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Immunogenicity of a cell culture-derived inactivated vaccine against a common virulent isolate of grass carp reovirus
Fish & Shellfish Immunology 54 (2016) 473e480
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Immunogenicity of a cell culture-derived inactivated vaccine against a common virulent isolate of grass carp reovirus Weiwei Zeng a, b, Qing Wang b, *, Yingying Wang b, Changchen Zhao b, Yingying Li b, Chunbin Shi b, Shuqin Wu b, Xinjian Song b, Qiwen Huang b, Shoujun Li a, ** a College of Veterinary Medicine, South China Agricultural University, Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, Guangzhou 510642, Guangdong, China b Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2016 Received in revised form 22 April 2016 Accepted 29 April 2016 Available online 30 April 2016
Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. There is no available antiviral therapy and vaccination is the primary method of disease control. In the present study, the immunological effects and protective efficacy of an inactivated HuNan1307 vaccine in grass carp were evaluated. The GCRV isolate HuNan1307 was produced by replication onto the grass carp PSF cell line, and inactivated with 1% bpropiolactone for 60 h at 4 C. Grass carp were injected with inactivated GCRV vaccine, followed by challenge with the isolate HuNan1307. The results showed that the minimum dosage of the inactivated vaccine was 105.5 TCID50/0.2 mL to induce immune protection. All grass carp immunized with the inactivated vaccine produced a high titer of serum antibodies and GCRV-specific neutralizing antibody. Moreover, the inactivated vaccine injection increased the expression of 6 immune-related genes in the spleen and head kidney, which indicated that a immune response was induced by the HuNan1307 vaccine. In addition, grass carp immunized with the inactivated vaccine showed a survival rate above 80% after the viral challenge, equal to that of grass carp immunized with a commercial attenuated vaccine, and the protection lasted at least for one year. The data in this study suggested that the inactivated HuNan1307 vaccine may represent an efficient method to induce immunity against GCRV infection and the induced disease in grass carp. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Inactivated vaccine Grass carp reovirus Humoral immune response
1. Introduction Grass carp (Ctenopharyngodon idella) is an economically important freshwater fish in China and its production accounted for 18.10% of the output of all freshwater fisheries in 2013 [1]. It is also the most commonly cultivated fish in the world and is cultured extensively in more than 40 countries [2]. However, hemorrhagic disease caused by grass carp reovirus (GCRV) has seriously hampered the development of grass carp aquaculture [3]. GCRV, a member of genus Aquareovirus in the family Reoviridae, was the first viral pathogen to be identified from aquatic animals in China in 1983 [4]. This pathogen can provoke severe hemorrhagic disease in
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Q. Wang), [email protected] (S. Li). http://dx.doi.org/10.1016/j.fsi.2016.04.133 1050-4648/© 2016 Elsevier Ltd. All rights reserved.
fingerling and yearling populations of grass carp, and causes a mortality rate of 60e100% during outbreaks [5]. GCRV was recognized to be the most virulent among Aquareovirus species so far [6]. In recent years, with the rapid development of aquaculture industry, diseases caused by GCRV have become increasingly significant and resulted in huge financial losses [7]. Attempts to control GCRV infection are hindered by a lack of thorough knowledge of the pathogenesis of the virus, the existence of diverse genetic make-ups, and the lack of anti-viral therapeutics [8,9]. Vaccination is considered the most effective way of protecting grass carp from this disease. To date, four vaccine types have been investigated for the control of GCRV: (i) inactivated vaccine; (ii) attenuated vaccine; (iii) recombinant subunit vaccine, and (iv) DNA vaccine. Inactivated vaccines are the most commonly applied method for the prevention of GCRV. In China, the first vaccine for grass carp hemorrhagic disease “inactivated tissue vaccine” was developed in the 1960s [10]. Subsequently, significant
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achievements have been obtained in the development of inactivated vaccines for GCRV through cell culture [11]. Currently, there is only 1 licensed GCRV vaccine available in the international market, which is an attenuated vaccine developed by attenuating the GCRV892 strain through serial passages in tissue culture [1]. With attenuated vaccines, the difference between vaccine virus isolates and wild-type viruses often results in a failure to produce effective immunity against the wild-type viruses. In some cases, fish populations immunized with attenuated vaccines have shown outbreaks; in these cases, safety considerations stopped further work [13]. The development and manufacture of subunit vaccine and DNA vaccine are expensive and time consuming, and difficulties in applying them in the aquatic environment have also resulted in many limitations [14]. Especially, the subunit vaccine is easily degraded during processing, delivery, and in the animals. So far, studies of recombinant subunit vaccine and DNA vaccine are still at an experimental stage. At present, it became necessary to develop a new GCRV vaccine due to the occurrence of rare serious adverse events following treatment with existing vaccines. The development of a new vaccine that reduces or eliminates the disadvantages of existing vaccines would be highly beneficial for grass carp aquaculture. In this study, aiming to produce a vaccine with an improved efficacy and safety profile, an inactivated vaccine was prepared by inactivating GCRV isolate HuNan1307, which is commonly known as a virulent isolate. The aim of this work was to evaluate the immunological effects and protective efficacy of this inactivated GCRV vaccine in grass carp.
flasks (Corning Inc., Corning, NY, USA) and infected using a multiplicity of infection (MOI) of 0.1. After 1 h of viral adsorption at 28 C, the cells were washed with sterile phosphate-buffered saline (PBS, pH 7.5) and maintained in M199 medium supplemented with 3% FBS at an incubation temperature of 28 C. At 5 days after infection, the GCRV-infected cell monolayers were harvested by scraping the sterile PBS-washed monolayer with 1 mL M199 followed by centrifugation of the cell suspension at 1500g and 4 C for 10 min. The titer of the virus was determined according to the 50% tissue culture infectious dose (TCID50) assay, which was performed as described previously with some modifications [16]. Ten-fold serial dilutions of the viral samples were prepared, and 100 mL aliquots of each dilution were inoculated in duplicate onto PSF cell monolayers in 24-well cluster plates. Virus adsorption was allowed to proceed for 3 days at 28 C in a 5% CO2 incubator. Then, the virus presents was assayed using indirect immunofluorescence assay (IFA) as described previously [17]. 2.4. Virus inactivation The GCRV preparations were inactivated with formaldehyde or
b-propiolactone (BPL; Sigma-Aldrich, St. Louis, MO, USA) and samples were removed at specific time intervals to determine the infectivity titer [18]. 0.05% or 1% (w/v) formaldehyde was added to live GCRV and the mixture was agitated at different temperatures. BPL was mixed with live GCRV to a final concentration of 0.1% (v/v) and the mixture was agitated at 4 C (Table 1). Virus inactivation by BPL was neutralized by the addition of 1 M sodium thiosulfate to a final concentration of 20 mM.
