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An oral nervous necrosis virus vaccine that induces protective immunity in larvae of grouper (Epinephelus coioides)
Aquaculture 268 (2007) 265 – 273 www.elsevier.com/locate/aqua-online
An oral nervous necrosis virus vaccine that induces protective immunity in larvae of grouper (Epinephelus coioides) Chin-Chiu Lin 1 , John Han-You Lin 1 , Ming-Shyan Chen, Huey-Lang Yang ⁎ Institute of Biotechnology and Research Center of Ocean Environment and Technology, National Cheng Kung University, Tainan, 701, Taiwan
Abstract Nervous necrosis virus (NNV) is a major viral pathogen that infects grouper and other fish at their larval stage. This infection often causes mortality rates higher than 99% and leads to total losses in the hatchery; hence, it is important to develop a preventive vaccine. However, the onset of this disease at the larval stage, with fish small and sensitive to handling makes the vaccination by injection or immersion impossible. In this report, we describe an oral NNV vaccine composed of Artemia-encapsulated recombinant E. coli expressing the NNV capsid protein gene. The NNV VP-containing Artemia were used to vaccinate grouper larvae. Immuno-histochemical analysis showed antigen to be delivered to, and absorbed in, the hindgut of grouper, and that it induced anti-NNV VP specific antibodies 7 days after vaccination, as assayed by ELISA. The vaccinated larvae showed a certain degree of protection after challenge with NNV achieving a Relative Percentage Survival of 64.2% and 69.5%. Oral NNV vaccine could effectively immunize grouper larvae. This method could be expanded to the development of other oral vaccines and for use in other fish species. © 2007 Elsevier B.V. All rights reserved. Keywords: Oral delivery; Fish vaccine; Recombinant subunit vaccine; Artemia
1. Introduction Viral nervous necrosis (VNN), characterized by a spiral swimming pattern and vacuolation of nerve and retina cells in infected fish, is caused by nervous necrosis virus (NNV), and is a major pathogen of several economically important fish species worldwide. Nervous necrosis virus is a member of the family Nodaviridae, on the basis of its nucleic acid, genomic structure, protein properties and serological relationships (Mori et al., 1992, 2003). The several NNVs that have been described share similar genomic structure ⁎ Corresponding author. E-mail address: [email protected] (H.-L. Yang). 1 These two authors have equal contribution in this work. 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.04.066
with some differences in nucleotide sequences (Nishizawa et al., 1995; Munday et al., 2002). Viral nervous necrosis occurs in the larvae of many fish species, such as the Japanese parrotfish, Oplegnathus fasciatus (Yoshikoshi and Inoue, 1990), Barramundi, Lates calcarifer Bloch, (Glazebrook and Heasman, 1990), European sea bass, Dicentrarchus labrax (L.) (Breuil et al., 2001), turbot, Scophthalmus maximus (L.), (Bloch et al., 1991), and redspotted grouper, Epinephelus akaara (Mori et al., 1991). Viral nervous necrosis causes high moralities in larvae and postlarva stage, resulting in rapid and complete loss of fish in the hatchery, leading to serious economic losses for the aquaculture industry. Since there is no simple and economic treatment for fish viral disease, it is important to have a vaccine for prophylaxis.
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Effective NNV vaccines must be administered at the early larval stage before infection with NNV. Due to the small size of grouper larvae and their sensitivity to stress, an oral vaccination is a more suitable means of immunization than injection or immersion. The major drawback of oral vaccination is the gastrointestinal digestion of antigen. To evaluate the efficacy of an “undigested” oral vaccine, Johnson and Amend (1983) obtained excellent protection against pathogen challenge with a single dose of antigen administered anally to bypass gastric digestion. They showed the level of protection to be even greater than that induced by immersion or injection vaccinations. This suggested that the success of an oral vaccine would depend on a method that protects the antigen from gastro-intestinal digestion and effectively delivers antigen to the hindgut of fish. Subsequently, several oral fish vaccines have been developed (Lillehaug, 1989; Kawai et al., 1989; Campbell et al., 1993; Joosten et al., 1995; Gomez-Gil et al., 1998). Among these, the bio-encapsulation of bacteria using live Artemia (Joosten et al., 1995; Gomez-Gil et al., 1998) appears to be one of the most promising. The advantage of this oral vaccination method is that the antigen is contained in a natural starter feed (Artemia) for fish larvae, ensuring the uptake of antigen. Despite its advantages, many attempts to develop oral vaccines based on this approach have failed (Ellis, 1988; Horne and Ellis, 1988), in that they have been shown to induce a serum antibody titer but have not resulted in protective immunity (Joosten et al., 1995). Because Artemia feed on bacteria, the method has been more frequently used for the development of vaccines against bacterial pathogens than for viral pathogens. In this paper, the preparation of an oral NNV vaccine that uses Artemia to deliver recombinant E. coli containing and expressing the NNV capsid gene is
Table 1 The DNA sequence of NNV capsid protein used in this study
described. The protection of the antigen against digestion, delivery of antigen to the hindgut and the protective immunity were evaluated. 2. Materials and methods 2.1. Virus The nervous necrosis virus strain used as a challenge pathogen in this study was isolated from groupers (Epinephelus coioides) in Taiwan. The virus was cultured in a grouper cell line (GL-1) established from grouper liver cells in our laboratory and cultured in L15 medium (Invitrogen Life Technologies, CA, USA) at 28 °C with 5% fetal bovine serum (Highveld Biological Ltd, Lyndhurst, RSA) and 5% carbon dioxide (CO2). The virus was collected from the supernatant of the infected cell culture, passing through a 0.22 μm syringe-driven filter unit (Millex-GS; Millpore Corporation, MA, USA) and stored at −80 °C until use. The TCID50 of the virus was tested before each use (Shieh and Chi, 2005). 2.2. Preparation of oral vaccine Nervous necrosis virus has five open reading frames from two single positive strand of RNA (Mori et al., 1992). A recombinant plasmid pET24a-NNV VP containing the capsid open reading frame (Table 1) was transformed into E. coli BL21 (DE3); the recombinant E. coli was grown in LB broth with kanamycin (50 μg ml− 1) at 37 °C with shaking, and the expression of NNV capsid protein was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). When the optical absorption in the spectrophotometer, set at 600 nm (OD600), reached 0.6, IPTG was added to produce a final concentration of 1 mM.
