min. The supernatant was removed, aliquoted in 1 ml of stock and stored at 80 C until use. For the challenge experiment, ten-fold serial dilutions of the filtrate were prepared using PBS and the WSSV stock was titered by in vivo experiments following the procedures described by van Hulten et al. [17] with some modifications. Briefly, 25 ml of the different virus dilutions were intramuscularly injected in shrimp of approximately 2e3 g in body weight. Mortality was recorded twice a day and dead shrimp were tested for the presence of WSSV either by PCR or a commercial detection kit (Shrimple, EnBioTec Lab. Co.
C.M.A. Caipang et al. Ltd., Tokyo). The timeemortality relationship obtained was used to determine the desired challenge pressure (approximately 50%) in the succeeding infection experiments. To produce, purify and analyze recombinant VP28, B. brevis carrying the recombinant plasmid pNOH3-Vp28 was grown for 3 days at 30 C in S2U media as described previously [18]. The culture was centrifuged and the cell pellet dissolved in PBS and sonicated. The homogeneous solution was centrifuged and the supernatant containing recombinant VP28 was concentrated five-fold using an ultrafiltration device (Amicon, Beverly, MA) through a 10,000 molecular weight cut-off filter. The concentrated solution was mixed with an equal volume of phosphate buffer (NaH2PO4 0.1 M, 300 mM NaCl, 20 mM imidazole, pH 8.2) and loaded into a packed column containing 40 ml of NiNTA agarose beads (Amersham Biosciences). Extensive washing was performed with phosphate buffer containing 20 mM imidazole and eluted with buffers containing 60 and 100 mM imidazole. Elution of the recombinant VP28 was completed with phosphate buffer containing 250 mM imidazole. The protein concentration in the eluted fractions was estimated spectrophotometrically at 280 nm wavelength and fractions containing the purified protein were pooled and concentrated using the Amicon ultrafiltration device (Millipore, Billerica, MA, USA). The concentrated recombinant VP28 was applied to a Sephadex G-100 (Amersham Biosciences AB) column (2 95 cm) equilibrated with PBS, pH 7.4. Protein concentration was determined and stored at 80 C until use. The purified protein was further analyzed by SDS-PAGE and Western blot. Briefly, B. brevis carrying recombinant plasmid or the purified recombinant VP28 were separated in 5e20% precast e-PAGEL (ATTO Co., Tokyo, Japan) under reducing conditions and stained with Coomassie brilliant blue R-250. The separated proteins in a second gel were electro-blotted on an AE6667 (ATTO Co., Tokyo, Japan) membrane, and then incubated with anti-his tag monoclonal antibody (Amersham Biosciences) or a rabbit polyclonal antibody anti-VP28 of shrimp white spot syndrome virus (Genesis Biotech Inc.). The second antibody was rabbit anti-mouse IgG or goat anti-rabbit IgG conjugated with alkaline phosphatase (Promega, Madison, WI, USA. and the last interaction revealed with the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP-NBT) alkaline phosphatase substrate (Sigma-Aldrich, St. Louis, MO, USA). The WSSV ORF encoding VP28 fused with a His6 tag was overexpressed in B. brevis. The band corresponding to the His6 tag fusion protein was observed at the expected molecular weight of approximately 23 kDa (Fig. 1a). The purified recombinant viral protein was confirmed to be Vp28 by Western analysis with anti-WSSV polyclonal antiserum (Fig. 1b). We then used this recombinant VP28 for the feeding experiments. Feed pellets were first produced by mixing finely ground commercial shrimp feed with the purified recombinant VP28 dissolved in PBS. The mixture was placed in a 50 ml syringe and slowly extruded to form feed pellets. The extruded moist pellets were placed in a dark room at 4 C and allowed to dry. The dried pellets were mixed with 1% fish oil (w/v) [12] to prevent dispersion of the feed particles in the water. Similar preparations using supernatant of
Survival of Penaeus (Marsupenaeus) japonicus from WSSV
Figure 1 (A) SDS-PAGE and (B) Western blot analysis of recombinant VP28 (rVP28) expressed in Brevibacillus brevis. M, protein standard; lane 1, B. brevis carrying recombinant plasmid pNOH3-VP28 and expressing rVP28 (indicate with an arrowhead); lane 2, purified rVP28. Purified rVP28 (enclosed in box) was detected using rabbit polyclonal antibody anti-VP28 of WSSV.
