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VP2 (PTA motif) encoding DNA vaccine confers protection against lethal challenge with infectious pancreatic necrosis virus (IPNV) in trout
Molecular Immunology 94 (2018) 61–67
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VP2 (PTA motif) encoding DNA vaccine confers protection against lethal challenge with infectious pancreatic necrosis virus (IPNV) in trout
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Sohrab Ahmadivanda, Mehdi Soltania,b, , Mahdi Behdanic, Øystein Evensend, Ehsan Alirahimic, Elahe Soltanie, Reza Hassanzadehf, Javad Ashrafi-Helang a
Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, P.O. Box 14155-6453, Tehran, Iran Centre of Excellence of Aquatic Animal Health, University of Tehran, Tehran, Iran c Biotechnology Research Center, Venom & Biotherapeutics Molecules Laboratory, Pasteur Institute of Iran, Tehran, Iran d Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo, Norway e Department of Microbiology, Faculty of Sciences, University of Tehran, Tehran, Iran f Central Veterinary Laboratory, Iran Veterinary Organization, Tehran, Iran g Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran b
A R T I C L E I N F O
A B S T R A C T
Keywords: IPNV Trout DNA vaccine Intramuscular VP2 PTA motif
IPNV in Atlantic salmon is represented by various strains with different virulence and immunogenicity linked to various motifs of the VP2 capsid. IPNV variant with P217, T221, A247 (PTA) motif is found to be avirulent in Atlantic salmon, but virulent in rainbow trout, and other salmonid species. This study describes a DNA vaccine delivered intramuscularly encoding the VP2 protein of infectious pancreatic necrosis virus (IPNV) with PTA motif that confers high protection in rainbow trout (Oncorhynchus mykiss). Intramuscular injection of 2, 5 and 10 μg of DNA (pcDNA3.1-VP2) in rainbow trout fry (4–5 g), confers relative protection of 75–83% in the different vaccine groups at 30 days post vaccination (450° days). The VP2 gene is expressed in spleen, kidney, muscle and liver at day 30 post-vaccination (RT-PCR), and IFN-1 and Mx-1 mRNA are upregulated at early time post vaccination, and so also for IgM, IgT, CD4 and CD8 in the head kidney of vaccinated fish compared to controls, 15 and 30 days post vaccination. Significant increase of serum anti-IPNV antibodies was found 30–90 days post-vaccination that was correlated with protection levels. Mortality corresponded with viral VP4 gene expression were significantly decreased in vaccinated and challenged fish. This shows for the first time that a VP2-encoding DNA vaccine delivered intramuscularly elicits a high level of protection alongside with high levels of circulating antibodies in rainbow trout and a lowered viral replication.
1. Introduction Infectious pancreatic necrosis virus (IPNV) belongs to Birnaviridae family and is the type strain of the genus Aquabirnavirus. It is one of the most widely distributed viruses in aquaculture and in the wild, affecting more than 63 marine aquatic animal species, including fish, mollusks and crustaceans causing high morbidity and mortality in fry and juveniles of farmed fish with asymptomatic adult carriers surviving the disease (Rodriguez Saint-Jean et al., 1991, 2003; Evensen and Santi, 2008). IPNV is a non-enveloped virus with a bi-segmented (A and B) dsRNA genome (∼5 kbp) (Dobos, 1976) where segment A encodes VP2 (Capsid protein) containing most of the neutralizing epitopes of the virus (Frost et al., 1995; Fridholm et al., 2007), VP3 (Structural protein), and the protease, VP4 (Dobos, 1976). It also encodes VP5, a non-structural
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protein of unknown function (Santi et al., 2005). Segment B only encodes an RNA-dependent RNA polymerase (VP1; Dobos, 1977). So far IPNV isolates have been grouped into 7 genogroups based on the VP2 sequences, which correlated with serotypes (A1–A9 and B) and geographical distribution (Hill and Way, 1995; Nishizawa et al., 2005). Depending upon the host species, viral strain and environmental conditions, IPN outbreak may result in mortality rates ranging from 5 to 100% (Evensen and Santi, 2008). VP2 and amino acid residues 217, 221 and 247 are the main molecular determinants of IPNV virulence and pathogenicity for Atlantic salmon (Salmo salar L.) (Santi et al., 2004; Song et al., 2005) and also correlate with immunogenicity (Munang’andu et al., 2013a). Highly virulent isolates from Atlantic salmon possess threonine and alanine at residues 217 and 221, respectively, while, Ala-to-Thr substitution at position 221 is indicative of a non-virulent or low virulent nature for
Corresponding author at: Faculty of Veterinary Medicine, University of Tehran, P.O. Box 14155-6453, Tehran, Iran. E-mail address: [email protected] (M. Soltani).
https://doi.org/10.1016/j.molimm.2017.12.015 Received 15 October 2017; Received in revised form 12 December 2017; Accepted 18 December 2017 0161-5890/ © 2017 Elsevier Ltd. All rights reserved.
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this fish species (Santi et al., 2004). Despite the avirulent nature for Atlantic salmon, IPNV variant with P217, T221, A247 (PTA) motif is found to be virulent in rainbow trout, and other salmonid species. This variant (PTA) is associated with high mortality in rainbow trout in Norway (Evensen, personal communication), and trout hatcheries in Iran (Raissy et al., 2010; Dadar et al., 2013; Ahmadivand et al., 2016), as well as in Finnish fish farms (Eriksson-Kallio et al., 2016), and in wild and farmed fish in Scotland (Bain et al., 2008). Prevention against mortality through vaccination is an important control strategy to avoid of IPN related losses. However, the current commercial IPN vaccines (Inactivated and subunit vaccines) induce an immune response biased towards humoral immunity and vaccinated fish may become carriers post infection (Bootland et al., 1995; Munang’andu et al., 2013c). DNA vaccines consist of antigen-encoding plasmid DNA delivered traditionally through the intramuscular(i.m) route in fish, and for some virus infections a strong and long-lasting immunity has been obtained under experimental (McLauchlan et al., 2003) and commercial use (Alonso and Leong, 2013). Recently, experimental VP2 encoding DNA vaccines delivered orally have been shown a lower virus infection level with a high protection against mortality in trout (Ballesteros et al., 2014; Ahmadivand et al., 2017). However, most plasmid-based IPN vaccines have been tested by intramuscular delivery, and those tested in laboratory trials the vaccines were not tested against real challenge since low mortality was obtained in non-vaccinated control groups (< 40%) and with disappointing levels of efficacy for VP2-encoding plasmid vaccines (Mikalsen et al., 2004). Other studies of VP2 plasmid vaccines testing by intramuscular delivery have not included lethal challenge post vaccination (de las Heras et al., 2009) and thus real efficacy cannot be decided. In this study, we have shown that an intramuscularly delivered DNA vaccine encoding the VP2 gene of a IPNV variant with PTA motif elicits a protective immune response in rainbow trout fry where the level of protection correlates with the level of circulating antibodies and the ability to limit virus replication in the internal organs post challenge.
concentration was measured using a spectrophotometer (NanoDrop 2000, Thermo scientific, Spain), and stored at −20 °C until use. 2.4. Vaccination and challenge Rainbow trout weighing 4–5 g were vaccinated by intramuscular (i.m.) injection using three doses, 2, 5 and 10 μg/fish of pcDNA3.1-VP2 in the left epaxial musculature in separate trials, each contains 90 fish. Two control groups (each group contains 90 fish) were also injected with 10 μg/fish empty plasmid (pcDNA3.1) and PBS. The vaccinated fish were then kept at 15 °C for 90 days. On day 30 post vaccination (450° days), 30 fish from each trail (15 fish per replicate) were challenged by IP injection of 0.2 ml/fish with IPNV at a concentration of 107 TCID50 mL−1. The relative percent survival (RPS) value of each experimental group was calculated by the formula [1 − (Cumulative percent mortality in vaccinated fish/cumulative percent mortality in non-vaccinated fish)] × 100. To demonstrate the viral load in the survivals the expression of IPNV-VP4 gene in the head kidney and spleen of fish (n = 5) were analyzed by RT-qPCR on day 45 post-challenge as described previously (Ballesteros et al., 2014; Ahmadivand et al., 2017). 2.5. Expression of VP2 gene from DNA vaccine On day 30 post-vaccination (day of challenge) three fish from each trial were sacrificed via overexposure to clove oil, and RNA was extracted from 20 mg of each homogenized tissues of spleen, muscle, head kidney and liver using the RiboEx SL Total RNA extraction kit (GeneAll, Korea). To eliminate genomic or plasmid DNA contamination, RNA samples were treated with RNase-free DNase (Promega), and then cDNA synthesis was carried out in a total volume of 25 μl from 5 μl of extracted RNA using HyperScript™ First Strand Synthesis Kit (GeneAll, Korea) according to the manufacturer’s recommendations. RT-PCR amplification of VP2 gene with an expected size of 405 bp was performed using SVP2-F and SVP2-R primer pairs according to Ahmadivand et al. (2017). The amplification products were resolved by electrophoresis using a 1% agarose under UV light.