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Bacterial cells were harvested after 150 min, washed twice with PBS, inactivated by re-suspending in PBS and treated with 3.7% formaldehyde, incubated at 4 °C for 16 h and finally dialyzed to remove formalin and stored at 4 °C until use. The inactivated bacteria were quantified by measuring OD600. Another recombinant plasmid pET24a containing plasmid only was used as a negative control. A recombinant plasmid pET24a-GFP containing green fluorescence protein open reading frame was used as an enrichment indicator. Both were treated in the same way. Artemia nauplii were hatched from cysts according to the supplier's instructions (INVE N.V., Belgium). The nauplii were suspended in aerated filtered sea water to a density of 500 Artemia ml− 1 before being fed the killed bacteria. The nauplii and bacteria mixture was gently aerated, at 25 °C for 120 min, centrifuged, and then washed with 0.22 μm filtered sea water to remove free bacteria. The Artemia containing bacteria were used live for oral vaccination or were frozen at − 20 °C for later use.
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2.3. Quality control of oral vaccine In order to quantify and control the quality of the NNV vaccine antigens, Artemia nauplii were added to a concentrated solution of E. coli expressing NNV VP protein to a final concentration of 108 bacteria ml− 1 and mixed with 107 ml− 1 of bacteria E. coli expressing GFP as indicator. In the placebo vaccine preparation, nauplii delivered E. coli expressing only pET-24a plasmid mixed with E. coli expressing GFP at the same concentration and ratio as for the NNV vaccine. The E. coli expressing GFP is a convenient indicator for the quality control and quantification of the oral vaccine preparation; this indicator was added with antigen in each oral vaccine preparation. The percentage of Artemia nauplii containing vaccine in each vaccine preparation was counted using fluorescence microscopy (Olympus IX70, Japan, 395 nm), and the amount of recombinant E. coli in each Artemia measured by assaying an aliquot of vaccine preparation using a fluorescent microtiter plate reader
Fig. 1. Oral vaccine incorporation of recombinant E. coli into different Artemia nauplii stage. Green fluorescence in the alimentary tract of nauplius II not seen in nauplius I or eggs. (a) Egg in light microscopy. (b) Egg in fluorescence microscopy. (c) Image combined from a to b. (d) Nauplius I in light microscopy. (e) Nauplius I in fluorescence microscopy. (f) Image combined from d to e. (g) Nauplius II in light microscopy. (h) Nauplius II in fluorescence microscopy. (i) Image combined from g and h.
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(Fluoroskan Ascent FL, Thermo Labsystem, Helsinki, Finland). The quantity of bacteria in each Artemia and the number of bacteria in each vaccine preparation were calculated based on a predetermined standard curve. 2.4. Experimental fish and vaccination Fertilized grouper eggs were obtained from a broodstock farm, and incubated in four 30 m3 tanks so that after hatching each contained about 10,000 larvae. The tanks were supplied with aerated, sand filtered, and UV treated sea water. Hatched larvae were fed live fry starter feed containing algae, rotifers and Artemia according to the procedure of Chi et al. (1999). The water was monitored daily for bacteria count and pH, as well as dissolved oxygen, ammonium, nitrite, and nitrate. Possible virus contamination in larvae and feed was evaluated by RT-PCR using a pair of NNV specific primers, (NNVLF 5′-CCAGCCGGGACAGGAACTGACG-3′ and NNVLR 5′-CCAGATGCCCCAGCGAAACCAG-3′) to confirm they were free from NNV. Larvae in 2 tanks were vaccinated orally and those in the other 2 tanks contained the unvaccinated control groups (placebo). A single dosage of the oral vaccine or placebo was given to fish, which were starved for 8 h, on 18 and 19 days-post-hatching (dph) for two days. At 35 dph, when the fish had reached about 1 cm body length and were still vulnerable to NNV, they were transferred to a laboratory aquarium for the challenge trail.
2.5. Immuno-histological evaluation of the uptake and stability of recombinant NNV antigen in the hindgut Twenty unvaccinated grouper (19 dph) were used to study the delivery of the NNV capsid protein to the hindgut. Ten fish were vaccinated with a single dose of oral vaccine delivered in Artemia fed on E. coli containing pET24a-NNV VP mixed with one tenth volume E. coli containing pET24a-GFP as indicator, as previously described. Ten control fish were given a dose of Artemia containing E. coli with pET24a. After 90 min, fish were killed by placing them on ice. Fish were dissected and their intestines observed under UV light at 395 nm. The alimentary canal was removed from each fish, fixed in buffered formalin for 24 h, rinsed, dehydrated with ethanol and embedded in paraffin. Paraffin sections of the stomach and anterior and posterior of intestines, were mounted on poly-L-lysinetreated slides (Sigma-Aldrich Co., St. Louis, USA). Location of the NNV VP antigen in the fish gut was identified using rabbit anti-NNV VP antibody that was prepared in our laboratory by immunizing rabbits with NNV viral particle and a goat anti-rabbit horseradish peroxidase (HRP) conjugate following the protocol suggested by the supplier (Leinco Technologies, Inc., St. Louis, MO, USA). Sections were counterstained with hematoxylin. Tissues containing NNV VP antigen stained reddish-brown.
Fig. 2. The tracing of fluorescent antigen in vaccinated fish as observed by UV light. a. Unvaccinated control fish (fed with placebo Artemia). b. Unvaccinated fish, with the alimentary tract exposed. c. Vaccinated fish. d. Vaccinated group fish with alimentary tract exposed. e. Figure showing the relative position of stomach and gut (modified from Powell and Tucker, 1992).