sonicated B. brevis dissolved in PBS and PBS only were used as negative controls in the experiment. The amount of recombinant protein in the diet was calculated based on
Table 1
317 the average body weight of the shrimp, dose, feeding rate (5%) and duration of feeding. Four feeding experiments were conducted (Table 1). The duration of protection by the recombinant VP28 in juvenile shrimp was determined in the first and second experimental runs. In the third experimental run, the effective dose of the recombinant VP28 to protect the shrimp against WSSV disease and the onset of protection were determined. Finally, on the fourth experimental run, the efficiency of using recombinant VP28 in protecting commercial size shrimp against WSSV after oral administration was determined. There were six groups in the first experimental run. One group was fed with shrimp pellets containing 50 mg/shrimp of purified recombinant VP28 for 2 weeks, then challenged with the virus at 3 days post-feeding. Another group was fed with a similar preparation of the recombinant VP28 for 2 weeks, followed by a 2-week feeding of non-treated shrimp pellets. Two groups fed with shrimp pellets containing supernatant of sonicated B. brevis (1 mg ml1) for 2 weeks followed by virus challenge at 3 and 14 days postfeeding, respectively. Another two groups fed with normal shrimp pellets served as controls. In the second experimental run, three groups (Treatment 1, 2 and 3) were fed with shrimp pellets containing purified recombinant VP28 (50 mg/shrimp) for 2 weeks followed by feeding with non-treated shrimp pellets for 3, 7 and 14 days, respectively. This was immediately followed by virus challenge. Another three groups were fed following the preceding feeding scheme using the preparation containing supernatant of sonicated B. brevis (1 mg ml1) and a group fed with non-treated shrimp pellets were used as controls.
Set-up of the vaccination experiments
Experimental run
Treatment no.
Feeding scheme and virus challenge
No. of shrimp
1a
1 2 3 4 5 6
2 weeks feeding 50 mg/shrimp, challenge 3 days post-feeding 2 weeks feeding, challenge 2 weeks post-feeding Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 1 Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 2 Commercial shrimp feed Commercial shrimp feed
18 15 21 16 30 10
2
1 2 3 4 5 6 7
2 weeks feeding 50 mg/shrimp, challenge 3 days post-feeding 2 weeks feeding, challenge 1 week post-feeding 2 weeks feeding, challenge 2 weeks post-feeding Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 1 Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 2 Sonicated Bacillus, 1 mg ml1, scheme same as Treatment 3 Commercial shrimp feed
30 21 33 30 27 27 32
3
1 2 3 4 5
2 weeks feeding 50 mg/shrimp, challenge 3 days post-feeding 2 weeks 10 mg/shrimp, challenge 3 days post-feeding 2 weeks 1 mg/shrimp, challenge 3 days post-feeding 1 day 50 mg/shrimp, challenge 1 day post-feeding Commercial shrimp feed
30 28 27 30 33
4
1 2 3
3 days feeding 50 mg/shrimp, challenge 1 day post-feeding 1 week feeding 50 mg/shrimp, challenge 1 day post-feeding Commercial shrimp feed
14 16 16
a
In Experiment 1, Treatment 5 served as the control for Treatments 1 and 3, while Treatment 6 was the control for Treatments 2 and 4.
318
Figure 2 Timeemortality relationship of experiment 1. Juvenile shrimp were fed recombinant VP28 for 2 weeks followed by viral challenge at (A) 3 days and (B) 2 weeks post-feeding. Cumulative mortality rates of shrimp from the experimental groups, as indicated in Table 1 are plotted against time (15day period) after challenge.