2. Materials and methods 2.5. Testing for remaining live virus 2.1. Experimental fish and virus Grass carp were kindly provided by a fish farm located in Foshan (Guangdong, China) with a mean total length and body weight of 12.0 ± 0.5 cm and 25.0 ± 0.5 g, respectively (data are presented as mean values ± SD). Fish were acclimatized at 28 C under laboratory conditions for 2 weeks before experimental manipulation, then were maintained in aerated water and fed daily with commercial dry feed pellets (Hello Fish Dry Pellets; CVM Products, Beijing, China). Possible viral contamination in fish and feed was checked by reverse transcription quantitative real-time PCR (RTqPCR) to verify that they were free from pathogens [7]. Care of animals was performed in compliance with the guidelines of the Animal Experiment Committee, South China Agricultural University. The protocol was approved by China Guangdong Province Science and Technology Department (permit number: SYXK(Yue) 2014e0136). The GCRV HuNan1307 that was used to prepare the inactivated vaccines in this study was isolated from infected grass carp in a fish farm located in Zhuzhou (Hunan, China). The complete genome sequences of HuNan1307 are publicly available (GenBank accession nos. KU254566 ~ KU254576). 2.2. Cell lines The proboscis snout into fibers (PSF) cell line, which is derived from grass carp, was used for the propagation of GCRV [15]. The cells were maintained at 28 C in M199 medium (Gibco, USA) containing 10% (v/v) fetal bovine serum (FBS; HyClone™, GE Healthcare Life Sciences, Logan, UT, USA). The concentration of FBS in the cell culture medium was reduced to 3% for virus propagation. 2.3. Virus replication Infectious GCRV was replicated onto PSF cells seeded in 150 cm2
Confirmation of virus inactivation in the vaccine preparations was performed by cell culture of PSF cells inoculated with the inactivated GCRV preparations. The titer of virus preparation after inactivation was determined using qPCR [7]. In addition, 1 mL of the inactivated viral suspension was initially inoculated into a 25 cm2 cell culture flask and incubated at 28 C/5% CO2. The cell monolayer was assessed daily using IFA. Every 7 days, 1 mL of the viral supernatant was removed from the prior flask and inoculated in a new flask, and the suspensions were evaluated for a total period of 21 days. The absence of live GCRV during this period confirmed that the viral inactivation had been successful. The sample was considered to be inactivated only when confirmed as inactive by two separate trials. The inactivated virus preparations were stored at 80 C. 2.6. Grass carp immunization Healthy grass carp (n ¼ 1800) were randomly divided into 3 groups (600 fish per group). The immunized group was administered with 5 104 TCID50 of the inactivated virus preparation in a volume of 200 mL by intraperitoneal injection. The negative control group was injected with 200 mL of PBS. The positive control group was immunized with a single dose of 1 103 TCID50/200 mL of the commercial attenuated GCRV vaccine (PuLin Biological Products Co., Ltd., Guangzhou, China). Subsequently, the fish in the control and treatment groups were transferred to different tanks and maintained as described above. 2.7. Measurement of antibody response by antibody ELISA An anti-IgM monoclonal antibodies (MAbs) preparation was made according to the methods described in our previous study [19]. For antibody determination, the immunized and control fish
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Table 1 Formaldehyde and BPL inactivation of GCRV.
Formaldehyde
Groups
Inactivation temperature ( C)
Concentration (%)
Inactivation time for HuNan1307 (h)
Infectivity
Protection rate (%)
1
37
0.05
24 36 48 60 72 24 36 48 60 72 24 36 48 60 72 24 36 48 60 72 5d 10d 15d 5d 10d 15d 24 36 48 60 72 96
þ þ þ ea e þ e e e e þ þ þ þ e þ þ e e e þ þ e þ e e þ þ þ ea e e
/ / /
2
BPL
a
37
0.1
3
28
0.05
4
28
0.1
5
4
0.05
6
4
0.1
1
4
0.1
80 70 / 60 50 50 30 / / / / 90 / / 90 90 80 / / 100 / 100 100 / / / 100 100 100
Serial blind passage confirmed the absence of residual GCRV in the inactivated virus preparations.
(ten fish per group) were sampled pre-vaccination and at 1, 2, 3, 4, 6, 8, 12, 24, 36, and 52 weeks post-immunization (WPI), respectively. Sera from immunized fish were examined by enzyme linked immunosorbent assay (ELISA) as described previously [20]. 2.8. Serum neutralization test (SNT) The SNT was carried out as described elsewhere [21]. Serum samples were filtered and heat-inactivated for 30 min at 42 C. Neutralizing antibody titers were determined using SNT with PSF cells. The GCRV neutralizing antibody titers were determined and expressed as the reciprocal of the highest dilution at which infection of the PSF cells was inhibited in 50% of the culture wells. 2.9. Quantitative real-time PCR (qPCR) Total RNA samples from spleen and head kidney were prepared by TRIzol® lysis according to the manufacturer’s instructions (TaKaRa Bio, Otsu, Japan). The cDNA were prepared as described previously [20]. The real-time qPCR assays were performed using a 7500 Real time PCR System (Applied Biosystems) and SYBR Premix Ex Taq™ II kit (TaKaRa Bio). Real-time PCR primers for grass carp bactin, interferon-induced Mx protein (Mx), interferon (IFN), type I interferon (IFN-I), immunoglobulin M (IgM), Toll-like receptor 3 (TLR3), and Toll-like receptor 7 (TLR7) were listed in Table 2. b-actin served as an internal reference gene for the normalization of the mRNA expression in different tissues. Real-time qPCR reactions for the amplification of each target gene were performed in a 25 mL final volume and run using the following cycling conditions: 1 cycle of 95 C for 10 min, followed by 40 cycles of denaturation at 95 C for 30 s, annealing at 60 C for 1 min, and 72 C for 30 s. All qPCR reactions were performed for 3 biological replicates, and the data
Table 2 Primers used in this investigation. Gene
Primer sequence (50 e30 )
Accessions
b-actin-F b-actin-R
50 -GGATGATGAAATTGCCGCACTGG-30 50 -ACCGACCATGACGCCCTGATGT-30 50 -GCAGGGGACAAAAAGAGATTATAGA-30 50 -AGCCAACTTAGGAATAGTAGCAAAAC-30 50 -GACACATACAGTAGGATATTCACTCGC-30 50 -TTGCCTGGGAAGTAGTTTTCTTG-30 50 -TCTACCTCCAACTCCACCACC-30 50 -TGTTTATTGTATTTGCCACCTGAT-30 50 -GAGAACAATCGTGACTCCCTGA-30 50 -CCAGTAGAGAACACAGCGAGGT-30 50 -GAGCATACAGTTGAGTAAACGCAC-30 50 -TCTCCAAGAATATCAGGACGATAA-30 50 -AAGCAACGAGTCTTTGAGCCT-30 50 -GCGTCCTGGAAATGACCT-30
M25013.1
Mx-F Mx-R IFN-F IFN-R IgM-F IgM-R TLR3-F TLR3-R TLR7-F TLR7-R IFN-I-F IFN-I-R
AY395698.1 DQ357216.1 DQ417927.1 DQ885910.1 AB553573.1 GU139255.1
for each sample were expressed relative to the expression level of b-actin by using the DDCt method [22].
2.10. Protection against lethal challenge At the time points of 2, 6, 8, 12, 24, 36, and 52 WPI, 20 immunized grass carp from each group were challenged by intraperitoneal injection with 1 104 TCID50/mL of GCRV. All fish were monitored for 14 days post-challenge, and morbidity and mortality were recorded. The results of the vaccine efficacy trials have been presented as the relative percent survival (RPS). RPS was calculated by the following formula: RPS ¼ [1 (% mortality of immunized group/% mortality of control group)] 100 [23].