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2.6. Measurement of anti-NNV VP antibody Thirty vaccinated fish (35 dph) and 30 control fish were killed by covering them with ice and used for humoral antibody determination. Fish extract was prepared by homogenizing three whole fish from each group, in 5 ml of chilled PBS containing 0.02% sodium azide, with a glass tissue homogenizer. The homogenate was centrifuged at 12,000 ×g for 15 min, and the supernatant collected and stored at − 20 °C until use. Purified recombinant NNV coat protein was used as the coating antigen (10 μg well− 1) on the micro-titer plate and fish extract was used directly as the primary antibody. Rabbit anti-grouper immunoglobulin serum was used as secondary antibody that was prepared in our laboratory by immunizing rabbits with a liquid chromatography-purified grouper immunoglobulin (grouper Ig) following the method of Watts et al. (2001). Commercial goat anti-rabbit antibody alkaline phosphatase conjugate (Bethyl Laboratories, Montgomery, TX, USA) was used for the tertiary antibody. The secondary and tertiary antibodies were diluted 1:1000 immediately before use with PBS containing 3% skimmed milk. Chromogen (0.1 ml of p-nitrophenyl phosphate [1 mgml− 1; pNPP, Sigma Chemical Co., St. Louis, MO, USA] in 10% diethanolamine buffer, pH 9.8) was added to each reaction well, and color allowed developing for 30 min at 37 °C. The resulting reaction was measured in a micro titer plate reader (Multiskan RC, Labsystems, Helsinki, Finland) at 405 nm.
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four weeks. The Relative Percentage Survival (RPS) was calculated as described by Amend (1981). 3. Results 3.1. Cloning NNV coat protein The major NNV protein is a viral capsid protein (NNV VP) which was cloned and used as antigen for our NNV oral vaccine. Our NNV VP nucleotide sequence was similar to that of the RGNNV (Genebank AY690596, AY744705) with a DNA sequence homology of 98.8%. 3.2. Optimum conditions for incorporating recombinant E. coli into Artemia The optimal stage of the nauplius for incorporating vaccine is important; first nauplii or later are most
2.7. Challenge test In each test, five groups of 25 unvaccinated grouper (35 dph) were used for the determination of the challenge dose of NNV. Virus was diluted in L15 medium and four groups were injected intra-peritoneally (IP) with 107, 105, 103 or 101 TCID50 fish− 1, respectively. One group was injected with L15 medium as a negative control. The survival rate was monitored for 4 weeks. The dose causing 60% mortality (LD60) was used for the subsequent challenge. The lethal dosage has been repeated twice to ensure its reproducibility. The LD60 was also checked in each batch of larvae before each trail. Two separate challenge trials were performed to evaluate the efficacy of the oral vaccination. About 25 vaccinated fish and 25 control fish (35 dph) were used in the first trial and 35 vaccinated fish and 50 nonvaccinated fish employed in the second trial. Fish were IP injected with NNV at a predetermined LD60 dose. Dead fish were counted and removed twice a day during
Fig. 3. The delivery of antigen to the intestine of vaccinated fish. Tissue section of hindgut from unvaccinated grouper (a) and vaccinated grouper (b), (bar = 20 μm).
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suitable for incorporation of recombinant E. coli (Fig. 1). There were 105 bacteria in each Artemia and 80–90% contained antigen after a 2 h incubation. 3.3. Delivery of NNV VP antigen to the hindgut of fish The delivery of NNV VP antigen to the hindgut of grouper larvae was demonstrated directly by fluorescence and indirectly by immuno-histology. In the study using Artemia containing E. coli expressing GFP protein, there was fluorescence in the mouth as soon as the larvae started feeding, fluorescent GFP was seen in the whole digestive canal within 45 min. Nervous necrosis virus VP appeared in the hindgut within 60 min, peaked at 90– 180 min after oral vaccination and was undetectable after 360 min (Fig. 2). Immune staining detected NNV-VP antigen on the epithelium of the villi at high magnification (Fig. 3). It indicated that antigen was taken up by epithelium cells of the hindgut as was observed in a similar study of oral vaccination in zebra fish (Lin et al., 2005b). No colorimetric background signal or fluorescence was observed in fish vaccinated with placebo. 3.4. Oral vaccination induced specific antibody Seven days post-vaccination, the absorbance in an ELISA test for specific anti-NNV VP antibody in the vaccinated fish was higher than those of the control fish (Fig. 4) indicating that oral vaccination could induce a specific immune reaction. There was a significant difference in mean absorbance in the ELISA assay in vaccinated fish (0.323) compared to control fish (0.137) (χ2 P b 0.01).
Fig. 5. Survival rates of grouper IP-challenged with different doses of NNV virus. (–○–, injected L15medium only, as negative control;–♦– 101 TCID50 fish− 1;–□–103 TCID50 fish− 1;–■–105 TCID50 fish− 1; –▵–107 TCID50 fish− 1).
3.5. Determination of challenge dose In the challenge using fish larvae at 35 dph, the onset of disease symptoms began 5–6 days post-IP challenge. The first clinical signs included spiral swimming behavior, darkened body color and loss of appetite, as seen in hatchery infections. Sick fish died within 7 days after the first appearance of symptoms. Maximum mortality appeared during weeks 2–4. Mortality was not observed after week 4. The final survival rate from different challenge doses was 20% in 107 TCID50 fish− 1, 28% in 105 TCID50 fish− 1, 56% in 103 TCID50 fish− 1, 76% in 101 TCID50 fish− 1, and 92% in control fish injected only with incubation medium (L15). Mortality was correlated with the dose. The 60% lethal dosage (LD60) was between 103 and 105 TCID50 (Fig. 5) a dosage of 105 TCID50 fish− 1 was used in our challenge trial according to the guideline of Amend (1981). Larvae of E. coioides after 45 dph were found resistant to the injection of 107 TCID50 fish− 1 (unpublished data). The determination of the
Table 2 Survival rate and relative percentage survival of grouper challenged with NNV virus Challenge Survival rate % (survival /total) test Vaccinated Control Relative percentage group group survival % A B Fig. 4. ELISA analysis for NNV-specific antibody in vaccinated and unvaccinated grouper.
80 (20/25) 86 (30/35)
44 (11/25) 54 (27/50)
64.2 69.5
After 8 weeks challenge test in vaccinated and unvaccinated grouper with the dose of 105 TCID50 fish− 1.