2. Materials and methods
2.6. Immune genes transcription (RT-qPCR)
2.1. Ethics statement
The relative expression of IFN-1 and Mx-1 mRNA was assessed on days 3, 7 and 15 post-vaccination. Also, IgM, IgT, CD4 and CD8 genes related to adaptive immune responses were quantified on days 15 and 30 post-vaccination. The head-kidney tissues of 5 fish in each trail were processed for gene expressions using Real-Time PCR (Applied Biosystems) and SYBR Green qPCR Master Mix as described previously (Ballesteros et al., 2014; Ahmadivand et al., 2017). The melting curve of each amplicon was examined, and the expression of the target genes was corrected based on the endogenous control expression (EF-1 α) calculated relative to empty plasmid according to the 2−ΔΔCt method (Livak and Schmittgen, 2001).
All applicable guidelines for the care and use of animals were followed according to the instructions given by the University of Tehran Ethics Committee for Animal Experimentation. 2.2. Virus and cell culture The vaccine strain (IPNV with PTA motif, genotype 5, serotype Sp) was used for immunoassays and challenge studies. The strain was selected based on epidemiological studies of the disease outbreaks in Iranian trout farms during 2009–2016 (GenBank Acc No: KX665156, KX665157, KX665158, KX665159, GU338037, KF279643, KC489465). The virus was propagated in CHSE-214 cell line (Fryer et al., 1965) and titrated according to Reed and Muench producer (1938).
2.7. Antibody titer by ELISA On days 15, 30, 45, 60 and 90 post-vaccination, five non-challenged fish from each vaccine group were anesthetized by clove oil and blood samples were collected from the caudal vein, clotted and sera were separated by centrifuging at 500g for 10 min. Level of antibodies was assessed by ELISA, as described previously by Ahmadivand et al. (2017). Briefly, 96-well ELISA plates were coated by with 100 μl (107) well−1 IPNV and incubated overnight at 4 °C. After washing with PBS-T (phosphate buffered saline containing 0.05% Tween 20), the wells were blocked for 2 h at 22 °C with 3% dried skimmed milk in PBS (300 μl/ well). Following a wash with PBS-T, 100 μl of each fish serum sample (serially diluted with PBST-5% BSA) was added in triplicate and
2.3. Vaccine construction The DNA vaccine (pcDNA3.1-VP2) was prepared as previously described by Ahmadivand et al. (2017). Briefly, the VP2 gene of IPNV was inserted in pcDNA3.1 (Invitrogen, USA) under the control of the CMV promoter, verified using HindIII and XhoI endonuclease analysis and then amplified in Escherichia coli (TOP10). The constructed plasmid DNA was then isolated with the Endofree Plasmid Mega Purification Kit (Qiagen, USA) according to the manufacturer’s instructions. The DNA 62
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post-vaccination in vaccinated trails compared to control, while at 15 dpv post-vaccination it was upregulated only in 10 μg trail. Also, CD4 mRNA expression in kidney samples was significantly higher in all trails compared to control on days 15 and 30 post-vaccination, except for 2 μg group on day 30 post-vaccination (Fig. 2). A significant increase was also seen in CD8 expression of the kidney samples of 10 μg group on day 15 post-vaccination.
incubated for 3 h at 22 °C. PBS was used as negative control. The plates were again washed with PBS-T, then 100 μl of the anti-rainbow trout IgM monoclonal antibody (Aquatic Diagnostics Ltd, Stirling, Scotland) were added to the wells and incubated for 60 min at 22 °C. After washing with PBS-T, the bound antibodies were detected by adding 100 μl well−1 anti-mouse IgG-HRP (Bio-Rad) diluted 1/1000 in conjugate buffer (1% BSA solution) and incubated for 60 min at 22 °C. After washing with PBS-T antibody binding was then visualized by adding 100 μl of tetramethylbenzidine dihydrochloride (TMB; BioLegend, USA) to each well and incubation for 10 min at 22 °C. The reactions were stopped by adding 50 μl of the stop solution (2 M H2SO4 in distilled water) to the wells. The plates were read at 450 nm in Epoch Microplate Spectrophotometer (BioTek).
3.3. IPNV challenge Challenge was performed on day 30 post-vaccination and mortality was recorded daily for 30 days. Cumulative mortalities following IPNV challenge were significantly lower in all vaccinated groups compared to both of non-vaccinated fish and fish vaccinated with empty-plasmid (pcDNA.3.1; Fig. 3). Cumulative mortalities of 53%, and 57% were obtained in empty plasmid and PBS controls, respectively. The 2 μg pcDNA3.1-VP2 group showed cumulative mortality of 13% with a RPS of 76%, while this was 88% for both 5 and 10 μg groups (Fig. 3).
2.8. Statistics analysis Data was statistically analyzed by one-way or two-way followed by Dunnett’s multiple comparisons test analysis of variance (ANOVA) using SPSS package (SPSS 1998) or GraphPad Prism version 7.00, GraphPad Software, La Jolla California USA. Differences were considered statistically significant at p < 0.05.
3.4. Virus load in fish tissues Virus load was determined by RT-qPCR targeting the IPNV-VP4 gene in the spleen and kidney of surviving fish on 45 days post-challenge (Fig. 4). The results showed that the surviving vaccinated fish had a significantly lower VP4 expression than unvaccinated and empty plasmid groups. The IPNV-VP4 expression in the kidney of non-vaccinated trout was 10, 25 and 41 folds higher than vaccinated with 2, 5 or 10 μg/fish of pcDNA-VP2 DNA vaccine, respectively. However, a lower expression was quantified in the spleen tissue of the surviving fish.
3. Results 3.1. In vivo transcription of VP2 gene RT-PCR was used to detect the expression of VP2 gene of IPNV in different tissues (spleen, kidney, muscle and liver) of vaccinated rainbow trout at 30 dpv (Fig. 1). RT-PCR products with an expected size of 405 bp specific to the IPNV-VP2 were obtained in all tested tissue samples of vaccinated trout that confirmed the expression of VP2 gene and systemic distribution of the administered plasmid in different tissues of fish. No amplification was detected in tissue samples of the control group (vaccinated fish with empty-plasmid).
3.5. Anti-IPNV serum antibodies Analysis for circulating antibody was undertaken by ELISA over the period 15–90 days post-vaccination (Fig. 5). Anti-IPNV antibody for the three vaccinated groups were detectable by 90 days post-vaccination. The titer of antibody in all vaccinated trails were significantly higher than control groups (p < 0.05). The levels of antibody in sera of immunized fish progressively increased, and was relatively consistent in 30–60 dpv. However, anti-IPNV antibodies level was dose-dependent and the highest titer was obtained in the 10 μg trail.
3.2. RT-qPCR analysis of immune-related genes The transcription levels of different innate (IFN-1 and MX-1), adaptive (IgM, IgT, CD4 and CD8) immune-relevant genes in vaccinated trout with different doses of pcDNAVP2 DNA vaccine were evaluated through RT-qPCR in head kidney of the fish. The transcription levels of most of the examined immune genes were significantly induced in all vaccinated groups related to control group (Fig. 2 and Supplementary Fig. 1). By RT-qPCR we found a significant induction of IFN-1 and MX-1 mRNA expression after vaccination with the three doses, 2, 5 and 10 μg/fish of DNA at 3, 7 and 15 dpv (Fig. 2). The peak of IFN-1 and MX-1 expression was on day 3 post-vaccination and declined gradually up to day 15 post-vaccination. Expression of IFN-1 and MX-1 genes was dose-dependent, and for the 10 μg/fish group there were a 22 and 52fold upregulation on day 3 post-vaccination, respectively (p < 0.05). Strong IgM gene expression was detected in the kidney of vaccinated fish on day 15 post-vaccination, and increased significantly by 30 dpv. Similarly, IgT expression was significantly higher on day 30
4. Discussion The results of this study clearly show that the DNA vaccine encoding the VP2 gene of IPNV (Genogroup5, Serotype Sp) with the P217, T221, A247 motif elicited a protective immune response against lethal challenge of IPNV in rainbow trout with a RPS of 88% for the two highest vaccine doses, 5 and 10 μg/fish. This finding corresponds with the expression of the VP2 gene in the spleen, kidney, muscle and liver tissues post vaccination and high circulating antibody levels from 30 days post vaccination and onwards. Our findings are in contrast with previous studies performed in Atlantic salmon using DNA vaccines to protect against IPN virus infection (Mikalsen et al., 2004). Here we found that a plasmid encoding the VP2 protein confers protective immunity towards lethal challenge of the virus, while in Atlantic salmon a protection was only seen when a plasmid encoding the entire segment A was used (Mikalsen et al., 2004). It is worth to say that the construct used by Mikalsen et al. (2004) was based on the N1 isolate which carries a P217, T221, A247 motif, a non-virulent variant in Atlantic salmon. The strain used in the challenge was not characterized in detail, but the level of mortality obtained would indicate a moderate IPNV variant and not the virulent T217, A221, T247 variant (Johansen and Sommer, 2001). However, the level virulent of these strains was not established in year 2001 as their data was first published in 2004 (Nishizawa et al., 2005).