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2.11. Statistical analysis The software program SPSS Statistics 19.0 was used for statistical analyses. Descriptive statistics (mean, standard error), normality (Shapiro-Wilk), and homoscedasticity (Bartlett’s test) were determined. Groups with a normal distribution were compared by 1-way analysis of variance and Tukey tests. In all cases, the differences were considered significant at p < 0.05 and extremely significant at p < 0.01. 3. Results 3.1. Virus inactivation The GCRV preparations were inactivated with 0.1% (v/v) BPL and 0.05% or 0.1% (w/v) formaldehyde (Table 1). The pre-inactivation titers of infectious GCRV were 107.5 TCID50/mL. Each of the inactivating agents resulted in a total loss of GCRV infectivity. Chemical treatment of GCRV with 0.1% formaldehyde abolished virus infectivity within 36 h at 37 C, whereas treatment with the same concentration of formaldehyde at 4 C rendered the virus innocuous within 10 days. At the same temperature, 0.1% formaldehyde abolished GCRV infectivity in a shorter time than 0.05% formaldehyde did. Treatment of GCRV with 0.1% BPL abolished virus infectivity within 60 h at 4 C. Furthermore, the immune protection efficiency of the GCRV vaccines prepared by inactivated HuNan1307 with the above inactivating agents was evaluated. The results showed that the vaccines inactivated by treatment with formaldehyde or 0.1% BPL at 4 C were highly efficacious in establishing immune resistance in immunized grass carp challenged with GCRV after immunization (Table 1). By considering the inactivation conditions and the protective effect of the inactivated vaccine, we chose to use 0.1% BPL treatment at 4 C for 60 h to inactivate the preparations of GCRV in the follow-up study.
Fig. 1. Detection of serum antibody levels induced by inactivated vaccine in grass carp. Blood samples from the immunized groups and the negative control group were collected pre-vaccination (pv) and at 1, 2, 3, 4, 6, 8, 12, 24, 36, and 52 weeks post infection (WPI). The antibody response was determined by indirect ELISA using plates coated with purified recombinant VP4 protein. The blood samples with an absorbance 0.20 OD450 were recorded as having a positive antibody response. The results are expressed as the mean specific absorbance ±SD. The asterisk “**” indicates significance at p < 0.01.
6 genes were all up-regulated initially and then declined over time, except that the mRNA expression of IFN in the head kidney of the inactivated immunized group suddenly increased at 24 WPI (Fig. 3A). The relative expression levels of the IgM, Mx, TLR3, and TLR7 genes were much higher than those of other genes (p < 0.01). In addition, the relative expression levels of immune genes in the attenuated vaccine-treated groups were significantly higher than those in the inactivated vaccine-treated groups (p < 0.01) (Fig. 3). The results showed that compared with the control group, inactivated vaccine or attenuated vaccine injection increased the mRNA expression of these 6 immune-related genes in the spleen and head kidney of grass carp, indicating that the vaccine stimulated the grass carp to generate innate and adaptive immune responses.
3.2. Detection of serum antibody
3.5. Protection of inactivated vaccine
The ELISA results showed that the antibody levels of the fish in the control group were negative for positivity (OD450 ¼ 0.20). The fish immunized with inactivated vaccine or attenuated vaccine developed significant antibody responses (p < 0.01). At 4 WPI, the antibody level reached a peak titer in the immunized group (p < 0.01). Then, the humoral immune response in the immunized group began to gradually attenuate. The antibody level in the attenuated vaccine group declined faster than that in the inactivated vaccine group (Fig. 1).
To evaluate the protective effectiveness of the inactivated vaccine, each group was challenged with a lethal dose of GCRV at different time points. The mortality and clinical signs of the viruschallenged fish were recorded daily for 2 weeks post-challenge. The results showed that the inactivated vaccine immunized groups were protected at a level of more than 80% of individual fish, and the attenuated vaccine showed a slightly higher level of protection (Table 3). The immunized grass carp had lower mortality rates in comparison with that of the control group, demonstrating
3.3. Neutralization assay The SNT results showed that at 1 WPI, grass carp immunized with inactivated vaccine or attenuated vaccine developed detectable neutralizing antibody titers. A further increase in neutralizing antibody titers was observed at 2 WPI. At 4 WPI, the levels of neutralizing antibody began to gradually decline until the fish did not develop neutralizing antibody above the threshold for positivity at 12 WPI. The grass carp immunized with PBS did not develop any antibody response against GCRV. The GCRV-specific neutralizing antibody level in the attenuated vaccine group was significantly higher than that in the inactivated vaccine-immunized group (p < 0.01) (Fig. 2). 3.4. Immune-related genes expression In the immunized groups, the relative expression levels of these
Fig. 2. Detection of GCRV-specific serum neutralizing antibody levels induced in the vaccinated grass carp. Pooled serum samples from each group were tested by serumvirus neutralization test to determine the titers of antibody against the virulent GCRV strain HuNan1307. **p < 0.01; *p < 0.05.
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Table 3 Mortality rate and relative percentage survival (RPS) of fish challenged with HuNan1307 virus. Challenge time (WPI)
2 6 8 12 24 36 52
Mortality rate (%)
RPS (%)
Inactivated vaccine
Attenuated vaccine
Control
Inactivated vaccine
Attenuated vaccine
Control
0 5.0 10.0 10.0 15.0 15.0 20.0
0 0 0 10.0 10.0 5.0 5.0
90.0 85.0 95.0 100 100 80.0 85.0
100 94.1 89.5 95.0 85.0 81.3 76.5
100 100 100 90.0 90.0 93.8 94.1
e e e e e e e
that the inactivated vaccine could protect the fish from hemorrhagic disease. The protection provided by the inactivated vaccine and attenuated vaccine lasted for up to 1 year. In challenge trials, the dead fish showed typical clinical symptoms of GCRV infection, and no pathogen other than GCRV was detected from dead fish. 4. Discussion GCRV infection causes a severe disease in grass carp [24]. Although the existing attenuated vaccines have many advantages, a major disadvantage of these vaccines is their poor safety under natural conditions, as they may undergo harmful changes in the ecological environment. So, in Japan and some European countries, specific provision has been made to ensure that fish can only be immunized with inactivated vaccines. It is important to develop novel GCRV vaccines capable of inducing high protection combined with good safety. Inactivated vaccines are often preferred for safety reasons. Inactivated vaccines with the same antigenic composition as the microorganisms causing the diseases have been found to provide acceptable to good protection [25,26]. Two main points should be addressed in the development of inactivated vaccines: (i) it is necessary to completely inactivate an infectious virus to ensure the safety of the vaccine and (ii) essential viral epitopes are retained after the inactivation process in order to obtain a high-quality antigen [27]. In the present study, the inactivation dynamics of cell-associated GCRV antigen by formaldehyde or BPL were evaluated. Using uncomplicated processes, we found that the treatment of GCRV with formaldehyde or BPL completely eliminated its infectivity. The use of BPL, which acts on the viral genome with little or no effect on viral proteins, is highly recommended for the generation of an effective vaccine [28]. BPL has been widely used for the inactivation of other viruses and has resulted in known commercial vaccines [29,30]. The preparation of BPL-inactivated vaccines requires precaution during handling due to the toxicity of BPL. There is now substantial research showing that sodium thiosulfate can be added to the vaccine at the end of the inactivation step, which results in the conversion of BPL to nontoxic fat metabolites [31]. To explore the potential of the inactivated vaccine against GCRV, its immunogenicity was investigated through the immunization of grass carp with the cell-associated GCRV. First, the immunogenicity assay aimed to determine the minimum number of doses able to provide solid protection. The immune dose of 105.5 TCID50/0.2 mL of the inactivated vaccine induced a specific immune response, which provided good protection for the fishes after challenge with the virus HuNan1307(data not shown). The use of a low concentration of antigens in a vaccine is always an important step in the
