Sara I. Pérez-Prieto

Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish.

DNA vaccines and oral DNA-based immunotherapy against infectious pancreatic necrosis virus (IPNV) have scarcely been studied in salmonid fish. Here, a vector with the capsid VP2 gene inserted was encapsulated in alginate microspheres to avoid the aggressive gastrointestinal conditions experienced following oral administration. Alginate microspheres were effective to protect the pDNA encoding VP2, which was expressed early in different organs of the vaccinated trout and that persisted for at least 60 days. The vaccine induces innate immune responses, raising the expression of IFN more than 10-fold relative to the fish vaccinated with the empty plasmid, at 7 and 15 days post-vaccination. Likewise, maximal expression of the IFN-induced antiviral Mx protein was recorded 15 days post-vaccination and neutralizing antibodies were also detected after 15 days, although their titre rose further at 21 days post-vaccination. Protection was high in the immunized fish, which showed around an 80% relative survival when challenged 15 and 30 days after vaccine delivery. Very low viral load with respect to the control group was detected in the vaccinated fish that survived 45 days after challenge. Thus, this study demonstrates the potential of the encapsulation technique for IPNV-DNA vaccine delivery and the relevance of the IPNV-VP2 gene for future plasmid constructs.

Infectious pancreatic necrosis virus: biology, pathogenesis, and diagnostic methods.

Publisher Summary Infectious pancreatic necrosis virus (IPNV) is the etiological agent of an acute contagious systemic disease of several species of freshwater and marine fish, molluscs, and crustacean. IPNV is the widespread of the piscine viruses. IPNV belongs to the genus Aquabirnavirus within the family Birnaviridae. The family contains three genera: genus Aquabirnavirus (type species, IPNV and yellowtail ascites virus) of fish, genus Avibirnavirus (type species, infectiousbursal disease virus, IBDV) of birds, and genus Entomobirnavirus (type species, Drosophila X virus, DXV) of insects. Several diagnostic methods for IPNV have been reported, including the fluorescent antibody technique, the immunostaphylococcus- protein A test (ISPA), the coagglutination test, the enzyme-linked immunosorbent assay (ELISA), immunoblots and Western blots, and immunoperoxidase phosphatase cell staining (IP). More recently, molecular probes for the detection of nucleic acids using the polymerase chain reaction have been developed and applied for the diagnosis of fish viruses. Techniques involving detection and characterization of the viral genome or polypeptides are also considered these include DNA-based techniques, mainly nucleic acid hybridization, and the polymerase chain reaction.

Viral coinfection in salmonids: infectious pancreatic necrosis virus interferes with infectious hematopoietic necrosis virus

SummaryCoinfection of farm-reared salmonids involving two viruses has been described, but there is no report on the interactions between viruses. Here we examine whether infectious pancreatic necrosis virus (IPNV) strain Sp interferes with the growth of infectious hematopoietic necrosis virus (IHNV) strain S46, a coinfected isolate from rainbow trout. When BF-2 cell culture was inoculated with S46 the infective titer of the IHNV fraction decreased by 3 log10 units compared to the growth curve of IHNV in the single infection. RT-PCR assay confirmed this reduction, which after successive passages of the co-infected sample led to a decrease in IHNV mRNA and the absence of the specific PCR product for IHNV. Flow cytometry showed that only 13% of the cells inoculated with S46 strain were infected with IHNV at 48–72 h post infection, in contrast to the 50–80% of cells that were positive for IPNV. Exposure of cells to IHNV for 24 h before infection with IPNV did not affect the infective titers of either virus or the PCR results obtained in simultaneous coinfections. Moreover IHNV was not inhibited when the IPNV inoculum was reduced. So, a multiplicity of infection dependence was demonstrated for IPNV-IHNV interference; the RT-PCR assay described here was found to be a suitable technique for identifying and studying dual viral infections

The pyloric caeca area is a major site for IgM+ and IgT+ B cell recruitment in response to oral vaccination in rainbow trout