Fig. 1. Expression of IPNV- VP2 gene in tissues of rainbow trout at day 30 post-vaccination. M: 100-bp DNA ladder; 1–2: spleen, 3–4: kidney, 5: liver, 6: muscle, P: positive control (pcDNA3.1-VP2), N: negative control (pcDNA3.1). RT-PCR products were analyzed on a 1% agarose gel.
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Fig. 2. IFN-1 and MX-1 (3, 7 and 15 dpv), and CD4, CD8, IgM and IgT (15 and 30 dpv) gene expression in the kidney tissue of IM vaccinated trout with 2, 5 and 10 μg/fish of pcDNA3.1-VP2, respectively. Data were normalized based on endogenous EF-1a gene and presented as mean fold increase relative to empty plasmid (2−ΔΔCT method; n = 5). Asterisks showing significant differences between vaccinated and unvaccinated fish.
2016), and other salmonid species (Bain et al., 2008; Eriksson-Kallio et al., 2016). In general, a best immune protection via vaccination is against homologous serotypes of IPNV (vaccine-challenge), and this is found across serotypes (Munang’andu et al., 2012). Thus, selection of vaccine strains for use in an area should be done based on the most prevalent and immunogenic serotype causing disease. Humoral and cell-mediated immune responses have been demonstrated after intramuscular delivery of DNA vaccines (Tonheim et al., 2008). Here, we found a significant up-regulation of IFN-1 and MX-1 genes in a similar dose-dependent kinetic, suggesting IFN response, the first line of defense against viral infections. Similar observations have been recorded in brown trout (Salmo trutta; de las Heras et al., 2010), and rainbow trout (O. mykiss; de las Heras et al., 2010; Ballesteros et al., 2014; Ahmadivand et al., 2017; Xu et al., 2017) following oral delivery of DNA Vaccines. T and B cell markers, CD8, CD4, IgM and IgT genes were also
Moreover, the constructed DNA vaccine encoding VP2 with PTA motif has induced high immunogenicity, indicating that the protection and antibody levels for non-replicating vaccines probably cannot be influenced by PTA motif (Munang’andu et al., 2012, 2013c;). It has previously been shown that the level of circulating antibodies against IPNV correlates with protection against mortality in Atlantic salmon (Munang’andu et al., 2013a). Therfore, the findings reported here are in concert with what has been shown earlier (Munang’andu et al., 2013a; Ahmadivand et al., 2017). IPNV strains associated with the disease outbreak in Atlantic salmon possess different levels of virulence and immunogenicity linked to various motifs of the VP2 capsid. For instance, the IPNV genogroup 5 is more virulent than genogroup 1 and 2 in salmonids (Eriksson-Kallio et al., 2016; Zhu et al., 2017), and the PTA strain is found to be avirulent in Atlantic salmon (Santi et al., 2004; Song et al., 2005), but has been reported virulent in rainbow trout (Raissy et al., 2010; Skjesol et al., 2011; Ahmadi et al., 2013; Dadar et al., 2013; Ahmadivand et al., 64
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Fig. 3. Cumulative mortalities of i.m. vaccinated rainbow trout with different doses of pcDNA3.1-VP2 and challenged via IP injection of IPNV at 2 × 106 TCID50/fish. Fish were challenged on 30 days post-vaccination and mortality was recorded daily for 30 days.
previous vaccination and challenge studies (Ballesteros et al., 2015; Ahmadivand et al., 2017), giving a good indication of the virus replication post-challenge. We found a significantly decreased VP4 expression in vaccinated and challenged fish, suggesting a marked reduction in virus propagation post challenge in vaccinated fish, especially in the 5 and 10 μg groups with higher survival rates.
significantly up-regulated, which is in line with previous studies showing that IPNV DNA vaccines induce adaptive cellular immune response (Ballesteros et al., 2014; Ahmadivand et al., 2017). In a previous study Munang’andu et al. (2013b) found a correlation between the mRNA expression of markers for CD4 and CD8 T cells, and protection against IPNV challenge in Atlantic salmon. Corresponding markers of T cell responses, like Gata-3 T-bet and FoxP3 were also studied and good correlation of observed protection against challenge was seen (Munang’andu et al., 2013b). Importantly, in the studied referred (Munang’andu et al., 2013b) an inactivated vaccine was used for primary immunization and responses were biased towards humoral responses (CD4 responses/GATA-3) while in this study, we find that both CD4 and CD8 mRNA expression is upregulated post immunization (Fig. 2) in line with the general understanding that DNA vaccines elicit both CD4 and CD8 responses as well as humoral responses (Seder and Hill, 2000). This observation also points the fact that immune responses in fish correspond well similar to those happen in higher vertebrates. Expression of IPNV-VP4 has been used as a marker of viral load in
5. Conclusion In conclusion, this study describes the development of an effective DNA vaccine encoding the VP2 gene of IPNV (Genogroup5, Serotype Sp) with P217, T221, A247 (PTA) motif, prevalent in several European countries as well as Iran. This is the first description of an intramuscularly delivered DNA vaccine encoding only the VP2 protein of infectious pancreatic necrosis virus (IPNV) that confers protection against lethal challenge in rainbow trout. Future studies should have focused on the vaccine efficacy through immersion or cohabitation challenge, resembling the natural outbreak of IPN.
Fig. 4. Relative expression of the IPNV-VP4 gene in kidney and spleen tissues of vaccinated and non-vaccinated trout infected with IPNV on day 45 post-challenge (n = 5). Data are represented as 2−ΔCT where ΔCT is the Ct (target gene) – Ct (EF-1a). Each point represents data from a single fish. Significant differences are indicated. Control = non-vaccinated, challenged controls; pcDNA3.1 are fish injected with empty plasmid, then challenged; others are different vaccine groups, then challenged.
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Fig. 5. Serum anti-IPNV antibodies in different vaccine groups during 15–90 days post vaccination. Serum samples were collected on days 15, 30, 45, 60 and 90 days post vaccination. Data are mean optical density (OD; at 450 nm) at serum dilution 1:100. Control = non-vaccinated; pcDNA3.1 are vaccinated with empty plasmid; other vaccine groups are as indicated.