Five experimental groups were used in the third experimental run. Three groups (Treatment 1, 2 and 3) were fed with shrimp pellets containing recombinant VP28 at a dose of 1, 10 and 50 mg/shrimp, respectively. The feeding scheme lasted for 2 weeks followed by feeding with nontreated shrimp pellets for 3 days before virus challenge. A group (Treatment 4) fed with shrimp pellets containing purified recombinant VP28 at a dose of 50 mg/shrimp for 1 day followed by virus challenge was included to determine whether the onset of protection is observed immediately after oral administration of the purified recombinant protein. Shrimp fed with the non-treated shrimp pellets were used in the control group (Treatment 5). To determine the efficiency of using the purified recombinant VP28 in protecting bigger-size shrimp against WSSV, commercial size shrimp (approximately 12e15 g body weight) were used in the fourth experimental run. Treatment 1 and 2 were composed of shrimp fed with shrimp pellets containing purified recombinant VP28 at a dose of 50 mg/shrimp for 3 and 7 days, respectively. The shrimp were challenged with the virus 1 day after the last feeding of the purified recombinant VP28-treated pellets. Treatment 3, which is composed of shrimp fed with the non-treated shrimp pellets, was used as a control. Adjustments were made at the start of the feeding scheme such that virus challenge would commence at the same time. The challenge tests were conducted by
C.M.A. Caipang et al. intramuscularly injecting individual shrimps in the experimental groups with 50 ml of the WSSV dilution (105 dilution of the virus stock solution) at a challenge pressure of at least 50%. Dead shrimp were collected twice a day to prevent cannibalism and mortality was monitored over a 15-day period. Statistical analysis was done using c2 test (SYSAT ver. 8, Chicago, Il.) at the 0.05 level of significance. The protection against WSSV disease after oral administration of the purified recombinant VP28 was assessed by calculating the relative percent survival [19]. The timeemortality relationships in the first experiment are shown in Fig. 2. In the first run (Fig. 2a), the groups fed with commercial shrimp pellets and those containing supernatant of sonicated B. brevis for 2 weeks followed by virus challenge at 3 days after last feeding, had cumulative mortalities of 76.7% and 90.5%, respectively. In contrast to the group fed with shrimp pellets containing purified recombinant VP28 (second run), a significantly lower cumulative mortality of 5.5% was observed (relative survival of 92.8%, Table 2). However, the relative survival decreased to 33.4% when the shrimp were challenged with the virus 14 days post-feeding, regardless of the treatment (Table 2, Fig. 2b). The relative survival values in the groups fed with sonicated B. brevis, and commercial feed only were 12.5% and 0%, respectively (Table 2). In the second experiment (Fig. 3), the group fed with purified recombinant protein for 2 weeks followed by WSSV
Table 2 Relative survival of shrimp in the different experimental runs Experimental run
Treatment no.
Survival (%)
Mortalitya
RPS (%)b
1
1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 1 2 3
94.5 60.0 9.5 56.2 23.3 50.0 70.0 57.1 27.3 6.7 14.8 18.5 3.1 83.3 67.9 66.7 76.7 15.2 78.6 81.2 18.2
5.5 40.0 90.5 43.8 76.7 50.0 30.0 42.9 72.7 93.3 85.2 81.5 96.9 16.7 32.1 33.3 23.3 84.8 21.4 18.8 81.2
92.8* 33.4 0 12.5 0 0 69.03* 55.8* 24.9 3.66 12.07 15.9 0 80.3* 62.09* 60.7* 72.5* 0 73.7* 76.9* 0
2
3
4
(1/18) (6/15) (19/21) (7/16) (23/30) (5/10) (9/30) (9/21) (24/33) (28/30) (23/27) (22/27) (31/32) (5/30) (9/28) (9/27) (7/30) (28/33) (3/14) (3/16) (13/16)
An asterisk denotes significantly different (p > 0.05) from the control group in each experimental run. a Values in parentheses indicate the ratio of dead shrimp over the total shrimp in each treatment. b Relative percentage survival (RPS) was calculated based on the formula of Amend, 1981 [19].
Survival of Penaeus (Marsupenaeus) japonicus from WSSV
319
challenge at 3 days after cessation of feeding resulted in a cumulative mortality of 30.0% (relative survival of 69.03%). Cumulative mortality increased when the groups were challenged with the virus at 7 and 14 days after the last day of feeding, with relative survival of 55.8% and 24.9%, respectively. The cumulative mortality in the sonicated B. brevis-fed groups ranged from 81.5% to 93.3%. The positive control (normal fed) group had 96.9% cumulative mortality, while the negative control group showed no mortality. To determine the effective dose that is required for protection against the viral disease, varying amounts (50, 10 and 1 mg/shrimp) of the purified recombinant VP28 were incorporated in the diet and fed to the shrimp for 2 weeks followed by virus challenge 3 days post-feeding. The lowest cumulative mortality (16.6%) was obtained in the group fed with 50 mg/shrimp of the purified recombinant VP28 (relative survival of 80.3%) (Fig. 4). The cumulative mortality in the group fed with 1 mg/shrimp of the purified recombinant VP28 was 33.3%, but its relative survival was 60.7%. Shrimp fed pellets containing 50 mg/shrimp of the purified recombinant VP28 for 1 day and then challenged with the virus 1 day after feeding showed a cumulative mortality of 23.3% (relative survival of 72.5%). The negative control group had a cumulative mortality of 84.8%.