Although previous studies have characterized some aspects of the immune response of the teleost gut in response to diverse pathogens or stimuli, most studies have focused on the posterior segments exclusively. However, there are still many details of how teleost intestinal immunity is regulated that remain unsolved, including the location of IgM+ and IgT+ B cells along the digestive tract and their role during the course of a local stimulus. Thus, in the current work, we have studied the B cell response in five different segments of the rainbow trout (Oncorhynchus mykiss) digestive tract in both naïve fish and fish orally vaccinated with an alginate-encapsulated DNA vaccine against infectious pancreatic necrosis virus (IPNV). IgM+ and IgT+ cells were identified all along the tract with the exception of the stomach in naïve fish. While IgM+ cells were mostly located in the lamina propria (LP), IgT+ cells were primarily localized as intraepithelial lymphocytes (IELs). Scattered IgM+ IELs were only detected in the pyloric caeca. In response to oral vaccination, the pyloric caeca region was the area of the digestive tract in which a major recruitment of B cells was demonstrated through both real time PCR and immunohistochemistry, observing a significant increase in the number of both IgM+ and IgT+ IELs. Our findings demonstrate that both IgM+ and IgT+ respond to oral stimulation and challenge the paradigm that teleost IELs are exclusively T cells. Unexpectedly, we have also detected B cells in the fat tissue associated to the digestive tract that respond to vaccination, suggesting that these cells surrounded by adipocytes also play a role in mucosal defense.

Oral immunization of rainbow trout to infectious pancreatic necrosis virus (Ipnv) induces different immune gene expression profiles in head kidney and pyloric ceca

Induction of neutralizing antibodies and protection by oral vaccination with DNA-alginates of rainbow trout Oncorhynchus mykiss against infectious pancreatic necrosis virus (IPNV) was recently reported. Because orally induced immune response transcript gene profiles had not been described yet neither in fish, nor after IPNV vaccination, we studied them in head kidney (an immune response internal organ) and a vaccine entry tissue (pyloric ceca). By using an oligo microarray enriched in immune-related genes validated by RTqPCR, the number of increased transcripts in head kidney was higher than in pyloric ceca while the number of decreased transcripts was higher in pyloric ceca than in head kidney. Confirming previous reports on intramuscular DNA vaccination or viral infection, mx genes increased their transcription in head kidney. Other transcript responses such as those corresponding to interferons, their receptors and induced proteins (n=91 genes), VHSV-induced genes (n=25), macrophage-related genes (n=125), complement component genes (n=176), toll-like receptors (n=31), tumor necrosis factors (n=32), chemokines and their receptors (n=121), interleukines and their receptors (n=119), antimicrobial peptides (n=59), and cluster differentiation antigens (n=58) showed a contrasting and often complementary behavior when head kidney and pyloric ceca were compared. For instance, classical complement component transcripts increased in head kidney while only alternative pathway transcripts increased in pyloric ceca, different β-defensins increased in head kidney but remained constant in pyloric ceca. The identification of new gene markers on head kidney/pyloric ceca could be used to follow up and/or to improve immunity during fish oral vaccination.

Virulence of infectious hematopoietic necrosis virus and Infectious pancreatic necrosis virus coinfection in rainbow trout ( Oncorhynchus mykiss) and nucleotide sequence analysis of the IHNV glycoprotein gene.

Summary. The outcomes of a coinfection of rainbow trout (Oncorhynchus mykiss) with Infectious hematopoietic necrosis virus (IHNV) strain S46 and Infectious pancreatic necrosis virus (IPNV) strain S46 was determined after waterborne infection. Trout infected with the IHNV/IPNV.S46 sample, (a mixed sample containing equal infectious titers of the viruses) showed 50% less mortality than fish infected with either of the reference viruses alone. Forty-five days after the coinfection, IPNV antigens were detected by flow cytometry in 49 to 63% of the leukocytes from the surviving trout; whereas, only 9–15.6% of the leukocytes expressed IHNV viral antigens. IPNV was easily detected by reverse transcription-polymerase chain reaction (RT-PCR), whereas, for IHNV, a second step of amplification of a 753 bp fragment corresponding to the internal sequences of the IHNV G gene was necessary to optimize viral detection. The sequence of the IHNV gene involved in virulence, the glycoprotein (G) gene, was determined for the IHNV.S46 and compared with other sequences available in the GenBank. Changes found were not located in the antigenic domains of the glycoprotein and were considered not significant.