Conflict of interest
Dobos, P., 1976. Size and structure of the genome of infectious pancreatic necrosis virus. Nucleic Acids Res. 3, 1903–1924. Dobos, P., 1977. Virus-specific protein synthesis in cells infected by infectious pancreatic necrosis virus. J. Virol. 21, 242–258. Eriksson-Kallio, A.M., Holopainen, R., Viljamaa-Dirks, S., Vennerstrom, P., KuukkaAnttila, H., Koski, P., Gadd, T., 2016. Infectious pancreatic necrosis virus (IPNV) strain with genetic properties associated with low pathogenicity at Finnish fish farms. Dis. Aquat. Organ. 118, 21–30. Evensen, O., Santi, N., 2008. Infectious pancreatic necrosis virus. In: Mahy, B.W.J., Van Regenmortel, M.H.V. (Eds.), Encyclopedia of Virology, 5 ed. Elsevier, Oxford, pp. 83–89. Fridholm, H., Eliasson, L., Everitt, E., 2007. Immunogenicity properties of authentic and heterologously synthesized structural protein VP2 of infectious pancreatic necrosis virus. Viral Immunol. 20, 635–648. Frost, P., Havarstein, L.S., Lygren, B., Stahl, S., Endresen, C., Christie, K.E., 1995. Mapping of neutralization epitopes on infectious pancreatic necrosis viruses. J. Gen. Virol. 76, 1165–1172. Fryer, J.L., Yusha, A., Pilcher, K.S., 1965. The in vitro cultivation of tissue and cells of Pacific salmon and steelhead trout. Ann. N.Y. Acad. Sci. 126, 566–586. Hill, B., Way, K., 1995. Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Ann. Rev. Fish Dis. 5, 55–77. Johansen, L.H., Sommer, A.I., 2001. Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida. Dis. Aquat. Organ. 47, 109–117. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCt method. Methods 25, 402–408. McLauchlan, P.E., Collet, B., Ingerslev, E., Secombes, C.J., Lorenzen, N., Ellis, A.E., 2003. DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size, dose, route of injection and duration of protection-early protection correlates with Mx expression. Fish Shellfish Immunol. 15, 39–50. Mikalsen, A.B., Torgersen, J., Alestrom, P., Hellemann, A.L., Koppang, E.O., Rimstad, E., 2004. Protection of atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination. Dis. Aquat. Organ. 60, 11–20. Munang'andu, H.M., Fredriksen, B.N., Mutoloki, S., Brudeseth, B., Kuo, T.Y., Marjara, I.S., Dalmo, R.A., Evensen, O., 2012. Comparison of vaccine efficacy for different antigen delivery systems for infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L.) in a cohabitation challenge model. Vaccine 30, 4007–4016. Munang’Andu, H.M., Fredriksen, B.N., Mutoloki, S., Dalmo, R.A., Evensen, O., 2013a. Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L). Vet. Res. 44, 7. Munang'andu, H.M., Fredriksen, B.N., Mutoloki, S., Dalmo, R.A., Evensen, O., 2013b. The kinetics of CD4+ and CD8+ T-cell gene expression correlate with protection in Atlantic salmon (Salmo salar L) vaccinated against infectious pancreatic necrosis. Vaccine 31, 1956–1963. Munang’andu, H.M., Sandtro, A., Mutoloki, S., Brudeseth, B.E., Santi, N., Evensen, O., 2013c. Immunogenicity and cross protective ability of the central VP2 amino acids of infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar L.). PLoS One 8, e54263. Nishizawa, T., Kinoshita, S., Yoshimizu, M., 2005. An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region. J. Gen. Virol. 86, 1973–1978. Raissy, M., Momtaz, H., Ansari, M., Moumeni, M., Hosseinifard, M., 2010. Distribution of Infectious Pancreatic Necrosis Virus (IPNV) in two major rainbow trout fry producing provinces of Iran with respect to clinically infected farms. J. Food Agric. Environ. 8, 614–615. Reed, L.J., Muench, H.A., 1938. Simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497. Rodriguez Saint-Jean, S., Vilas Minondo, M.P., Palacios, A., Perez-Prieto, S., 1991. Detection of infectious pancreatic necrosis virus in a carrier population of rainbow trout (Oncorhynchus mykiss, Richardson) by flow cytometry. J. Fish Dis. 14, 545–553. Rodriguez Saint-Jean, S., Borrego, J.J., Perez-Prieto, S.I., 2003. Infectious pancreatic necrosis virus: biology, pathogenesis, and diagnostic methods. Adv. Virus Res. 62, 113–165.
Authors declare that they do not have any conflict of interest. Acknowledgments This study was financially funded by Research Council of University of Tehran and Centre of Excellence of Aquatic Animal Health, University of Tehran, Iran. We thank Mr. Mehran Ahmadpoor for his helpful assistance. We are grateful for the generous collaboration of Pasteur Institute, Iran and Central Veterinary Laboratory of Iran Veterinary Organization. Øystein Evensen received funding from the Research Council of Norway, project no. 239140. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.molimm.2017.12.015. References Ahmadi, N., Oryan, A., Akhlaghi, M., Hosseini, A., 2013. Tissue distribution of infectious pancreatic necrosis virus serotype Sp in naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum): an immunohistochemical and nested-PCR study. J. Fish Dis. 36, 629–637. Ahmadivand, S., Soltani, M., Mardani, K., Shokrpoor, S., Rahmati-Holasoo, H., Mokhtari, A., Hasanzadeh, R., 2016. Isolation and identification of viral hemorrhagic septicemia virus (VHSV) from farmed rainbow trout (Oncorhynchus mykiss) in Iran. Acta Trop. 156, 30–36. Ahmadivand, S., Soltani, M., Behdani, M., Evensen, O., Alirahimi, E., Hassanzadeh, R., Soltani, E., 2017. Oral DNA vaccines based on CS-TPP nanoparticles and alginate microparticles confer high protection against infectious pancreatic necrosis virus (IPNV) infection in trout. Dev. Comp. Immunol. 74, 178–189. Alonso, M., Leong, J.A., 2013. Licensed DNA Vaccines against Infectious Hematopoietic Necrosis Virus (IHNV). Recent Pat DNA Gene Seq. 7, 62–65. Bain, N., Gregory, A., Raynard, R.S., 2008. Genetic analysis of infectious pancreatic necrosis virus from Scotland. J. Fish Dis. 31, 37–47. Ballesteros, N.A., Rodriguez Saint-Jean, S., Perez-Prieto, S.I., 2014. Food pellets as an effective delivery method for a DNA vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss, Walbaum). Fish Shellfish Immunol. 37, 220–228. Ballesteros, N.A., Rodriguez Saint-Jean, S., Perez-Prieto, S.I., 2015. Immune responses to oral pcDNA-VP2 vaccine in relation to infectious pancreatic necrosis virus carrier state in rainbow trout Oncorhynchus mykiss. Vet. Immunol. Immunopathol. 165, 127–137. Bootland, L.M., Dobos, P., Stevenson, R.M.W., 1995. Immunization of adult brook trout, Salvelinus fontinalis (Mitchill), fails to prevent the infectious pancreatic necrosis virus (IPNV) carrier state. J. Fish Dis. 18, 449–458. Dadar, M., Peyghan, R., Memari, H.R., Shapouri, M.R., Hasanzadeh, R., Goudarzi, L.M., Vakharia, V.N., 2013. Sequence analysis of infectious pancreatic necrosis virus isolated from Iranian reared rainbow trout (Oncorhynchus mykiss) in 2012. Virus Genes 47, 574–578. de las Heras, A.I., Pérez-Prieto, S.I., Rodríguez Saint-Jean, S., 2009. In vitro and in vivo immune responses induced by a DNA vaccine encoding the VP2 gene of infectious pancreatic necrosis virus. Fish Shellfish Immunol. 27, 120–129. de las Heras, A.I., Rodríguez Saint-Jean, S., Perez-prieto, S.I., 2010. Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish. Fish Shellfish Immunol. 28, 562–570.
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infectious pancreatic necrosis virus virulence and cell culture adaptation. J. Virol. 79, 10289–10299. Tonheim, T., Ch Bøgwald, J., Dalmo, R.A., 2008. What happens to the DNA vaccine in fish? A review of current knowledge. Fish Shellfish Immunol. 25, 1–18. Xu, L., Zhao, J., Liu, M., Ren, G., Jian, F., Yin, J., Feng, J., Liu, H., Lu, T., 2017. Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout. Sci. Rep. 7, 5700. Zhu, L., Wang, X., Wang, K., Yang, Q., He, J., Qin, Z., Geng, Y., Ouyang, P., Huangc, X., 2017. Outbreak of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout in China. Acta Trop. 170, 63–69.