The relative survival of larger shrimp (12e15 g) fed with recombinant VP28 for 3 and 7 days, immediately followed by virus challenge a day post-feeding was 73.6 and 76.9%, respectively (Fig. 5). The negative control group had relative survival of 91.3%. For each shrimp a total of 50 mg over a 2-week period followed by virus challenge at 3 days post-feeding gave the highest relative survival (92.8%). The duration of protection against the disease was 1 week post-feeding with recombinant VP28, if the shrimp were fed for 2 weeks at a dose of 50 mg/shrimp. The minimum dose of recombinant VP28 that is sufficient to provide protection against white spot syndrome is 1 mg/shrimp, if the shrimp are fed for at least 2 weeks. If shrimp are fed with recombinant VP28 at a dose of 50 mg/shrimp, they are effectively protected against the disease even at 1 day post-feeding. These results exhibit a positive correlation between protection and dosage, even if the treatment is administered for a short period of time (1e3 days), and show that the level of protection in shrimp against the disease significantly decreased at 2 weeks post-feeding regardless of the dose and duration of feeding. These results are similar to those of previous studies that showed decreased survival in shrimp at least 3 weeks post-feeding with recombinant VP28 [8,9] Such protection was suggested to be caused by the blocking of host cell receptors for WSSV of envelope proteins preventing viral infection and therefore maintenance of protection for about 2 weeks [20]. If this is the case, then the 50 mg/shrimp VP28 dose used in this study might have been enough to cover all receptors for WSSV, such that short-term treatment can already confer protection. Such a mechanism is supported by the finding that a low VP28 dose (1 mg/shrimp) can also induce protection if administered for a longer period (at least 2 weeks). We also actually analyzed whether inactivated bacteria induce a non-specific immune response when sonicated B. bacillus was used as one of the controls. Results showed that shrimp fed with the sonicated bacteria were not protected from subsequent infection. Similar findings were also obtained by Witteveldt and co-workers [8] using E. coli. We believe that feeding shrimp with inactivated bacteria does not trigger an immune response that would protect the shrimp against WSSV.
Figure 4 Timeemortality relationship of experiment 3. Shrimp were fed with varying levels of recombinant VP28 for 2 weeks and challenged with the virus at 3 days post-feeding. Cumulative mortality rates of shrimp from the experimental groups, as indicated in Table 1 are plotted against time (15day period) after challenge.
Figure 5 Timeemortality relationship of experiment 4. Marketable-sized shrimp (12e15 g body weight) were fed with recombinant VP28 at certain duration and challenged with WSSV. Cumulative mortality rates of shrimp from the experimental groups, as indicated in Table 1 are plotted against time (15-day period) after challenge.
Figure 3 Timeemortality relationship of experiment 2. Cumulative mortality rates of shrimp from the experimental groups, as indicated in Table 1 are plotted against time (15day period) after challenge.
320 By using B. brevis, a Gram-positive bacterium, to produce VP28, we were able to generate a higher relative survival than was achieved in previous studies that utilized Escherichia coli, a Gram-negative bacterium [8,9,12]. This is probably because Gram-positive bacteria secrete functional extracellular proteins directly into the culture medium, and are less toxic, i.e., they do not produce lipopolysaccharides (endotoxins) from the cell wall [16]. Taken together, our results show that oral administration of recombinant VP28 expressed in B. brevis was successful in protecting shrimp against WSSV, confirming previous reports of the efficacy of VP28 protein as a protective antigen in shrimp [8,9,21e23]. The exact mechanism that would explain the efficacy of VP28 is still not clear, although it is now known that VP28 is located on the surface of WSSV, which allows the virus particle to bind to shrimp cells and eventually enter the cytoplasm [17,24]. In conclusion, purified recombinant VP28 expressed in a Gram-positive bacterium, B. brevis, clearly protected shrimp against white spot syndrome disease. The use of this bacterial species in the production of recombinant protein eliminates the possibility of endotoxin contamination, which is common in Gram-negative bacteria. Hence, this study is a first step in addressing safety issues in producing vaccines for the shrimp aquaculture industry. Future studies will focus on optimizing the combination of the following parameters: dose of the recombinant protein, size of the shrimp, feeding duration and start of virus challenge, as these will pave the way in the application of this feeding scheme in protecting shrimp against WSSV infections on a large-scale basis.
Acknowledgements This study was supported in part by a grant from the Japan Science and Technology Corporation (JST): Creation and Support Program for Start-ups from Universities.
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