establishment of preclinical and clinical studies, because it is associated with the technical and economic viability of the commercial product [32]. The results of ELISA indicated that specific serum antibodies in the inactivated vaccine or attenuated vaccine immunized fish could be detected during 1e52 weeks post-vaccination. In general, the concentration of antibodies against an antigen produced following vaccination usually corresponds with the protection/survival rate conferred by the vaccine. In the subsequent challenge experiment with a virulent strain of GCRV, the 2 immunized groups showed higher protection rates than the control group did. The results for the antibody levels of the immunized fish show good agreement with the results of our challenge studies indicating the protective efficacy of the antigens. The neutralizing antibody is the main correlate of protection against infection with GCRV [14]. SNT is a widely accepted method and has been used for evaluating the humoral response induced by other vaccines [21]. From 8 weeks post vaccination, the inactivated vaccine and attenuated vaccine immunized groups were still protected at levels of more than 80% despite the lack of detectable NAb. This lack of correlation between neutralizing antibody levels and protection indicated that the effectiveness of these vaccines might be partly due to the induction of a cellular response and not exclusively a humoral response through NAb. This speculation will require further experiments for verification. It is well known that vaccination with an antigen stimulates the expression of certain immune-related genes. IgM is regarded as an indicator of specific immune responses and the major Ig isotype of teleost fish [33]. It has been demonstrated that TLR3 can trigger immune responses against GCRV in grass carp [34]. Mx proteins are one of the key components of the antivirals induced by interferons in many species. A unique property of some Mx proteins is their antiviral activity against a wide range of viruses [35]. TLR3 is important for resistance to dsRNA analogs and viruses in fish. A previous study confirmed that an increased mRNA expression of TLR7 is triggered by GCRV challenge both in vivo and in vitro [36]. In our study, the expression of the IgM, Mx, TLR3, and TRL7 genes was highly increased in immunized grass carp from 1 week after immunization and maintained for almost 2 months. In addition to these 4 genes, the expression of 2 genes related to the innate immune response, which encode the proteins IFN and IFN-I, respectively, was also found to be slightly increased by treatment with the inactivated vaccine. All of the 6 tested genes were considered to be important in innate immunity and inflammatory or antiviral responses. Compared to the negative group, the expression of six immunerelated genes in the spleen and head kidney of inactivated vaccine
Fig. 3. qRT-PCR analysis of the expression of immune-related genes in grass carp vaccinated with inactivated vaccine. (A) IFN; (B) IFN-I; (C) IgM; (D) Mx; (E) TLR3; (F) TLR7. Samples were collected from the grass carp at 1, 2, 3, 4, 6, 8, 12, 24, 36, and 52 weeks post infection (WPI). Data are the means of 3 assays and are presented as the mean ± SD. ***p < 0.01; **p < 0.01; *p < 0.05.
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treated grass carp were significantly enhanced at the early stage of immune (mainly during weeks 2 through 6). These results indicated that the inactivated vaccine could induce cellular immune response. Both in two vaccine-immunized groups, relative expressions of genes were all up-regulated at first and then declined as time went on. The results are consistent with another study, which scientists investigated the impact of the injection of inactivated vaccine for hemorrhage of grass carp on the expressions of other main immune-related genes in spleen, including major histocompatibility complex I (MHC I), complement 3 (C3), interleukin1b (IL-1b), and intelectin genes [37]. Their results also revealed that inactivated vaccine injection increased mRNA expressions of the immune-related genes in the spleen. It is noteworthy that in our whole experimental process, the expression of these genes between inactivated vaccine and control groups showed less significant changes than those between attenuated vaccine and control groups. Although relative genes expression of inactivated vaccine immunized grass carp reduced to normal levels in the late stage of immune, the specific antibody levels of inactivated vaccine group remained high. This indicated that the inactivated HuNan1307 vaccine may mainly depend on inducing humoral immunity to provide good immunoprotection. In summary, the present study demonstrated that the inactivated HuNan1307 vaccine induced a strong humoral immune response and conferred effective protection against GCRV infection in grass carp. Although antibody levels and the relative mRNA levels of immune-relevant genes induced by the inactivated HuNan1307 vaccine were lower than those induced by the commercial attenuated vaccine, these 2 vaccines conferred equal protection against infection with a virulent strain of GCRV in grass carp. Considering its security and high protective effectiveness, the inactivated vaccine could serve as a potential vaccine candidate for the prevention of GCRV and has good development prospects. In addition, the duration of immunity and the development of immunological memory are both crucial for vaccine efficacy. In commercial aquaculture, the duration of protection needed is directly related to the length of the production cycle. In our study, a duration of protection of 1 year was achieved in the inactivated HuNan1307 immunization group, which could be suitable for use in commercial grass carp aquaculture considering the production cycle of grass carp. To further validate our findings, we will perform preclinical studies to evaluate the protective efficacy of the inactivated Hunan1307 vaccine in preventing the progression of GCRV infection to clinical disease and in conferring long-term immunity. Conflict of interest The authors have no conflict of interest to declare. Acknowledgements Funding for this research was provided by the National Key Technology R&D Program (No. 2012BAD25B02), the Science and Technology Planning Project of Jiangxi Province (No. 20152ACF60021), and Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2016ZD0503). References [1] Y. Rao, J. Su, Insights into the antiviral immunity against grass carp (Ctenopharyngodon idella) reovirus (GCRV) in grass carp, J. Immunol. Res. 2015 (2015) 670437. [2] Z.D. Dong, J. Zhang, X.S. Ji, F.N. Zhou, Y. Fu, W. Chen, et al., Molecular cloning, characterization and expression of cathepsin D from grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. 33 (5) (2012) 1207e1214.