Santi, N., Vakharia, V.N., Evensen, O., 2004. Identification of putative motifs involved in the virulence of infectious pancreatic necrosis virus. Virology 322, 31–40. Santi, N., Sandtro, A., Sindre, H., Song, H., Hong, J.R., Thu, B., Wu, J.L., Vakharia, V.N., Evensen, O., 2005. Infectious pancreatic necrosis virus induces apoptosis in vitro and in vivo independent of VP5 expression. Virology 342, 13–25. Seder, R.A., Hill, A.V., 2000. Vaccines against intracellular infections requiring cellular immunity. Nature 406, 793–798. Skjesol, A., Skjaeveland, I., Elnaes, M., Timmerhaus, G., Fredriksen, B.N., Jorgensen, S.M., Krasnov, A., Jorgensen, J.B., 2011. IPNV with high and low virulence: host immune responses and viral mutations during infection. Virol. J. 8, 396. Song, H., Santi, N., Evensen, O., Vakharia, V.N., 2005. Molecular determinants of
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VP2 (PTA motif) encoding DNA vaccine confers protection against lethal challenge with infectious pancreatic necrosis virus (IPNV) in trout
T
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Sohrab Ahmadivanda, Mehdi Soltania,b, , Mahdi Behdanic, Øystein Evensend, Ehsan Alirahimic, Elahe Soltanie, Reza Hassanzadehf, Javad Ashrafi-Helang a
Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, P.O. Box 14155-6453, Tehran, Iran Centre of Excellence of Aquatic Animal Health, University of Tehran, Tehran, Iran c Biotechnology Research Center, Venom & Biotherapeutics Molecules Laboratory, Pasteur Institute of Iran, Tehran, Iran d Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo, Norway e Department of Microbiology, Faculty of Sciences, University of Tehran, Tehran, Iran f Central Veterinary Laboratory, Iran Veterinary Organization, Tehran, Iran g Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran b
A R T I C L E I N F O
A B S T R A C T
Keywords: IPNV Trout DNA vaccine Intramuscular VP2 PTA motif
IPNV in Atlantic salmon is represented by various strains with different virulence and immunogenicity linked to various motifs of the VP2 capsid. IPNV variant with P217, T221, A247 (PTA) motif is found to be avirulent in Atlantic salmon, but virulent in rainbow trout, and other salmonid species. This study describes a DNA vaccine delivered intramuscularly encoding the VP2 protein of infectious pancreatic necrosis virus (IPNV) with PTA motif that confers high protection in rainbow trout (Oncorhynchus mykiss). Intramuscular injection of 2, 5 and 10 μg of DNA (pcDNA3.1-VP2) in rainbow trout fry (4–5 g), confers relative protection of 75–83% in the different vaccine groups at 30 days post vaccination (450° days). The VP2 gene is expressed in spleen, kidney, muscle and liver at day 30 post-vaccination (RT-PCR), and IFN-1 and Mx-1 mRNA are upregulated at early time post vaccination, and so also for IgM, IgT, CD4 and CD8 in the head kidney of vaccinated fish compared to controls, 15 and 30 days post vaccination. Significant increase of serum anti-IPNV antibodies was found 30–90 days post-vaccination that was correlated with protection levels. Mortality corresponded with viral VP4 gene expression were significantly decreased in vaccinated and challenged fish. This shows for the first time that a VP2-encoding DNA vaccine delivered intramuscularly elicits a high level of protection alongside with high levels of circulating antibodies in rainbow trout and a lowered viral replication.
1. Introduction Infectious pancreatic necrosis virus (IPNV) belongs to Birnaviridae family and is the type strain of the genus Aquabirnavirus. It is one of the most widely distributed viruses in aquaculture and in the wild, affecting more than 63 marine aquatic animal species, including fish, mollusks and crustaceans causing high morbidity and mortality in fry and juveniles of farmed fish with asymptomatic adult carriers surviving the disease (Rodriguez Saint-Jean et al., 1991, 2003; Evensen and Santi, 2008). IPNV is a non-enveloped virus with a bi-segmented (A and B) dsRNA genome (∼5 kbp) (Dobos, 1976) where segment A encodes VP2 (Capsid protein) containing most of the neutralizing epitopes of the virus (Frost et al., 1995; Fridholm et al., 2007), VP3 (Structural protein), and the protease, VP4 (Dobos, 1976). It also encodes VP5, a non-structural
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protein of unknown function (Santi et al., 2005). Segment B only encodes an RNA-dependent RNA polymerase (VP1; Dobos, 1977). So far IPNV isolates have been grouped into 7 genogroups based on the VP2 sequences, which correlated with serotypes (A1–A9 and B) and geographical distribution (Hill and Way, 1995; Nishizawa et al., 2005). Depending upon the host species, viral strain and environmental conditions, IPN outbreak may result in mortality rates ranging from 5 to 100% (Evensen and Santi, 2008). VP2 and amino acid residues 217, 221 and 247 are the main molecular determinants of IPNV virulence and pathogenicity for Atlantic salmon (Salmo salar L.) (Santi et al., 2004; Song et al., 2005) and also correlate with immunogenicity (Munang’andu et al., 2013a). Highly virulent isolates from Atlantic salmon possess threonine and alanine at residues 217 and 221, respectively, while, Ala-to-Thr substitution at position 221 is indicative of a non-virulent or low virulent nature for
Corresponding author at: Faculty of Veterinary Medicine, University of Tehran, P.O. Box 14155-6453, Tehran, Iran. E-mail address: [email protected] (M. Soltani).
https://doi.org/10.1016/j.molimm.2017.12.015 Received 15 October 2017; Received in revised form 12 December 2017; Accepted 18 December 2017 0161-5890/ © 2017 Elsevier Ltd. All rights reserved.
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this fish species (Santi et al., 2004). Despite the avirulent nature for Atlantic salmon, IPNV variant with P217, T221, A247 (PTA) motif is found to be virulent in rainbow trout, and other salmonid species. This variant (PTA) is associated with high mortality in rainbow trout in Norway (Evensen, personal communication), and trout hatcheries in Iran (Raissy et al., 2010; Dadar et al., 2013; Ahmadivand et al., 2016), as well as in Finnish fish farms (Eriksson-Kallio et al., 2016), and in wild and farmed fish in Scotland (Bain et al., 2008). Prevention against mortality through vaccination is an important control strategy to avoid of IPN related losses. However, the current commercial IPN vaccines (Inactivated and subunit vaccines) induce an immune response biased towards humoral immunity and vaccinated fish may become carriers post infection (Bootland et al., 1995; Munang’andu et al., 2013c). DNA vaccines consist of antigen-encoding plasmid DNA delivered traditionally through the intramuscular(i.m) route in fish, and for some virus infections a strong and long-lasting immunity has been obtained under experimental (McLauchlan et al., 2003) and commercial use (Alonso and Leong, 2013). Recently, experimental VP2 encoding DNA vaccines delivered orally have been shown a lower virus infection level with a high protection against mortality in trout (Ballesteros et al., 2014; Ahmadivand et al., 2017). However, most plasmid-based IPN vaccines have been tested by intramuscular delivery, and those tested in laboratory trials the vaccines were not tested against real challenge since low mortality was obtained in non-vaccinated control groups (< 40%) and with disappointing levels of efficacy for VP2-encoding plasmid vaccines (Mikalsen et al., 2004). Other studies of VP2 plasmid vaccines testing by intramuscular delivery have not included lethal challenge post vaccination (de las Heras et al., 2009) and thus real efficacy cannot be decided. In this study, we have shown that an intramuscularly delivered DNA vaccine encoding the VP2 gene of a IPNV variant with PTA motif elicits a protective immune response in rainbow trout fry where the level of protection correlates with the level of circulating antibodies and the ability to limit virus replication in the internal organs post challenge.
concentration was measured using a spectrophotometer (NanoDrop 2000, Thermo scientific, Spain), and stored at −20 °C until use. 2.4. Vaccination and challenge Rainbow trout weighing 4–5 g were vaccinated by intramuscular (i.m.) injection using three doses, 2, 5 and 10 μg/fish of pcDNA3.1-VP2 in the left epaxial musculature in separate trials, each contains 90 fish. Two control groups (each group contains 90 fish) were also injected with 10 μg/fish empty plasmid (pcDNA3.1) and PBS. The vaccinated fish were then kept at 15 °C for 90 days. On day 30 post vaccination (450° days), 30 fish from each trail (15 fish per replicate) were challenged by IP injection of 0.2 ml/fish with IPNV at a concentration of 107 TCID50 mL−1. The relative percent survival (RPS) value of each experimental group was calculated by the formula [1 − (Cumulative percent mortality in vaccinated fish/cumulative percent mortality in non-vaccinated fish)] × 100. To demonstrate the viral load in the survivals the expression of IPNV-VP4 gene in the head kidney and spleen of fish (n = 5) were analyzed by RT-qPCR on day 45 post-challenge as described previously (Ballesteros et al., 2014; Ahmadivand et al., 2017). 2.5. Expression of VP2 gene from DNA vaccine On day 30 post-vaccination (day of challenge) three fish from each trial were sacrificed via overexposure to clove oil, and RNA was extracted from 20 mg of each homogenized tissues of spleen, muscle, head kidney and liver using the RiboEx SL Total RNA extraction kit (GeneAll, Korea). To eliminate genomic or plasmid DNA contamination, RNA samples were treated with RNase-free DNase (Promega), and then cDNA synthesis was carried out in a total volume of 25 μl from 5 μl of extracted RNA using HyperScript™ First Strand Synthesis Kit (GeneAll, Korea) according to the manufacturer’s recommendations. RT-PCR amplification of VP2 gene with an expected size of 405 bp was performed using SVP2-F and SVP2-R primer pairs according to Ahmadivand et al. (2017). The amplification products were resolved by electrophoresis using a 1% agarose under UV light.