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challenge dosage was repeated twice. The dosage was found reproducible as long as the quality and age of larvae were carefully monitored (Lin et al., 2005a). 3.6. Oral vaccine induced protective immunity Protection conferred by oral vaccination was evaluated with a replicated challenge. Fish were injected IP with NNV virus at 105 TCID50 fish− 1. Survival rate was evaluated for 4 weeks. The final survival rate for the vaccinated fish was 80% and 86%. For control fish it was 44% and 54% while the RPS was 64.2% and 69.5%, respectively (Table 2). The results demonstrate that oral NNV vaccine induces protective immunity in grouper larvae challenged with NNV virus. 4. Discussion This study demonstrates that an oral recombinant subunit vaccine using Artemia as a delivery vehicle induces a protective immune response. The improved efficacy of this vaccine over other oral vaccines may be due to several factors: first, using the natural starter feed for fish larvae facilitated the uptake of vaccine by grouper larvae; second, two biolayers, the E. coli cell wall and the Artemia cuticle, protected the antigen from gastrointestinal degradation and enabled delivery of sufficient antigen to the hindgut; third, the quantity of selected antigen expressed in each recombinant E. coli is much larger than that in a natural pathogen, often 1000 fold, and consequently increased the quantity of antigen in each Artemia-based oral vaccine dose (Lin et al., 2005b); fourth, most of the antigen expressed in the recombinant bacterium is in insoluble inclusion form, which might be more resistant to digestion, hence providing a slow release of antigen into the hindgut; and finally, some of the incorporated E. coli, when digested and unwrapped gradually in the fish intestine, might be still intact and recognized by macrophages in the mucosal layer of the hindgut producing an innate immune response. Although numerous reports suggest that oral vaccines may only confer local mucosal immunity but not produce a serum antibody titer (Anderson and Ross, 1972; Baudin-Laurencin and Tangtronpiros, 1980; Kawai et al., 1981), several other reports suggest that oral vaccination leads to a systemic immune reaction as indicated by the presence of antibodies in mucus and bile obtained from the gut and serum (Kusuda et al., 1978; Kawai and Kusuda, 1985; Dec et al., 1990). Zebra fish treated with a similar Artemia oral vaccine containing rPE antigen, developed protection against PE toxin
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injected into muscles, indicating that an oral vaccine might induce systemic immunity (Lin et al., 2005b). The specific antibody ELISA assay using fish serum, further demonstrated that oral vaccination in grouper induces a systemic immune response. Although the antibody titer was lower than in fish immunized by injection, the results of a challenge test revealed it provided sufficient protection. The protection might be due to both humoral and innate immunity (Quentel and Vigneulle, 1997). This NNV oral vaccine protected grouper larvae from VNN during the susceptible period for NNV infection including the larvae and post-larva stage (14– 40 dph). The duration of protective immunity induced by this oral vaccine is difficult to be determined due to larvae developing resistance to NNV infection after 45 dph. Lacking any previous experience as guidance, the schedule of vaccination at the larvae stage was based on the following information: histology and histochemical enzyme-staining patterns of major immune organs (Lin et al., 2005a) and detection of expression of rag-1 gene as an immune development marker (unpublished data) in various organs of E. malabaricus indicated that the immune system of grouper larvae could function as early as 10 dph. Nervous necrosis virus symptoms and mortality commonly started at 35–42 dph in the hatchery in Taiwan, and larvae began feeding on small Artemia nauplii at 18–20 dph. Our vaccination schedule was tentatively decided as feeding vaccine or placebo for two consecutive days beginning at 18 dph, then allowing 17 days for the immune response to develop, followed by challenge with NNV at 35 dph. To establish the virus challenge method the infection of grouper larvae with NNVeither by IP or by immersion was evaluated (data not shown). The injection method was usually more reliable in producing symptoms and in mortality with the predetermined lethal dose. With this challenge method, symptoms of VNN occurred in 7– 28 days, whereas the symptoms of VNN resulting from the immersion method were observed around 7–42 days. Both methods resulted in similar pathological features: the typical spiral swimming pattern and vacuolation in the retina, brain, and nerve tissues as observed in naturally-infected fish. Based on these data, we decided to use the injection challenge. We have also found that larvae become resistant to the injected challenge at 107 TCID50 after 45 dph, and hatchery farmers have observed that less VNN occurs after 60 dph. This interesting mechanism of resistance in larger larvae merits further investigation. Early vaccination of fish larvae is desirable. Attempts to encapsulate the recombinant E. coli in Artemia and stype rotifer to allow earlier vaccination has yielded
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promising results (data not shown). However, earlier immunization might induce tolerance before maturation of the grouper's immune system. The development of the immune system, the time of first feeding of Artemia and period of susceptibility to NNV might be different in each fish species, therefore the optimization of the vaccination schedule for a specific fish is required. For example, grouper, such as E. lanceolatus, showed to be susceptible to NNV later than E. coioides and lasted even up to 500 g. It probably could be vaccinated successfully at a later fingerling stage. Regarding the time of vaccination and challenge, length, mass and age (dpf, or day post-hatching; or dph), and appearance of stripes have been used to define the developmental stages of various species of fish larvae (Chantanachookhin et al., 1991; Padros and Crespo, 1996; Schroder et al., 1998).This is particularly important for commercially hatched larvae, as the development of fish from hatching to juvenile will depend on seasonal and nutritional conditions. Although we used dph as the index of age in this study, we suggest that the time of vaccination should be determined at least based on these three indicators. Any selected gene of a specific pathogen can be cloned and expressed in E. coli or other suitable bacterial host using recombinant technology, a feature that is particularly useful for the development of oral subunit vaccines for fish. Furthermore, a multi-valent oral vaccine against several pathogens could be developed using the same technology, which is safer, non-stressful, user-friendly, and more versatile than other methods currently available for vaccinating fish. Acknowledgments This study was supported by grants of No.92-2317-B006-002 (2003), and 93-2317-B-006-001 (2004) from National Program on Agricultural Biotechnology, National Science Council, Executive Yuan, Taiwan. We also like to thank Dr. H-M Su and Mr. W-M Lee for their kind advice and help with grouper larva culture and deeply appreciate Dr. Alan Pike and Ms. Kathleen Hills of the Lucidus Consultancy for editing of this manuscript. References Amend, D.F., 1981. Potency Testing of Fish Vaccines. Karger, Basel. 447–454 pp. Anderson, D.P., Ross, A.J., 1972. Comparative study of Hagerman redmouth disease oral bacterin. Prog. Fish-Cult. 34, 226–228. Baudin-Laurencin, F., Tangtronpiros, J., 1980. Some results of vaccination against vibriosis in Brittany. Springer-Verlag, Berlin. 60–68 pp.