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Na, J. Yulin, L. Xiangmei, Detection of grass carp reovirus (GCRV) with monoclonal antibodies, Arch. Virol. 159 (4) (2014) 649e655. [20] B. Zhu, G.L. Liu, Y.X. Gong, F. Ling, G.X. Wang, Protective immunity of grass carp immunized with DNA vaccine encoding the vp7 gene of grass carp reovirus using carbon nanotubes as a carrier molecule, Fish Shellfish Immunol. 42 (2) (2015) 325e334. [21] M. Li, Y.F. Wang, Y. Wang, H. Gao, N. Li, Y. Sun, et al., Immune responses induced by a BacMam virus expressing the E2 protein of classical swine fever virus in mice, Immunol. Lett. 125 (2) (2009) 145e150. [22] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method, Methods 25 (4) (2001) 402e408. [23] D.F. Amend, Potency testing of fish vaccines. International symposium on fish biologics: serodiagnostics and vaccines, Dev. Biol. Stand. 49 (1981) 447e454. [24] FAO Fishery and Aquaculture, 2012. http://www.fao.org/fishery/statistics/en. [25] H.Y. Huang, Y.C. Chen, P.C. Wang, M.A. Tsai, S.C. Yeh, H.J. Liang, et al., Efficacy of a formalin-inactivated vaccine against Streptococcus iniae infection in the farmed grouper Epinephelus coioides by intraperitoneal immunization, Vaccine 32 (51) (2014) 7014e7020. [26] S.K. Nayak, Y. Shibasaki, T. Nakanishi, Immune responses to live and inactivated Nocardia seriolae and protective effect of recombinant interferon gamma (rIFNg) against nocardiosis in ginbuna crucian carp, Carassius auratus langsdorfii, Fish Shellfish Immunol. 39 (2) (2014) 354e364. [27] R.C. Pereira, A.N. Silva, M.C. Souza, M.V. Silva, P.P. Neves, A.A. Silva, et al., An inactivated yellow fever 17DD vaccine cultivated in Vero cell cultures, Vaccine 33 (35) (2015) 4261e4268. [28] I. Delrue, D. Verzele, A. Madder, H.J. Nauwynck, Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges, Expert Rev. Vaccines 11 (6) (2012) 695e719. [29] O. Perez, C.C. Paolazzi, Production methods for rabies vaccine, J. Ind. Microbiol. 18 (5) (1997) 340e347. [30] F. Stauffer, T. El-Bacha, A.T. Da Poian, Advances in the development of inactivated virus vaccines, Recent Pat. Antiinfect. Drug Discov. 1 (3) (2006) 291e296. [31] H.G. Bahnemann, Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine, Vaccine 8 (4) (1990) 299e303. [32] A.P. Durbin, S.S. Whitehead, Dengue vaccine candidates in development, Curr. Top. Microbiol. Immunol. 338 (2010) 129e143. [33] B. Schaerlinger, M. Bascove, J.P. Frippiat, A new isotype of immunoglobulin heavy chain in the urodele amphibian Pleurodeles waltl predominantly
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expressed in larvae, Mol. Immunol. 45 (3) (2008) 776e786. [34] J. Su, S. Jang, C. Yang, Y. Wang, Z. Zhu, Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. 27 (3) (2009) 433e439. [35] O. Haller, P. Staeheli, G. Kochs, Interferon-induced Mx proteins in antiviral host defense, Biochimie 89 (6e7) (2007) 812e818. [36] L. Chen, Q. Li, J. Su, C. Yang, Y. Li, Y. Rao, Trunk kidney of grass carp
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Immunogenicity of a cell culture-derived inactivated vaccine against a common virulent isolate of grass carp reovirus Weiwei Zeng a, b, Qing Wang b, *, Yingying Wang b, Changchen Zhao b, Yingying Li b, Chunbin Shi b, Shuqin Wu b, Xinjian Song b, Qiwen Huang b, Shoujun Li a, ** a College of Veterinary Medicine, South China Agricultural University, Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, Guangzhou 510642, Guangdong, China b Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2016 Received in revised form 22 April 2016 Accepted 29 April 2016 Available online 30 April 2016
Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. There is no available antiviral therapy and vaccination is the primary method of disease control. In the present study, the immunological effects and protective efficacy of an inactivated HuNan1307 vaccine in grass carp were evaluated. The GCRV isolate HuNan1307 was produced by replication onto the grass carp PSF cell line, and inactivated with 1% bpropiolactone for 60 h at 4 C. Grass carp were injected with inactivated GCRV vaccine, followed by challenge with the isolate HuNan1307. The results showed that the minimum dosage of the inactivated vaccine was 105.5 TCID50/0.2 mL to induce immune protection. All grass carp immunized with the inactivated vaccine produced a high titer of serum antibodies and GCRV-specific neutralizing antibody. Moreover, the inactivated vaccine injection increased the expression of 6 immune-related genes in the spleen and head kidney, which indicated that a immune response was induced by the HuNan1307 vaccine. In addition, grass carp immunized with the inactivated vaccine showed a survival rate above 80% after the viral challenge, equal to that of grass carp immunized with a commercial attenuated vaccine, and the protection lasted at least for one year. The data in this study suggested that the inactivated HuNan1307 vaccine may represent an efficient method to induce immunity against GCRV infection and the induced disease in grass carp. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Inactivated vaccine Grass carp reovirus Humoral immune response
1. Introduction Grass carp (Ctenopharyngodon idella) is an economically important freshwater fish in China and its production accounted for 18.10% of the output of all freshwater fisheries in 2013 [1]. It is also the most commonly cultivated fish in the world and is cultured extensively in more than 40 countries [2]. However, hemorrhagic disease caused by grass carp reovirus (GCRV) has seriously hampered the development of grass carp aquaculture [3]. GCRV, a member of genus Aquareovirus in the family Reoviridae, was the first viral pathogen to be identified from aquatic animals in China in 1983 [4]. This pathogen can provoke severe hemorrhagic disease in
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Q. Wang), [email protected] (S. Li). http://dx.doi.org/10.1016/j.fsi.2016.04.133 1050-4648/© 2016 Elsevier Ltd. All rights reserved.
fingerling and yearling populations of grass carp, and causes a mortality rate of 60e100% during outbreaks [5]. GCRV was recognized to be the most virulent among Aquareovirus species so far [6]. In recent years, with the rapid development of aquaculture industry, diseases caused by GCRV have become increasingly significant and resulted in huge financial losses [7]. Attempts to control GCRV infection are hindered by a lack of thorough knowledge of the pathogenesis of the virus, the existence of diverse genetic make-ups, and the lack of anti-viral therapeutics [8,9]. Vaccination is considered the most effective way of protecting grass carp from this disease. To date, four vaccine types have been investigated for the control of GCRV: (i) inactivated vaccine; (ii) attenuated vaccine; (iii) recombinant subunit vaccine, and (iv) DNA vaccine. Inactivated vaccines are the most commonly applied method for the prevention of GCRV. In China, the first vaccine for grass carp hemorrhagic disease “inactivated tissue vaccine” was developed in the 1960s [10]. Subsequently, significant
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achievements have been obtained in the development of inactivated vaccines for GCRV through cell culture [11]. Currently, there is only 1 licensed GCRV vaccine available in the international market, which is an attenuated vaccine developed by attenuating the GCRV892 strain through serial passages in tissue culture [1]. With attenuated vaccines, the difference between vaccine virus isolates and wild-type viruses often results in a failure to produce effective immunity against the wild-type viruses. In some cases, fish populations immunized with attenuated vaccines have shown outbreaks; in these cases, safety considerations stopped further work [13]. The development and manufacture of subunit vaccine and DNA vaccine are expensive and time consuming, and difficulties in applying them in the aquatic environment have also resulted in many limitations [14]. Especially, the subunit vaccine is easily degraded during processing, delivery, and in the animals. So far, studies of recombinant subunit vaccine and DNA vaccine are still at an experimental stage. At present, it became necessary to develop a new GCRV vaccine due to the occurrence of rare serious adverse events following treatment with existing vaccines. The development of a new vaccine that reduces or eliminates the disadvantages of existing vaccines would be highly beneficial for grass carp aquaculture. In this study, aiming to produce a vaccine with an improved efficacy and safety profile, an inactivated vaccine was prepared by inactivating GCRV isolate HuNan1307, which is commonly known as a virulent isolate. The aim of this work was to evaluate the immunological effects and protective efficacy of this inactivated GCRV vaccine in grass carp.
flasks (Corning Inc., Corning, NY, USA) and infected using a multiplicity of infection (MOI) of 0.1. After 1 h of viral adsorption at 28 C, the cells were washed with sterile phosphate-buffered saline (PBS, pH 7.5) and maintained in M199 medium supplemented with 3% FBS at an incubation temperature of 28 C. At 5 days after infection, the GCRV-infected cell monolayers were harvested by scraping the sterile PBS-washed monolayer with 1 mL M199 followed by centrifugation of the cell suspension at 1500g and 4 C for 10 min. The titer of the virus was determined according to the 50% tissue culture infectious dose (TCID50) assay, which was performed as described previously with some modifications [16]. Ten-fold serial dilutions of the viral samples were prepared, and 100 mL aliquots of each dilution were inoculated in duplicate onto PSF cell monolayers in 24-well cluster plates. Virus adsorption was allowed to proceed for 3 days at 28 C in a 5% CO2 incubator. Then, the virus presents was assayed using indirect immunofluorescence assay (IFA) as described previously [17]. 2.4. Virus inactivation The GCRV preparations were inactivated with formaldehyde or
b-propiolactone (BPL; Sigma-Aldrich, St. Louis, MO, USA) and samples were removed at specific time intervals to determine the infectivity titer [18]. 0.05% or 1% (w/v) formaldehyde was added to live GCRV and the mixture was agitated at different temperatures. BPL was mixed with live GCRV to a final concentration of 0.1% (v/v) and the mixture was agitated at 4 C (Table 1). Virus inactivation by BPL was neutralized by the addition of 1 M sodium thiosulfate to a final concentration of 20 mM.