2. Materials and methods
2.6. Immune genes transcription (RT-qPCR)
2.1. Ethics statement
The relative expression of IFN-1 and Mx-1 mRNA was assessed on days 3, 7 and 15 post-vaccination. Also, IgM, IgT, CD4 and CD8 genes related to adaptive immune responses were quantified on days 15 and 30 post-vaccination. The head-kidney tissues of 5 fish in each trail were processed for gene expressions using Real-Time PCR (Applied Biosystems) and SYBR Green qPCR Master Mix as described previously (Ballesteros et al., 2014; Ahmadivand et al., 2017). The melting curve of each amplicon was examined, and the expression of the target genes was corrected based on the endogenous control expression (EF-1 α) calculated relative to empty plasmid according to the 2−ΔΔCt method (Livak and Schmittgen, 2001).
All applicable guidelines for the care and use of animals were followed according to the instructions given by the University of Tehran Ethics Committee for Animal Experimentation. 2.2. Virus and cell culture The vaccine strain (IPNV with PTA motif, genotype 5, serotype Sp) was used for immunoassays and challenge studies. The strain was selected based on epidemiological studies of the disease outbreaks in Iranian trout farms during 2009–2016 (GenBank Acc No: KX665156, KX665157, KX665158, KX665159, GU338037, KF279643, KC489465). The virus was propagated in CHSE-214 cell line (Fryer et al., 1965) and titrated according to Reed and Muench producer (1938).
2.7. Antibody titer by ELISA On days 15, 30, 45, 60 and 90 post-vaccination, five non-challenged fish from each vaccine group were anesthetized by clove oil and blood samples were collected from the caudal vein, clotted and sera were separated by centrifuging at 500g for 10 min. Level of antibodies was assessed by ELISA, as described previously by Ahmadivand et al. (2017). Briefly, 96-well ELISA plates were coated by with 100 μl (107) well−1 IPNV and incubated overnight at 4 °C. After washing with PBS-T (phosphate buffered saline containing 0.05% Tween 20), the wells were blocked for 2 h at 22 °C with 3% dried skimmed milk in PBS (300 μl/ well). Following a wash with PBS-T, 100 μl of each fish serum sample (serially diluted with PBST-5% BSA) was added in triplicate and
2.3. Vaccine construction The DNA vaccine (pcDNA3.1-VP2) was prepared as previously described by Ahmadivand et al. (2017). Briefly, the VP2 gene of IPNV was inserted in pcDNA3.1 (Invitrogen, USA) under the control of the CMV promoter, verified using HindIII and XhoI endonuclease analysis and then amplified in Escherichia coli (TOP10). The constructed plasmid DNA was then isolated with the Endofree Plasmid Mega Purification Kit (Qiagen, USA) according to the manufacturer’s instructions. The DNA 62
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post-vaccination in vaccinated trails compared to control, while at 15 dpv post-vaccination it was upregulated only in 10 μg trail. Also, CD4 mRNA expression in kidney samples was significantly higher in all trails compared to control on days 15 and 30 post-vaccination, except for 2 μg group on day 30 post-vaccination (Fig. 2). A significant increase was also seen in CD8 expression of the kidney samples of 10 μg group on day 15 post-vaccination.
incubated for 3 h at 22 °C. PBS was used as negative control. The plates were again washed with PBS-T, then 100 μl of the anti-rainbow trout IgM monoclonal antibody (Aquatic Diagnostics Ltd, Stirling, Scotland) were added to the wells and incubated for 60 min at 22 °C. After washing with PBS-T, the bound antibodies were detected by adding 100 μl well−1 anti-mouse IgG-HRP (Bio-Rad) diluted 1/1000 in conjugate buffer (1% BSA solution) and incubated for 60 min at 22 °C. After washing with PBS-T antibody binding was then visualized by adding 100 μl of tetramethylbenzidine dihydrochloride (TMB; BioLegend, USA) to each well and incubation for 10 min at 22 °C. The reactions were stopped by adding 50 μl of the stop solution (2 M H2SO4 in distilled water) to the wells. The plates were read at 450 nm in Epoch Microplate Spectrophotometer (BioTek).
3.3. IPNV challenge Challenge was performed on day 30 post-vaccination and mortality was recorded daily for 30 days. Cumulative mortalities following IPNV challenge were significantly lower in all vaccinated groups compared to both of non-vaccinated fish and fish vaccinated with empty-plasmid (pcDNA.3.1; Fig. 3). Cumulative mortalities of 53%, and 57% were obtained in empty plasmid and PBS controls, respectively. The 2 μg pcDNA3.1-VP2 group showed cumulative mortality of 13% with a RPS of 76%, while this was 88% for both 5 and 10 μg groups (Fig. 3).
2.8. Statistics analysis Data was statistically analyzed by one-way or two-way followed by Dunnett’s multiple comparisons test analysis of variance (ANOVA) using SPSS package (SPSS 1998) or GraphPad Prism version 7.00, GraphPad Software, La Jolla California USA. Differences were considered statistically significant at p < 0.05.
3.4. Virus load in fish tissues Virus load was determined by RT-qPCR targeting the IPNV-VP4 gene in the spleen and kidney of surviving fish on 45 days post-challenge (Fig. 4). The results showed that the surviving vaccinated fish had a significantly lower VP4 expression than unvaccinated and empty plasmid groups. The IPNV-VP4 expression in the kidney of non-vaccinated trout was 10, 25 and 41 folds higher than vaccinated with 2, 5 or 10 μg/fish of pcDNA-VP2 DNA vaccine, respectively. However, a lower expression was quantified in the spleen tissue of the surviving fish.
3. Results 3.1. In vivo transcription of VP2 gene RT-PCR was used to detect the expression of VP2 gene of IPNV in different tissues (spleen, kidney, muscle and liver) of vaccinated rainbow trout at 30 dpv (Fig. 1). RT-PCR products with an expected size of 405 bp specific to the IPNV-VP2 were obtained in all tested tissue samples of vaccinated trout that confirmed the expression of VP2 gene and systemic distribution of the administered plasmid in different tissues of fish. No amplification was detected in tissue samples of the control group (vaccinated fish with empty-plasmid).
3.5. Anti-IPNV serum antibodies Analysis for circulating antibody was undertaken by ELISA over the period 15–90 days post-vaccination (Fig. 5). Anti-IPNV antibody for the three vaccinated groups were detectable by 90 days post-vaccination. The titer of antibody in all vaccinated trails were significantly higher than control groups (p < 0.05). The levels of antibody in sera of immunized fish progressively increased, and was relatively consistent in 30–60 dpv. However, anti-IPNV antibodies level was dose-dependent and the highest titer was obtained in the 10 μg trail.
3.2. RT-qPCR analysis of immune-related genes The transcription levels of different innate (IFN-1 and MX-1), adaptive (IgM, IgT, CD4 and CD8) immune-relevant genes in vaccinated trout with different doses of pcDNAVP2 DNA vaccine were evaluated through RT-qPCR in head kidney of the fish. The transcription levels of most of the examined immune genes were significantly induced in all vaccinated groups related to control group (Fig. 2 and Supplementary Fig. 1). By RT-qPCR we found a significant induction of IFN-1 and MX-1 mRNA expression after vaccination with the three doses, 2, 5 and 10 μg/fish of DNA at 3, 7 and 15 dpv (Fig. 2). The peak of IFN-1 and MX-1 expression was on day 3 post-vaccination and declined gradually up to day 15 post-vaccination. Expression of IFN-1 and MX-1 genes was dose-dependent, and for the 10 μg/fish group there were a 22 and 52fold upregulation on day 3 post-vaccination, respectively (p < 0.05). Strong IgM gene expression was detected in the kidney of vaccinated fish on day 15 post-vaccination, and increased significantly by 30 dpv. Similarly, IgT expression was significantly higher on day 30
4. Discussion The results of this study clearly show that the DNA vaccine encoding the VP2 gene of IPNV (Genogroup5, Serotype Sp) with the P217, T221, A247 motif elicited a protective immune response against lethal challenge of IPNV in rainbow trout with a RPS of 88% for the two highest vaccine doses, 5 and 10 μg/fish. This finding corresponds with the expression of the VP2 gene in the spleen, kidney, muscle and liver tissues post vaccination and high circulating antibody levels from 30 days post vaccination and onwards. Our findings are in contrast with previous studies performed in Atlantic salmon using DNA vaccines to protect against IPN virus infection (Mikalsen et al., 2004). Here we found that a plasmid encoding the VP2 protein confers protective immunity towards lethal challenge of the virus, while in Atlantic salmon a protection was only seen when a plasmid encoding the entire segment A was used (Mikalsen et al., 2004). It is worth to say that the construct used by Mikalsen et al. (2004) was based on the N1 isolate which carries a P217, T221, A247 motif, a non-virulent variant in Atlantic salmon. The strain used in the challenge was not characterized in detail, but the level of mortality obtained would indicate a moderate IPNV variant and not the virulent T217, A221, T247 variant (Johansen and Sommer, 2001). However, the level virulent of these strains was not established in year 2001 as their data was first published in 2004 (Nishizawa et al., 2005).