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An oral nervous necrosis virus vaccine that induces protective immunity in larvae of grouper (Epinephelus coioides) Chin-Chiu Lin 1 , John Han-You Lin 1 , Ming-Shyan Chen, Huey-Lang Yang ⁎ Institute of Biotechnology and Research Center of Ocean Environment and Technology, National Cheng Kung University, Tainan, 701, Taiwan
Abstract Nervous necrosis virus (NNV) is a major viral pathogen that infects grouper and other fish at their larval stage. This infection often causes mortality rates higher than 99% and leads to total losses in the hatchery; hence, it is important to develop a preventive vaccine. However, the onset of this disease at the larval stage, with fish small and sensitive to handling makes the vaccination by injection or immersion impossible. In this report, we describe an oral NNV vaccine composed of Artemia-encapsulated recombinant E. coli expressing the NNV capsid protein gene. The NNV VP-containing Artemia were used to vaccinate grouper larvae. Immuno-histochemical analysis showed antigen to be delivered to, and absorbed in, the hindgut of grouper, and that it induced anti-NNV VP specific antibodies 7 days after vaccination, as assayed by ELISA. The vaccinated larvae showed a certain degree of protection after challenge with NNV achieving a Relative Percentage Survival of 64.2% and 69.5%. Oral NNV vaccine could effectively immunize grouper larvae. This method could be expanded to the development of other oral vaccines and for use in other fish species. © 2007 Elsevier B.V. All rights reserved. Keywords: Oral delivery; Fish vaccine; Recombinant subunit vaccine; Artemia
1. Introduction Viral nervous necrosis (VNN), characterized by a spiral swimming pattern and vacuolation of nerve and retina cells in infected fish, is caused by nervous necrosis virus (NNV), and is a major pathogen of several economically important fish species worldwide. Nervous necrosis virus is a member of the family Nodaviridae, on the basis of its nucleic acid, genomic structure, protein properties and serological relationships (Mori et al., 1992, 2003). The several NNVs that have been described share similar genomic structure ⁎ Corresponding author. E-mail address: [email protected] (H.-L. Yang). 1 These two authors have equal contribution in this work. 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.04.066
with some differences in nucleotide sequences (Nishizawa et al., 1995; Munday et al., 2002). Viral nervous necrosis occurs in the larvae of many fish species, such as the Japanese parrotfish, Oplegnathus fasciatus (Yoshikoshi and Inoue, 1990), Barramundi, Lates calcarifer Bloch, (Glazebrook and Heasman, 1990), European sea bass, Dicentrarchus labrax (L.) (Breuil et al., 2001), turbot, Scophthalmus maximus (L.), (Bloch et al., 1991), and redspotted grouper, Epinephelus akaara (Mori et al., 1991). Viral nervous necrosis causes high moralities in larvae and postlarva stage, resulting in rapid and complete loss of fish in the hatchery, leading to serious economic losses for the aquaculture industry. Since there is no simple and economic treatment for fish viral disease, it is important to have a vaccine for prophylaxis.
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Effective NNV vaccines must be administered at the early larval stage before infection with NNV. Due to the small size of grouper larvae and their sensitivity to stress, an oral vaccination is a more suitable means of immunization than injection or immersion. The major drawback of oral vaccination is the gastrointestinal digestion of antigen. To evaluate the efficacy of an “undigested” oral vaccine, Johnson and Amend (1983) obtained excellent protection against pathogen challenge with a single dose of antigen administered anally to bypass gastric digestion. They showed the level of protection to be even greater than that induced by immersion or injection vaccinations. This suggested that the success of an oral vaccine would depend on a method that protects the antigen from gastro-intestinal digestion and effectively delivers antigen to the hindgut of fish. Subsequently, several oral fish vaccines have been developed (Lillehaug, 1989; Kawai et al., 1989; Campbell et al., 1993; Joosten et al., 1995; Gomez-Gil et al., 1998). Among these, the bio-encapsulation of bacteria using live Artemia (Joosten et al., 1995; Gomez-Gil et al., 1998) appears to be one of the most promising. The advantage of this oral vaccination method is that the antigen is contained in a natural starter feed (Artemia) for fish larvae, ensuring the uptake of antigen. Despite its advantages, many attempts to develop oral vaccines based on this approach have failed (Ellis, 1988; Horne and Ellis, 1988), in that they have been shown to induce a serum antibody titer but have not resulted in protective immunity (Joosten et al., 1995). Because Artemia feed on bacteria, the method has been more frequently used for the development of vaccines against bacterial pathogens than for viral pathogens. In this paper, the preparation of an oral NNV vaccine that uses Artemia to deliver recombinant E. coli containing and expressing the NNV capsid gene is
Table 1 The DNA sequence of NNV capsid protein used in this study
described. The protection of the antigen against digestion, delivery of antigen to the hindgut and the protective immunity were evaluated. 2. Materials and methods 2.1. Virus The nervous necrosis virus strain used as a challenge pathogen in this study was isolated from groupers (Epinephelus coioides) in Taiwan. The virus was cultured in a grouper cell line (GL-1) established from grouper liver cells in our laboratory and cultured in L15 medium (Invitrogen Life Technologies, CA, USA) at 28 °C with 5% fetal bovine serum (Highveld Biological Ltd, Lyndhurst, RSA) and 5% carbon dioxide (CO2). The virus was collected from the supernatant of the infected cell culture, passing through a 0.22 μm syringe-driven filter unit (Millex-GS; Millpore Corporation, MA, USA) and stored at −80 °C until use. The TCID50 of the virus was tested before each use (Shieh and Chi, 2005). 2.2. Preparation of oral vaccine Nervous necrosis virus has five open reading frames from two single positive strand of RNA (Mori et al., 1992). A recombinant plasmid pET24a-NNV VP containing the capsid open reading frame (Table 1) was transformed into E. coli BL21 (DE3); the recombinant E. coli was grown in LB broth with kanamycin (50 μg ml− 1) at 37 °C with shaking, and the expression of NNV capsid protein was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). When the optical absorption in the spectrophotometer, set at 600 nm (OD600), reached 0.6, IPTG was added to produce a final concentration of 1 mM.