2. Materials and methods 2.5. Testing for remaining live virus 2.1. Experimental fish and virus Grass carp were kindly provided by a fish farm located in Foshan (Guangdong, China) with a mean total length and body weight of 12.0 ± 0.5 cm and 25.0 ± 0.5 g, respectively (data are presented as mean values ± SD). Fish were acclimatized at 28 C under laboratory conditions for 2 weeks before experimental manipulation, then were maintained in aerated water and fed daily with commercial dry feed pellets (Hello Fish Dry Pellets; CVM Products, Beijing, China). Possible viral contamination in fish and feed was checked by reverse transcription quantitative real-time PCR (RTqPCR) to verify that they were free from pathogens [7]. Care of animals was performed in compliance with the guidelines of the Animal Experiment Committee, South China Agricultural University. The protocol was approved by China Guangdong Province Science and Technology Department (permit number: SYXK(Yue) 2014e0136). The GCRV HuNan1307 that was used to prepare the inactivated vaccines in this study was isolated from infected grass carp in a fish farm located in Zhuzhou (Hunan, China). The complete genome sequences of HuNan1307 are publicly available (GenBank accession nos. KU254566 ~ KU254576). 2.2. Cell lines The proboscis snout into fibers (PSF) cell line, which is derived from grass carp, was used for the propagation of GCRV [15]. The cells were maintained at 28 C in M199 medium (Gibco, USA) containing 10% (v/v) fetal bovine serum (FBS; HyClone™, GE Healthcare Life Sciences, Logan, UT, USA). The concentration of FBS in the cell culture medium was reduced to 3% for virus propagation. 2.3. Virus replication Infectious GCRV was replicated onto PSF cells seeded in 150 cm2
Confirmation of virus inactivation in the vaccine preparations was performed by cell culture of PSF cells inoculated with the inactivated GCRV preparations. The titer of virus preparation after inactivation was determined using qPCR [7]. In addition, 1 mL of the inactivated viral suspension was initially inoculated into a 25 cm2 cell culture flask and incubated at 28 C/5% CO2. The cell monolayer was assessed daily using IFA. Every 7 days, 1 mL of the viral supernatant was removed from the prior flask and inoculated in a new flask, and the suspensions were evaluated for a total period of 21 days. The absence of live GCRV during this period confirmed that the viral inactivation had been successful. The sample was considered to be inactivated only when confirmed as inactive by two separate trials. The inactivated virus preparations were stored at 80 C. 2.6. Grass carp immunization Healthy grass carp (n ¼ 1800) were randomly divided into 3 groups (600 fish per group). The immunized group was administered with 5 104 TCID50 of the inactivated virus preparation in a volume of 200 mL by intraperitoneal injection. The negative control group was injected with 200 mL of PBS. The positive control group was immunized with a single dose of 1 103 TCID50/200 mL of the commercial attenuated GCRV vaccine (PuLin Biological Products Co., Ltd., Guangzhou, China). Subsequently, the fish in the control and treatment groups were transferred to different tanks and maintained as described above. 2.7. Measurement of antibody response by antibody ELISA An anti-IgM monoclonal antibodies (MAbs) preparation was made according to the methods described in our previous study [19]. For antibody determination, the immunized and control fish
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Table 1 Formaldehyde and BPL inactivation of GCRV.
Formaldehyde
Groups
Inactivation temperature ( C)
Concentration (%)
Inactivation time for HuNan1307 (h)
Infectivity
Protection rate (%)
1
37
0.05
24 36 48 60 72 24 36 48 60 72 24 36 48 60 72 24 36 48 60 72 5d 10d 15d 5d 10d 15d 24 36 48 60 72 96
þ þ þ ea e þ e e e e þ þ þ þ e þ þ e e e þ þ e þ e e þ þ þ ea e e
/ / /
2
BPL
a
37
0.1
3
28
0.05
4
28
0.1
5
4
0.05
6
4
0.1
1
4
0.1
80 70 / 60 50 50 30 / / / / 90 / / 90 90 80 / / 100 / 100 100 / / / 100 100 100
Serial blind passage confirmed the absence of residual GCRV in the inactivated virus preparations.
(ten fish per group) were sampled pre-vaccination and at 1, 2, 3, 4, 6, 8, 12, 24, 36, and 52 weeks post-immunization (WPI), respectively. Sera from immunized fish were examined by enzyme linked immunosorbent assay (ELISA) as described previously [20]. 2.8. Serum neutralization test (SNT) The SNT was carried out as described elsewhere [21]. Serum samples were filtered and heat-inactivated for 30 min at 42 C. Neutralizing antibody titers were determined using SNT with PSF cells. The GCRV neutralizing antibody titers were determined and expressed as the reciprocal of the highest dilution at which infection of the PSF cells was inhibited in 50% of the culture wells. 2.9. Quantitative real-time PCR (qPCR) Total RNA samples from spleen and head kidney were prepared by TRIzol® lysis according to the manufacturer’s instructions (TaKaRa Bio, Otsu, Japan). The cDNA were prepared as described previously [20]. The real-time qPCR assays were performed using a 7500 Real time PCR System (Applied Biosystems) and SYBR Premix Ex Taq™ II kit (TaKaRa Bio). Real-time PCR primers for grass carp bactin, interferon-induced Mx protein (Mx), interferon (IFN), type I interferon (IFN-I), immunoglobulin M (IgM), Toll-like receptor 3 (TLR3), and Toll-like receptor 7 (TLR7) were listed in Table 2. b-actin served as an internal reference gene for the normalization of the mRNA expression in different tissues. Real-time qPCR reactions for the amplification of each target gene were performed in a 25 mL final volume and run using the following cycling conditions: 1 cycle of 95 C for 10 min, followed by 40 cycles of denaturation at 95 C for 30 s, annealing at 60 C for 1 min, and 72 C for 30 s. All qPCR reactions were performed for 3 biological replicates, and the data
Table 2 Primers used in this investigation. Gene
Primer sequence (50 e30 )
Accessions
b-actin-F b-actin-R
50 -GGATGATGAAATTGCCGCACTGG-30 50 -ACCGACCATGACGCCCTGATGT-30 50 -GCAGGGGACAAAAAGAGATTATAGA-30 50 -AGCCAACTTAGGAATAGTAGCAAAAC-30 50 -GACACATACAGTAGGATATTCACTCGC-30 50 -TTGCCTGGGAAGTAGTTTTCTTG-30 50 -TCTACCTCCAACTCCACCACC-30 50 -TGTTTATTGTATTTGCCACCTGAT-30 50 -GAGAACAATCGTGACTCCCTGA-30 50 -CCAGTAGAGAACACAGCGAGGT-30 50 -GAGCATACAGTTGAGTAAACGCAC-30 50 -TCTCCAAGAATATCAGGACGATAA-30 50 -AAGCAACGAGTCTTTGAGCCT-30 50 -GCGTCCTGGAAATGACCT-30
M25013.1
Mx-F Mx-R IFN-F IFN-R IgM-F IgM-R TLR3-F TLR3-R TLR7-F TLR7-R IFN-I-F IFN-I-R
AY395698.1 DQ357216.1 DQ417927.1 DQ885910.1 AB553573.1 GU139255.1
for each sample were expressed relative to the expression level of b-actin by using the DDCt method [22].