Fig. 1. Expression of IPNV- VP2 gene in tissues of rainbow trout at day 30 post-vaccination. M: 100-bp DNA ladder; 1–2: spleen, 3–4: kidney, 5: liver, 6: muscle, P: positive control (pcDNA3.1-VP2), N: negative control (pcDNA3.1). RT-PCR products were analyzed on a 1% agarose gel.
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Fig. 2. IFN-1 and MX-1 (3, 7 and 15 dpv), and CD4, CD8, IgM and IgT (15 and 30 dpv) gene expression in the kidney tissue of IM vaccinated trout with 2, 5 and 10 μg/fish of pcDNA3.1-VP2, respectively. Data were normalized based on endogenous EF-1a gene and presented as mean fold increase relative to empty plasmid (2−ΔΔCT method; n = 5). Asterisks showing significant differences between vaccinated and unvaccinated fish.
2016), and other salmonid species (Bain et al., 2008; Eriksson-Kallio et al., 2016). In general, a best immune protection via vaccination is against homologous serotypes of IPNV (vaccine-challenge), and this is found across serotypes (Munang’andu et al., 2012). Thus, selection of vaccine strains for use in an area should be done based on the most prevalent and immunogenic serotype causing disease. Humoral and cell-mediated immune responses have been demonstrated after intramuscular delivery of DNA vaccines (Tonheim et al., 2008). Here, we found a significant up-regulation of IFN-1 and MX-1 genes in a similar dose-dependent kinetic, suggesting IFN response, the first line of defense against viral infections. Similar observations have been recorded in brown trout (Salmo trutta; de las Heras et al., 2010), and rainbow trout (O. mykiss; de las Heras et al., 2010; Ballesteros et al., 2014; Ahmadivand et al., 2017; Xu et al., 2017) following oral delivery of DNA Vaccines. T and B cell markers, CD8, CD4, IgM and IgT genes were also
Moreover, the constructed DNA vaccine encoding VP2 with PTA motif has induced high immunogenicity, indicating that the protection and antibody levels for non-replicating vaccines probably cannot be influenced by PTA motif (Munang’andu et al., 2012, 2013c;). It has previously been shown that the level of circulating antibodies against IPNV correlates with protection against mortality in Atlantic salmon (Munang’andu et al., 2013a). Therfore, the findings reported here are in concert with what has been shown earlier (Munang’andu et al., 2013a; Ahmadivand et al., 2017). IPNV strains associated with the disease outbreak in Atlantic salmon possess different levels of virulence and immunogenicity linked to various motifs of the VP2 capsid. For instance, the IPNV genogroup 5 is more virulent than genogroup 1 and 2 in salmonids (Eriksson-Kallio et al., 2016; Zhu et al., 2017), and the PTA strain is found to be avirulent in Atlantic salmon (Santi et al., 2004; Song et al., 2005), but has been reported virulent in rainbow trout (Raissy et al., 2010; Skjesol et al., 2011; Ahmadi et al., 2013; Dadar et al., 2013; Ahmadivand et al., 64
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Fig. 3. Cumulative mortalities of i.m. vaccinated rainbow trout with different doses of pcDNA3.1-VP2 and challenged via IP injection of IPNV at 2 × 106 TCID50/fish. Fish were challenged on 30 days post-vaccination and mortality was recorded daily for 30 days.
previous vaccination and challenge studies (Ballesteros et al., 2015; Ahmadivand et al., 2017), giving a good indication of the virus replication post-challenge. We found a significantly decreased VP4 expression in vaccinated and challenged fish, suggesting a marked reduction in virus propagation post challenge in vaccinated fish, especially in the 5 and 10 μg groups with higher survival rates.
significantly up-regulated, which is in line with previous studies showing that IPNV DNA vaccines induce adaptive cellular immune response (Ballesteros et al., 2014; Ahmadivand et al., 2017). In a previous study Munang’andu et al. (2013b) found a correlation between the mRNA expression of markers for CD4 and CD8 T cells, and protection against IPNV challenge in Atlantic salmon. Corresponding markers of T cell responses, like Gata-3 T-bet and FoxP3 were also studied and good correlation of observed protection against challenge was seen (Munang’andu et al., 2013b). Importantly, in the studied referred (Munang’andu et al., 2013b) an inactivated vaccine was used for primary immunization and responses were biased towards humoral responses (CD4 responses/GATA-3) while in this study, we find that both CD4 and CD8 mRNA expression is upregulated post immunization (Fig. 2) in line with the general understanding that DNA vaccines elicit both CD4 and CD8 responses as well as humoral responses (Seder and Hill, 2000). This observation also points the fact that immune responses in fish correspond well similar to those happen in higher vertebrates. Expression of IPNV-VP4 has been used as a marker of viral load in
5. Conclusion In conclusion, this study describes the development of an effective DNA vaccine encoding the VP2 gene of IPNV (Genogroup5, Serotype Sp) with P217, T221, A247 (PTA) motif, prevalent in several European countries as well as Iran. This is the first description of an intramuscularly delivered DNA vaccine encoding only the VP2 protein of infectious pancreatic necrosis virus (IPNV) that confers protection against lethal challenge in rainbow trout. Future studies should have focused on the vaccine efficacy through immersion or cohabitation challenge, resembling the natural outbreak of IPN.
Fig. 4. Relative expression of the IPNV-VP4 gene in kidney and spleen tissues of vaccinated and non-vaccinated trout infected with IPNV on day 45 post-challenge (n = 5). Data are represented as 2−ΔCT where ΔCT is the Ct (target gene) – Ct (EF-1a). Each point represents data from a single fish. Significant differences are indicated. Control = non-vaccinated, challenged controls; pcDNA3.1 are fish injected with empty plasmid, then challenged; others are different vaccine groups, then challenged.
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Fig. 5. Serum anti-IPNV antibodies in different vaccine groups during 15–90 days post vaccination. Serum samples were collected on days 15, 30, 45, 60 and 90 days post vaccination. Data are mean optical density (OD; at 450 nm) at serum dilution 1:100. Control = non-vaccinated; pcDNA3.1 are vaccinated with empty plasmid; other vaccine groups are as indicated.