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Bacterial cells were harvested after 150 min, washed twice with PBS, inactivated by re-suspending in PBS and treated with 3.7% formaldehyde, incubated at 4 °C for 16 h and finally dialyzed to remove formalin and stored at 4 °C until use. The inactivated bacteria were quantified by measuring OD600. Another recombinant plasmid pET24a containing plasmid only was used as a negative control. A recombinant plasmid pET24a-GFP containing green fluorescence protein open reading frame was used as an enrichment indicator. Both were treated in the same way. Artemia nauplii were hatched from cysts according to the supplier's instructions (INVE N.V., Belgium). The nauplii were suspended in aerated filtered sea water to a density of 500 Artemia ml− 1 before being fed the killed bacteria. The nauplii and bacteria mixture was gently aerated, at 25 °C for 120 min, centrifuged, and then washed with 0.22 μm filtered sea water to remove free bacteria. The Artemia containing bacteria were used live for oral vaccination or were frozen at − 20 °C for later use.
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2.3. Quality control of oral vaccine In order to quantify and control the quality of the NNV vaccine antigens, Artemia nauplii were added to a concentrated solution of E. coli expressing NNV VP protein to a final concentration of 108 bacteria ml− 1 and mixed with 107 ml− 1 of bacteria E. coli expressing GFP as indicator. In the placebo vaccine preparation, nauplii delivered E. coli expressing only pET-24a plasmid mixed with E. coli expressing GFP at the same concentration and ratio as for the NNV vaccine. The E. coli expressing GFP is a convenient indicator for the quality control and quantification of the oral vaccine preparation; this indicator was added with antigen in each oral vaccine preparation. The percentage of Artemia nauplii containing vaccine in each vaccine preparation was counted using fluorescence microscopy (Olympus IX70, Japan, 395 nm), and the amount of recombinant E. coli in each Artemia measured by assaying an aliquot of vaccine preparation using a fluorescent microtiter plate reader
Fig. 1. Oral vaccine incorporation of recombinant E. coli into different Artemia nauplii stage. Green fluorescence in the alimentary tract of nauplius II not seen in nauplius I or eggs. (a) Egg in light microscopy. (b) Egg in fluorescence microscopy. (c) Image combined from a to b. (d) Nauplius I in light microscopy. (e) Nauplius I in fluorescence microscopy. (f) Image combined from d to e. (g) Nauplius II in light microscopy. (h) Nauplius II in fluorescence microscopy. (i) Image combined from g and h.
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(Fluoroskan Ascent FL, Thermo Labsystem, Helsinki, Finland). The quantity of bacteria in each Artemia and the number of bacteria in each vaccine preparation were calculated based on a predetermined standard curve. 2.4. Experimental fish and vaccination Fertilized grouper eggs were obtained from a broodstock farm, and incubated in four 30 m3 tanks so that after hatching each contained about 10,000 larvae. The tanks were supplied with aerated, sand filtered, and UV treated sea water. Hatched larvae were fed live fry starter feed containing algae, rotifers and Artemia according to the procedure of Chi et al. (1999). The water was monitored daily for bacteria count and pH, as well as dissolved oxygen, ammonium, nitrite, and nitrate. Possible virus contamination in larvae and feed was evaluated by RT-PCR using a pair of NNV specific primers, (NNVLF 5′-CCAGCCGGGACAGGAACTGACG-3′ and NNVLR 5′-CCAGATGCCCCAGCGAAACCAG-3′) to confirm they were free from NNV. Larvae in 2 tanks were vaccinated orally and those in the other 2 tanks contained the unvaccinated control groups (placebo). A single dosage of the oral vaccine or placebo was given to fish, which were starved for 8 h, on 18 and 19 days-post-hatching (dph) for two days. At 35 dph, when the fish had reached about 1 cm body length and were still vulnerable to NNV, they were transferred to a laboratory aquarium for the challenge trail.
2.5. Immuno-histological evaluation of the uptake and stability of recombinant NNV antigen in the hindgut Twenty unvaccinated grouper (19 dph) were used to study the delivery of the NNV capsid protein to the hindgut. Ten fish were vaccinated with a single dose of oral vaccine delivered in Artemia fed on E. coli containing pET24a-NNV VP mixed with one tenth volume E. coli containing pET24a-GFP as indicator, as previously described. Ten control fish were given a dose of Artemia containing E. coli with pET24a. After 90 min, fish were killed by placing them on ice. Fish were dissected and their intestines observed under UV light at 395 nm. The alimentary canal was removed from each fish, fixed in buffered formalin for 24 h, rinsed, dehydrated with ethanol and embedded in paraffin. Paraffin sections of the stomach and anterior and posterior of intestines, were mounted on poly-L-lysinetreated slides (Sigma-Aldrich Co., St. Louis, USA). Location of the NNV VP antigen in the fish gut was identified using rabbit anti-NNV VP antibody that was prepared in our laboratory by immunizing rabbits with NNV viral particle and a goat anti-rabbit horseradish peroxidase (HRP) conjugate following the protocol suggested by the supplier (Leinco Technologies, Inc., St. Louis, MO, USA). Sections were counterstained with hematoxylin. Tissues containing NNV VP antigen stained reddish-brown.
Fig. 2. The tracing of fluorescent antigen in vaccinated fish as observed by UV light. a. Unvaccinated control fish (fed with placebo Artemia). b. Unvaccinated fish, with the alimentary tract exposed. c. Vaccinated fish. d. Vaccinated group fish with alimentary tract exposed. e. Figure showing the relative position of stomach and gut (modified from Powell and Tucker, 1992).