2.10. Protection against lethal challenge At the time points of 2, 6, 8, 12, 24, 36, and 52 WPI, 20 immunized grass carp from each group were challenged by intraperitoneal injection with 1 104 TCID50/mL of GCRV. All fish were monitored for 14 days post-challenge, and morbidity and mortality were recorded. The results of the vaccine efficacy trials have been presented as the relative percent survival (RPS). RPS was calculated by the following formula: RPS ¼ [1 (% mortality of immunized group/% mortality of control group)] 100 [23].
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2.11. Statistical analysis The software program SPSS Statistics 19.0 was used for statistical analyses. Descriptive statistics (mean, standard error), normality (Shapiro-Wilk), and homoscedasticity (Bartlett’s test) were determined. Groups with a normal distribution were compared by 1-way analysis of variance and Tukey tests. In all cases, the differences were considered significant at p < 0.05 and extremely significant at p < 0.01. 3. Results 3.1. Virus inactivation The GCRV preparations were inactivated with 0.1% (v/v) BPL and 0.05% or 0.1% (w/v) formaldehyde (Table 1). The pre-inactivation titers of infectious GCRV were 107.5 TCID50/mL. Each of the inactivating agents resulted in a total loss of GCRV infectivity. Chemical treatment of GCRV with 0.1% formaldehyde abolished virus infectivity within 36 h at 37 C, whereas treatment with the same concentration of formaldehyde at 4 C rendered the virus innocuous within 10 days. At the same temperature, 0.1% formaldehyde abolished GCRV infectivity in a shorter time than 0.05% formaldehyde did. Treatment of GCRV with 0.1% BPL abolished virus infectivity within 60 h at 4 C. Furthermore, the immune protection efficiency of the GCRV vaccines prepared by inactivated HuNan1307 with the above inactivating agents was evaluated. The results showed that the vaccines inactivated by treatment with formaldehyde or 0.1% BPL at 4 C were highly efficacious in establishing immune resistance in immunized grass carp challenged with GCRV after immunization (Table 1). By considering the inactivation conditions and the protective effect of the inactivated vaccine, we chose to use 0.1% BPL treatment at 4 C for 60 h to inactivate the preparations of GCRV in the follow-up study.
Fig. 1. Detection of serum antibody levels induced by inactivated vaccine in grass carp. Blood samples from the immunized groups and the negative control group were collected pre-vaccination (pv) and at 1, 2, 3, 4, 6, 8, 12, 24, 36, and 52 weeks post infection (WPI). The antibody response was determined by indirect ELISA using plates coated with purified recombinant VP4 protein. The blood samples with an absorbance 0.20 OD450 were recorded as having a positive antibody response. The results are expressed as the mean specific absorbance ±SD. The asterisk “**” indicates significance at p < 0.01.
6 genes were all up-regulated initially and then declined over time, except that the mRNA expression of IFN in the head kidney of the inactivated immunized group suddenly increased at 24 WPI (Fig. 3A). The relative expression levels of the IgM, Mx, TLR3, and TLR7 genes were much higher than those of other genes (p < 0.01). In addition, the relative expression levels of immune genes in the attenuated vaccine-treated groups were significantly higher than those in the inactivated vaccine-treated groups (p < 0.01) (Fig. 3). The results showed that compared with the control group, inactivated vaccine or attenuated vaccine injection increased the mRNA expression of these 6 immune-related genes in the spleen and head kidney of grass carp, indicating that the vaccine stimulated the grass carp to generate innate and adaptive immune responses.
3.2. Detection of serum antibody
3.5. Protection of inactivated vaccine
The ELISA results showed that the antibody levels of the fish in the control group were negative for positivity (OD450 ¼ 0.20). The fish immunized with inactivated vaccine or attenuated vaccine developed significant antibody responses (p < 0.01). At 4 WPI, the antibody level reached a peak titer in the immunized group (p < 0.01). Then, the humoral immune response in the immunized group began to gradually attenuate. The antibody level in the attenuated vaccine group declined faster than that in the inactivated vaccine group (Fig. 1).
To evaluate the protective effectiveness of the inactivated vaccine, each group was challenged with a lethal dose of GCRV at different time points. The mortality and clinical signs of the viruschallenged fish were recorded daily for 2 weeks post-challenge. The results showed that the inactivated vaccine immunized groups were protected at a level of more than 80% of individual fish, and the attenuated vaccine showed a slightly higher level of protection (Table 3). The immunized grass carp had lower mortality rates in comparison with that of the control group, demonstrating
3.3. Neutralization assay The SNT results showed that at 1 WPI, grass carp immunized with inactivated vaccine or attenuated vaccine developed detectable neutralizing antibody titers. A further increase in neutralizing antibody titers was observed at 2 WPI. At 4 WPI, the levels of neutralizing antibody began to gradually decline until the fish did not develop neutralizing antibody above the threshold for positivity at 12 WPI. The grass carp immunized with PBS did not develop any antibody response against GCRV. The GCRV-specific neutralizing antibody level in the attenuated vaccine group was significantly higher than that in the inactivated vaccine-immunized group (p < 0.01) (Fig. 2). 3.4. Immune-related genes expression In the immunized groups, the relative expression levels of these
Fig. 2. Detection of GCRV-specific serum neutralizing antibody levels induced in the vaccinated grass carp. Pooled serum samples from each group were tested by serumvirus neutralization test to determine the titers of antibody against the virulent GCRV strain HuNan1307. **p < 0.01; *p < 0.05.