Conflict of interest
Dobos, P., 1976. Size and structure of the genome of infectious pancreatic necrosis virus. Nucleic Acids Res. 3, 1903–1924. Dobos, P., 1977. Virus-specific protein synthesis in cells infected by infectious pancreatic necrosis virus. J. Virol. 21, 242–258. Eriksson-Kallio, A.M., Holopainen, R., Viljamaa-Dirks, S., Vennerstrom, P., KuukkaAnttila, H., Koski, P., Gadd, T., 2016. Infectious pancreatic necrosis virus (IPNV) strain with genetic properties associated with low pathogenicity at Finnish fish farms. Dis. Aquat. Organ. 118, 21–30. Evensen, O., Santi, N., 2008. Infectious pancreatic necrosis virus. In: Mahy, B.W.J., Van Regenmortel, M.H.V. (Eds.), Encyclopedia of Virology, 5 ed. Elsevier, Oxford, pp. 83–89. Fridholm, H., Eliasson, L., Everitt, E., 2007. Immunogenicity properties of authentic and heterologously synthesized structural protein VP2 of infectious pancreatic necrosis virus. Viral Immunol. 20, 635–648. Frost, P., Havarstein, L.S., Lygren, B., Stahl, S., Endresen, C., Christie, K.E., 1995. Mapping of neutralization epitopes on infectious pancreatic necrosis viruses. J. Gen. Virol. 76, 1165–1172. Fryer, J.L., Yusha, A., Pilcher, K.S., 1965. The in vitro cultivation of tissue and cells of Pacific salmon and steelhead trout. Ann. N.Y. Acad. Sci. 126, 566–586. Hill, B., Way, K., 1995. Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Ann. Rev. Fish Dis. 5, 55–77. Johansen, L.H., Sommer, A.I., 2001. Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida. Dis. Aquat. Organ. 47, 109–117. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCt method. Methods 25, 402–408. McLauchlan, P.E., Collet, B., Ingerslev, E., Secombes, C.J., Lorenzen, N., Ellis, A.E., 2003. DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size, dose, route of injection and duration of protection-early protection correlates with Mx expression. Fish Shellfish Immunol. 15, 39–50. Mikalsen, A.B., Torgersen, J., Alestrom, P., Hellemann, A.L., Koppang, E.O., Rimstad, E., 2004. Protection of atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination. Dis. Aquat. Organ. 60, 11–20. Munang'andu, H.M., Fredriksen, B.N., Mutoloki, S., Brudeseth, B., Kuo, T.Y., Marjara, I.S., Dalmo, R.A., Evensen, O., 2012. Comparison of vaccine efficacy for different antigen delivery systems for infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L.) in a cohabitation challenge model. Vaccine 30, 4007–4016. Munang’Andu, H.M., Fredriksen, B.N., Mutoloki, S., Dalmo, R.A., Evensen, O., 2013a. Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L). Vet. Res. 44, 7. Munang'andu, H.M., Fredriksen, B.N., Mutoloki, S., Dalmo, R.A., Evensen, O., 2013b. The kinetics of CD4+ and CD8+ T-cell gene expression correlate with protection in Atlantic salmon (Salmo salar L) vaccinated against infectious pancreatic necrosis. Vaccine 31, 1956–1963. Munang’andu, H.M., Sandtro, A., Mutoloki, S., Brudeseth, B.E., Santi, N., Evensen, O., 2013c. Immunogenicity and cross protective ability of the central VP2 amino acids of infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar L.). PLoS One 8, e54263. Nishizawa, T., Kinoshita, S., Yoshimizu, M., 2005. An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region. J. Gen. Virol. 86, 1973–1978. Raissy, M., Momtaz, H., Ansari, M., Moumeni, M., Hosseinifard, M., 2010. Distribution of Infectious Pancreatic Necrosis Virus (IPNV) in two major rainbow trout fry producing provinces of Iran with respect to clinically infected farms. J. Food Agric. Environ. 8, 614–615. Reed, L.J., Muench, H.A., 1938. Simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497. Rodriguez Saint-Jean, S., Vilas Minondo, M.P., Palacios, A., Perez-Prieto, S., 1991. Detection of infectious pancreatic necrosis virus in a carrier population of rainbow trout (Oncorhynchus mykiss, Richardson) by flow cytometry. J. Fish Dis. 14, 545–553. Rodriguez Saint-Jean, S., Borrego, J.J., Perez-Prieto, S.I., 2003. Infectious pancreatic necrosis virus: biology, pathogenesis, and diagnostic methods. Adv. Virus Res. 62, 113–165.
Authors declare that they do not have any conflict of interest. Acknowledgments This study was financially funded by Research Council of University of Tehran and Centre of Excellence of Aquatic Animal Health, University of Tehran, Iran. We thank Mr. Mehran Ahmadpoor for his helpful assistance. We are grateful for the generous collaboration of Pasteur Institute, Iran and Central Veterinary Laboratory of Iran Veterinary Organization. Øystein Evensen received funding from the Research Council of Norway, project no. 239140. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.molimm.2017.12.015. References Ahmadi, N., Oryan, A., Akhlaghi, M., Hosseini, A., 2013. Tissue distribution of infectious pancreatic necrosis virus serotype Sp in naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum): an immunohistochemical and nested-PCR study. J. Fish Dis. 36, 629–637. Ahmadivand, S., Soltani, M., Mardani, K., Shokrpoor, S., Rahmati-Holasoo, H., Mokhtari, A., Hasanzadeh, R., 2016. Isolation and identification of viral hemorrhagic septicemia virus (VHSV) from farmed rainbow trout (Oncorhynchus mykiss) in Iran. Acta Trop. 156, 30–36. Ahmadivand, S., Soltani, M., Behdani, M., Evensen, O., Alirahimi, E., Hassanzadeh, R., Soltani, E., 2017. Oral DNA vaccines based on CS-TPP nanoparticles and alginate microparticles confer high protection against infectious pancreatic necrosis virus (IPNV) infection in trout. Dev. Comp. Immunol. 74, 178–189. Alonso, M., Leong, J.A., 2013. Licensed DNA Vaccines against Infectious Hematopoietic Necrosis Virus (IHNV). Recent Pat DNA Gene Seq. 7, 62–65. Bain, N., Gregory, A., Raynard, R.S., 2008. Genetic analysis of infectious pancreatic necrosis virus from Scotland. J. Fish Dis. 31, 37–47. Ballesteros, N.A., Rodriguez Saint-Jean, S., Perez-Prieto, S.I., 2014. Food pellets as an effective delivery method for a DNA vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss, Walbaum). Fish Shellfish Immunol. 37, 220–228. Ballesteros, N.A., Rodriguez Saint-Jean, S., Perez-Prieto, S.I., 2015. Immune responses to oral pcDNA-VP2 vaccine in relation to infectious pancreatic necrosis virus carrier state in rainbow trout Oncorhynchus mykiss. Vet. Immunol. Immunopathol. 165, 127–137. Bootland, L.M., Dobos, P., Stevenson, R.M.W., 1995. Immunization of adult brook trout, Salvelinus fontinalis (Mitchill), fails to prevent the infectious pancreatic necrosis virus (IPNV) carrier state. J. Fish Dis. 18, 449–458. Dadar, M., Peyghan, R., Memari, H.R., Shapouri, M.R., Hasanzadeh, R., Goudarzi, L.M., Vakharia, V.N., 2013. Sequence analysis of infectious pancreatic necrosis virus isolated from Iranian reared rainbow trout (Oncorhynchus mykiss) in 2012. Virus Genes 47, 574–578. de las Heras, A.I., Pérez-Prieto, S.I., Rodríguez Saint-Jean, S., 2009. In vitro and in vivo immune responses induced by a DNA vaccine encoding the VP2 gene of infectious pancreatic necrosis virus. Fish Shellfish Immunol. 27, 120–129. de las Heras, A.I., Rodríguez Saint-Jean, S., Perez-prieto, S.I., 2010. Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish. Fish Shellfish Immunol. 28, 562–570.
66
Molecular Immunology 94 (2018) 61–67
S. Ahmadivand et al.
infectious pancreatic necrosis virus virulence and cell culture adaptation. J. Virol. 79, 10289–10299. Tonheim, T., Ch Bøgwald, J., Dalmo, R.A., 2008. What happens to the DNA vaccine in fish? A review of current knowledge. Fish Shellfish Immunol. 25, 1–18. Xu, L., Zhao, J., Liu, M., Ren, G., Jian, F., Yin, J., Feng, J., Liu, H., Lu, T., 2017. Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout. Sci. Rep. 7, 5700. Zhu, L., Wang, X., Wang, K., Yang, Q., He, J., Qin, Z., Geng, Y., Ouyang, P., Huangc, X., 2017. Outbreak of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout in China. Acta Trop. 170, 63–69.
Santi, N., Vakharia, V.N., Evensen, O., 2004. Identification of putative motifs involved in the virulence of infectious pancreatic necrosis virus. Virology 322, 31–40. Santi, N., Sandtro, A., Sindre, H., Song, H., Hong, J.R., Thu, B., Wu, J.L., Vakharia, V.N., Evensen, O., 2005. Infectious pancreatic necrosis virus induces apoptosis in vitro and in vivo independent of VP5 expression. Virology 342, 13–25. Seder, R.A., Hill, A.V., 2000. Vaccines against intracellular infections requiring cellular immunity. Nature 406, 793–798. Skjesol, A., Skjaeveland, I., Elnaes, M., Timmerhaus, G., Fredriksen, B.N., Jorgensen, S.M., Krasnov, A., Jorgensen, J.B., 2011. IPNV with high and low virulence: host immune responses and viral mutations during infection. Virol. J. 8, 396. Song, H., Santi, N., Evensen, O., Vakharia, V.N., 2005. Molecular determinants of
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