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2.6. Measurement of anti-NNV VP antibody Thirty vaccinated fish (35 dph) and 30 control fish were killed by covering them with ice and used for humoral antibody determination. Fish extract was prepared by homogenizing three whole fish from each group, in 5 ml of chilled PBS containing 0.02% sodium azide, with a glass tissue homogenizer. The homogenate was centrifuged at 12,000 ×g for 15 min, and the supernatant collected and stored at − 20 °C until use. Purified recombinant NNV coat protein was used as the coating antigen (10 μg well− 1) on the micro-titer plate and fish extract was used directly as the primary antibody. Rabbit anti-grouper immunoglobulin serum was used as secondary antibody that was prepared in our laboratory by immunizing rabbits with a liquid chromatography-purified grouper immunoglobulin (grouper Ig) following the method of Watts et al. (2001). Commercial goat anti-rabbit antibody alkaline phosphatase conjugate (Bethyl Laboratories, Montgomery, TX, USA) was used for the tertiary antibody. The secondary and tertiary antibodies were diluted 1:1000 immediately before use with PBS containing 3% skimmed milk. Chromogen (0.1 ml of p-nitrophenyl phosphate [1 mgml− 1; pNPP, Sigma Chemical Co., St. Louis, MO, USA] in 10% diethanolamine buffer, pH 9.8) was added to each reaction well, and color allowed developing for 30 min at 37 °C. The resulting reaction was measured in a micro titer plate reader (Multiskan RC, Labsystems, Helsinki, Finland) at 405 nm.
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four weeks. The Relative Percentage Survival (RPS) was calculated as described by Amend (1981). 3. Results 3.1. Cloning NNV coat protein The major NNV protein is a viral capsid protein (NNV VP) which was cloned and used as antigen for our NNV oral vaccine. Our NNV VP nucleotide sequence was similar to that of the RGNNV (Genebank AY690596, AY744705) with a DNA sequence homology of 98.8%. 3.2. Optimum conditions for incorporating recombinant E. coli into Artemia The optimal stage of the nauplius for incorporating vaccine is important; first nauplii or later are most
2.7. Challenge test In each test, five groups of 25 unvaccinated grouper (35 dph) were used for the determination of the challenge dose of NNV. Virus was diluted in L15 medium and four groups were injected intra-peritoneally (IP) with 107, 105, 103 or 101 TCID50 fish− 1, respectively. One group was injected with L15 medium as a negative control. The survival rate was monitored for 4 weeks. The dose causing 60% mortality (LD60) was used for the subsequent challenge. The lethal dosage has been repeated twice to ensure its reproducibility. The LD60 was also checked in each batch of larvae before each trail. Two separate challenge trials were performed to evaluate the efficacy of the oral vaccination. About 25 vaccinated fish and 25 control fish (35 dph) were used in the first trial and 35 vaccinated fish and 50 nonvaccinated fish employed in the second trial. Fish were IP injected with NNV at a predetermined LD60 dose. Dead fish were counted and removed twice a day during
Fig. 3. The delivery of antigen to the intestine of vaccinated fish. Tissue section of hindgut from unvaccinated grouper (a) and vaccinated grouper (b), (bar = 20 μm).
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suitable for incorporation of recombinant E. coli (Fig. 1). There were 105 bacteria in each Artemia and 80–90% contained antigen after a 2 h incubation. 3.3. Delivery of NNV VP antigen to the hindgut of fish The delivery of NNV VP antigen to the hindgut of grouper larvae was demonstrated directly by fluorescence and indirectly by immuno-histology. In the study using Artemia containing E. coli expressing GFP protein, there was fluorescence in the mouth as soon as the larvae started feeding, fluorescent GFP was seen in the whole digestive canal within 45 min. Nervous necrosis virus VP appeared in the hindgut within 60 min, peaked at 90– 180 min after oral vaccination and was undetectable after 360 min (Fig. 2). Immune staining detected NNV-VP antigen on the epithelium of the villi at high magnification (Fig. 3). It indicated that antigen was taken up by epithelium cells of the hindgut as was observed in a similar study of oral vaccination in zebra fish (Lin et al., 2005b). No colorimetric background signal or fluorescence was observed in fish vaccinated with placebo. 3.4. Oral vaccination induced specific antibody Seven days post-vaccination, the absorbance in an ELISA test for specific anti-NNV VP antibody in the vaccinated fish was higher than those of the control fish (Fig. 4) indicating that oral vaccination could induce a specific immune reaction. There was a significant difference in mean absorbance in the ELISA assay in vaccinated fish (0.323) compared to control fish (0.137) (χ2 P b 0.01).
Fig. 5. Survival rates of grouper IP-challenged with different doses of NNV virus. (–○–, injected L15medium only, as negative control;–♦– 101 TCID50 fish− 1;–□–103 TCID50 fish− 1;–■–105 TCID50 fish− 1; –▵–107 TCID50 fish− 1).
3.5. Determination of challenge dose In the challenge using fish larvae at 35 dph, the onset of disease symptoms began 5–6 days post-IP challenge. The first clinical signs included spiral swimming behavior, darkened body color and loss of appetite, as seen in hatchery infections. Sick fish died within 7 days after the first appearance of symptoms. Maximum mortality appeared during weeks 2–4. Mortality was not observed after week 4. The final survival rate from different challenge doses was 20% in 107 TCID50 fish− 1, 28% in 105 TCID50 fish− 1, 56% in 103 TCID50 fish− 1, 76% in 101 TCID50 fish− 1, and 92% in control fish injected only with incubation medium (L15). Mortality was correlated with the dose. The 60% lethal dosage (LD60) was between 103 and 105 TCID50 (Fig. 5) a dosage of 105 TCID50 fish− 1 was used in our challenge trial according to the guideline of Amend (1981). Larvae of E. coioides after 45 dph were found resistant to the injection of 107 TCID50 fish− 1 (unpublished data). The determination of the
Table 2 Survival rate and relative percentage survival of grouper challenged with NNV virus Challenge Survival rate % (survival /total) test Vaccinated Control Relative percentage group group survival % A B Fig. 4. ELISA analysis for NNV-specific antibody in vaccinated and unvaccinated grouper.
80 (20/25) 86 (30/35)
44 (11/25) 54 (27/50)
64.2 69.5
After 8 weeks challenge test in vaccinated and unvaccinated grouper with the dose of 105 TCID50 fish− 1.