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Table 3 Mortality rate and relative percentage survival (RPS) of fish challenged with HuNan1307 virus. Challenge time (WPI)
2 6 8 12 24 36 52
Mortality rate (%)
RPS (%)
Inactivated vaccine
Attenuated vaccine
Control
Inactivated vaccine
Attenuated vaccine
Control
0 5.0 10.0 10.0 15.0 15.0 20.0
0 0 0 10.0 10.0 5.0 5.0
90.0 85.0 95.0 100 100 80.0 85.0
100 94.1 89.5 95.0 85.0 81.3 76.5
100 100 100 90.0 90.0 93.8 94.1
e e e e e e e
that the inactivated vaccine could protect the fish from hemorrhagic disease. The protection provided by the inactivated vaccine and attenuated vaccine lasted for up to 1 year. In challenge trials, the dead fish showed typical clinical symptoms of GCRV infection, and no pathogen other than GCRV was detected from dead fish. 4. Discussion GCRV infection causes a severe disease in grass carp [24]. Although the existing attenuated vaccines have many advantages, a major disadvantage of these vaccines is their poor safety under natural conditions, as they may undergo harmful changes in the ecological environment. So, in Japan and some European countries, specific provision has been made to ensure that fish can only be immunized with inactivated vaccines. It is important to develop novel GCRV vaccines capable of inducing high protection combined with good safety. Inactivated vaccines are often preferred for safety reasons. Inactivated vaccines with the same antigenic composition as the microorganisms causing the diseases have been found to provide acceptable to good protection [25,26]. Two main points should be addressed in the development of inactivated vaccines: (i) it is necessary to completely inactivate an infectious virus to ensure the safety of the vaccine and (ii) essential viral epitopes are retained after the inactivation process in order to obtain a high-quality antigen [27]. In the present study, the inactivation dynamics of cell-associated GCRV antigen by formaldehyde or BPL were evaluated. Using uncomplicated processes, we found that the treatment of GCRV with formaldehyde or BPL completely eliminated its infectivity. The use of BPL, which acts on the viral genome with little or no effect on viral proteins, is highly recommended for the generation of an effective vaccine [28]. BPL has been widely used for the inactivation of other viruses and has resulted in known commercial vaccines [29,30]. The preparation of BPL-inactivated vaccines requires precaution during handling due to the toxicity of BPL. There is now substantial research showing that sodium thiosulfate can be added to the vaccine at the end of the inactivation step, which results in the conversion of BPL to nontoxic fat metabolites [31]. To explore the potential of the inactivated vaccine against GCRV, its immunogenicity was investigated through the immunization of grass carp with the cell-associated GCRV. First, the immunogenicity assay aimed to determine the minimum number of doses able to provide solid protection. The immune dose of 105.5 TCID50/0.2 mL of the inactivated vaccine induced a specific immune response, which provided good protection for the fishes after challenge with the virus HuNan1307(data not shown). The use of a low concentration of antigens in a vaccine is always an important step in the
establishment of preclinical and clinical studies, because it is associated with the technical and economic viability of the commercial product [32]. The results of ELISA indicated that specific serum antibodies in the inactivated vaccine or attenuated vaccine immunized fish could be detected during 1e52 weeks post-vaccination. In general, the concentration of antibodies against an antigen produced following vaccination usually corresponds with the protection/survival rate conferred by the vaccine. In the subsequent challenge experiment with a virulent strain of GCRV, the 2 immunized groups showed higher protection rates than the control group did. The results for the antibody levels of the immunized fish show good agreement with the results of our challenge studies indicating the protective efficacy of the antigens. The neutralizing antibody is the main correlate of protection against infection with GCRV [14]. SNT is a widely accepted method and has been used for evaluating the humoral response induced by other vaccines [21]. From 8 weeks post vaccination, the inactivated vaccine and attenuated vaccine immunized groups were still protected at levels of more than 80% despite the lack of detectable NAb. This lack of correlation between neutralizing antibody levels and protection indicated that the effectiveness of these vaccines might be partly due to the induction of a cellular response and not exclusively a humoral response through NAb. This speculation will require further experiments for verification. It is well known that vaccination with an antigen stimulates the expression of certain immune-related genes. IgM is regarded as an indicator of specific immune responses and the major Ig isotype of teleost fish [33]. It has been demonstrated that TLR3 can trigger immune responses against GCRV in grass carp [34]. Mx proteins are one of the key components of the antivirals induced by interferons in many species. A unique property of some Mx proteins is their antiviral activity against a wide range of viruses [35]. TLR3 is important for resistance to dsRNA analogs and viruses in fish. A previous study confirmed that an increased mRNA expression of TLR7 is triggered by GCRV challenge both in vivo and in vitro [36]. In our study, the expression of the IgM, Mx, TLR3, and TRL7 genes was highly increased in immunized grass carp from 1 week after immunization and maintained for almost 2 months. In addition to these 4 genes, the expression of 2 genes related to the innate immune response, which encode the proteins IFN and IFN-I, respectively, was also found to be slightly increased by treatment with the inactivated vaccine. All of the 6 tested genes were considered to be important in innate immunity and inflammatory or antiviral responses. Compared to the negative group, the expression of six immunerelated genes in the spleen and head kidney of inactivated vaccine
Fig. 3. qRT-PCR analysis of the expression of immune-related genes in grass carp vaccinated with inactivated vaccine. (A) IFN; (B) IFN-I; (C) IgM; (D) Mx; (E) TLR3; (F) TLR7. Samples were collected from the grass carp at 1, 2, 3, 4, 6, 8, 12, 24, 36, and 52 weeks post infection (WPI). Data are the means of 3 assays and are presented as the mean ± SD. ***p < 0.01; **p < 0.01; *p < 0.05.
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treated grass carp were significantly enhanced at the early stage of immune (mainly during weeks 2 through 6). These results indicated that the inactivated vaccine could induce cellular immune response. Both in two vaccine-immunized groups, relative expressions of genes were all up-regulated at first and then declined as time went on. The results are consistent with another study, which scientists investigated the impact of the injection of inactivated vaccine for hemorrhage of grass carp on the expressions of other main immune-related genes in spleen, including major histocompatibility complex I (MHC I), complement 3 (C3), interleukin1b (IL-1b), and intelectin genes [37]. Their results also revealed that inactivated vaccine injection increased mRNA expressions of the immune-related genes in the spleen. It is noteworthy that in our whole experimental process, the expression of these genes between inactivated vaccine and control groups showed less significant changes than those between attenuated vaccine and control groups. Although relative genes expression of inactivated vaccine immunized grass carp reduced to normal levels in the late stage of immune, the specific antibody levels of inactivated vaccine group remained high. This indicated that the inactivated HuNan1307 vaccine may mainly depend on inducing humoral immunity to provide good immunoprotection. In summary, the present study demonstrated that the inactivated HuNan1307 vaccine induced a strong humoral immune response and conferred effective protection against GCRV infection in grass carp. Although antibody levels and the relative mRNA levels of immune-relevant genes induced by the inactivated HuNan1307 vaccine were lower than those induced by the commercial attenuated vaccine, these 2 vaccines conferred equal protection against infection with a virulent strain of GCRV in grass carp. Considering its security and high protective effectiveness, the inactivated vaccine could serve as a potential vaccine candidate for the prevention of GCRV and has good development prospects. In addition, the duration of immunity and the development of immunological memory are both crucial for vaccine efficacy. In commercial aquaculture, the duration of protection needed is directly related to the length of the production cycle. In our study, a duration of protection of 1 year was achieved in the inactivated HuNan1307 immunization group, which could be suitable for use in commercial grass carp aquaculture considering the production cycle of grass carp. To further validate our findings, we will perform preclinical studies to evaluate the protective efficacy of the inactivated Hunan1307 vaccine in preventing the progression of GCRV infection to clinical disease and in conferring long-term immunity. Conflict of interest The authors have no conflict of interest to declare. Acknowledgements Funding for this research was provided by the National Key Technology R&D Program (No. 2012BAD25B02), the Science and Technology Planning Project of Jiangxi Province (No. 20152ACF60021), and Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2016ZD0503). References [1] Y. Rao, J. Su, Insights into the antiviral immunity against grass carp (Ctenopharyngodon idella) reovirus (GCRV) in grass carp, J. Immunol. Res. 2015 (2015) 670437. [2] Z.D. Dong, J. Zhang, X.S. Ji, F.N. Zhou, Y. Fu, W. Chen, et al., Molecular cloning, characterization and expression of cathepsin D from grass carp (Ctenopharyngodon idella), Fish Shellfish Immunol. 33 (5) (2012) 1207e1214.
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