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challenge dosage was repeated twice. The dosage was found reproducible as long as the quality and age of larvae were carefully monitored (Lin et al., 2005a). 3.6. Oral vaccine induced protective immunity Protection conferred by oral vaccination was evaluated with a replicated challenge. Fish were injected IP with NNV virus at 105 TCID50 fish− 1. Survival rate was evaluated for 4 weeks. The final survival rate for the vaccinated fish was 80% and 86%. For control fish it was 44% and 54% while the RPS was 64.2% and 69.5%, respectively (Table 2). The results demonstrate that oral NNV vaccine induces protective immunity in grouper larvae challenged with NNV virus. 4. Discussion This study demonstrates that an oral recombinant subunit vaccine using Artemia as a delivery vehicle induces a protective immune response. The improved efficacy of this vaccine over other oral vaccines may be due to several factors: first, using the natural starter feed for fish larvae facilitated the uptake of vaccine by grouper larvae; second, two biolayers, the E. coli cell wall and the Artemia cuticle, protected the antigen from gastrointestinal degradation and enabled delivery of sufficient antigen to the hindgut; third, the quantity of selected antigen expressed in each recombinant E. coli is much larger than that in a natural pathogen, often 1000 fold, and consequently increased the quantity of antigen in each Artemia-based oral vaccine dose (Lin et al., 2005b); fourth, most of the antigen expressed in the recombinant bacterium is in insoluble inclusion form, which might be more resistant to digestion, hence providing a slow release of antigen into the hindgut; and finally, some of the incorporated E. coli, when digested and unwrapped gradually in the fish intestine, might be still intact and recognized by macrophages in the mucosal layer of the hindgut producing an innate immune response. Although numerous reports suggest that oral vaccines may only confer local mucosal immunity but not produce a serum antibody titer (Anderson and Ross, 1972; Baudin-Laurencin and Tangtronpiros, 1980; Kawai et al., 1981), several other reports suggest that oral vaccination leads to a systemic immune reaction as indicated by the presence of antibodies in mucus and bile obtained from the gut and serum (Kusuda et al., 1978; Kawai and Kusuda, 1985; Dec et al., 1990). Zebra fish treated with a similar Artemia oral vaccine containing rPE antigen, developed protection against PE toxin
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injected into muscles, indicating that an oral vaccine might induce systemic immunity (Lin et al., 2005b). The specific antibody ELISA assay using fish serum, further demonstrated that oral vaccination in grouper induces a systemic immune response. Although the antibody titer was lower than in fish immunized by injection, the results of a challenge test revealed it provided sufficient protection. The protection might be due to both humoral and innate immunity (Quentel and Vigneulle, 1997). This NNV oral vaccine protected grouper larvae from VNN during the susceptible period for NNV infection including the larvae and post-larva stage (14– 40 dph). The duration of protective immunity induced by this oral vaccine is difficult to be determined due to larvae developing resistance to NNV infection after 45 dph. Lacking any previous experience as guidance, the schedule of vaccination at the larvae stage was based on the following information: histology and histochemical enzyme-staining patterns of major immune organs (Lin et al., 2005a) and detection of expression of rag-1 gene as an immune development marker (unpublished data) in various organs of E. malabaricus indicated that the immune system of grouper larvae could function as early as 10 dph. Nervous necrosis virus symptoms and mortality commonly started at 35–42 dph in the hatchery in Taiwan, and larvae began feeding on small Artemia nauplii at 18–20 dph. Our vaccination schedule was tentatively decided as feeding vaccine or placebo for two consecutive days beginning at 18 dph, then allowing 17 days for the immune response to develop, followed by challenge with NNV at 35 dph. To establish the virus challenge method the infection of grouper larvae with NNVeither by IP or by immersion was evaluated (data not shown). The injection method was usually more reliable in producing symptoms and in mortality with the predetermined lethal dose. With this challenge method, symptoms of VNN occurred in 7– 28 days, whereas the symptoms of VNN resulting from the immersion method were observed around 7–42 days. Both methods resulted in similar pathological features: the typical spiral swimming pattern and vacuolation in the retina, brain, and nerve tissues as observed in naturally-infected fish. Based on these data, we decided to use the injection challenge. We have also found that larvae become resistant to the injected challenge at 107 TCID50 after 45 dph, and hatchery farmers have observed that less VNN occurs after 60 dph. This interesting mechanism of resistance in larger larvae merits further investigation. Early vaccination of fish larvae is desirable. Attempts to encapsulate the recombinant E. coli in Artemia and stype rotifer to allow earlier vaccination has yielded
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promising results (data not shown). However, earlier immunization might induce tolerance before maturation of the grouper's immune system. The development of the immune system, the time of first feeding of Artemia and period of susceptibility to NNV might be different in each fish species, therefore the optimization of the vaccination schedule for a specific fish is required. For example, grouper, such as E. lanceolatus, showed to be susceptible to NNV later than E. coioides and lasted even up to 500 g. It probably could be vaccinated successfully at a later fingerling stage. Regarding the time of vaccination and challenge, length, mass and age (dpf, or day post-hatching; or dph), and appearance of stripes have been used to define the developmental stages of various species of fish larvae (Chantanachookhin et al., 1991; Padros and Crespo, 1996; Schroder et al., 1998).This is particularly important for commercially hatched larvae, as the development of fish from hatching to juvenile will depend on seasonal and nutritional conditions. Although we used dph as the index of age in this study, we suggest that the time of vaccination should be determined at least based on these three indicators. Any selected gene of a specific pathogen can be cloned and expressed in E. coli or other suitable bacterial host using recombinant technology, a feature that is particularly useful for the development of oral subunit vaccines for fish. Furthermore, a multi-valent oral vaccine against several pathogens could be developed using the same technology, which is safer, non-stressful, user-friendly, and more versatile than other methods currently available for vaccinating fish. Acknowledgments This study was supported by grants of No.92-2317-B006-002 (2003), and 93-2317-B-006-001 (2004) from National Program on Agricultural Biotechnology, National Science Council, Executive Yuan, Taiwan. We also like to thank Dr. H-M Su and Mr. W-M Lee for their kind advice and help with grouper larva culture and deeply appreciate Dr. Alan Pike and Ms. Kathleen Hills of the Lucidus Consultancy for editing of this manuscript. References Amend, D.F., 1981. Potency Testing of Fish Vaccines. Karger, Basel. 447–454 pp. Anderson, D.P., Ross, A.J., 1972. Comparative study of Hagerman redmouth disease oral bacterin. Prog. Fish-Cult. 34, 226–228. Baudin-Laurencin, F., Tangtronpiros, J., 1980. Some results of vaccination against vibriosis in Brittany. Springer-Verlag, Berlin. 60–68 pp.
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