An active DNA vaccine against infectious pancreatic necrosis virus (IPNV) with a different mode of action than fish rhabdovirus DNA vaccines

Although there are some commercial vaccines available against infectious pancreatic necrosis virus (IPNV), the disease still continues to be a major problem for aquaculture development worldwide. In the current work, we constructed a DNA vaccine against IPNV (pIPNV-PP) by cloning the long open reading frame of the polyprotein encoded by the viral RNA segment A. In vitro, the vaccine is properly translated giving the functional IPNV polyprotein since preVP2, VP2 and VP3 proteins were detected because of the VP4-protease cleavage. EPC cells transfected with the vaccine plasmid expressed the viral proteins and induced the expression of type I interferon (IFN)-induced Mx genes. Furthermore, IPNV synthesized proteins seemed to assemble in virus-like particles as evidenced by electron microscopy. In vivo, rainbow trout specimens were intramuscularly injected with the vaccine and expression of immune-relevant genes, the presence of neutralizing antibodies and effect on viral load was determined. The pIPNV-PP vaccine was expressed at the injection site and up-regulated MHC Ialpha, MHC IIalpha, type-I interferon (IFN), Mx, CD4 and CD8alpha gene expression in the muscle, head kidney or spleen, although to a much lower extent than the up-regulations observed in response to an effective DNA vaccine against viral hemorrhagic septicaemia virus (VHSV). However, the IPNV vaccine was also very effective in terms of acquired immunity since it elicited neutralizing antibodies (in 6 out of 8 trout fingerlings) and decreased 665-fold the viral load after IPNV infection. The effectiveness of this new IPNV DNA vaccine and its possible mechanism of action are discussed and compared to other viral vaccines.

An oral DNA vaccine against infectious haematopoietic necrosis virus (IHNV) encapsulated in alginate microspheres induces dose- dependent immune responses and significant protection in rainbow trout (Oncorrhynchus mykiss)

Administered by intramuscular injection, a DNA vaccine (pIRF1A-G) containing the promoter regions upstream of the rainbow trout interferon regulatory factor 1A gene (IRF1A) driven the expression of the infectious hematopoietic necrosis virus (IHNV) glycoprotein (G) elicited protective immune responses in rainbow trout (Oncorhynchus mykiss). However, less laborious and cost-effective routes of DNA vaccine delivery are required to vaccinate large numbers of susceptible farmed fish. In this study, the pIRF1A-G vaccine was encapsulated into alginate microspheres and orally administered to rainbow trout. At 1, 3, 5, and 7 d post-vaccination, IHNV G transcripts were detected by quantitative real-time PCR in gills, spleen, kidney and intestinal tissues of vaccinated fish. This result suggested that the encapsulation of pIRF1A-G in alginate microparticles protected the DNA vaccine from degradation in the fish stomach and ensured vaccine early delivery to the hindgut, vaccine passage through the intestinal mucosa and its distribution thought internal and external organs of vaccinated fish. We also observed that the oral route required approximately 20-fold more plasmid DNA than the injection route to induce the expression of significant levels of IHNV G transcripts in kidney and spleen of vaccinated fish. Despite this limitation, increased IFN-1, TLR-7 and IgM gene expression was detected by qRT-PCR in kidney of vaccinated fish when a 10 μg dose of the oral pIRF1A-G vaccine was administered. In contrast, significant Mx-1, Vig-1, Vig-2, TLR-3 and TLR-8 gene expression was only detected when higher doses of pIRF1A-G (50 and 100 μg) were orally administered. The pIRF1A-G vaccine also induced the expression of several markers of the adaptive immune response (CD4, CD8, IgM and IgT) in kidney and spleen of immunized fish in a dose-dependent manner. When vaccinated fish were challenged by immersion with live IHNV, evidence of a dose-response effect of the oral vaccine could also be observed. Although the protective effects of the oral pIRF1A-G vaccine after a challenge with IHNV were partial, significant differences in cumulative percent mortalities among the orally vaccinated fish and the unvaccinated or empty-plasmid vaccinated fish were observed. Similar levels of protection were obtained after the intramuscular administration of 5 μg of pIRF1A-G or after the oral administration of a high dose of pIRF1A-G vaccine (100 μg); with 70 and 56 relative percent survival values, respectively. When fish were vaccinated with alginate microspheres containing high doses of the pIRF1A-G vaccine (50 or 100 μg), a significant increase in the production of anti-IHNV antibodies was detected in serum samples of the vaccinated fish compared with that in unvaccinated fish. At 10 days post-challenge, IHNV N gene expression was nearly undetectable in kidney and spleen of orally vaccinated fish which suggested that the vaccine effectively reduced the amount of virus in tissues of vaccinated fish that survived the challenge. In conclusion, our results demonstrated a significant increase in fish immune responses and resistance to an IHNV infection after the oral administration of increasing concentrations of a DNA vaccine against IHNV encapsulated into alginate microspheres.

Dextrans produced by lactic acid bacteria exhibit antiviral and immunomodulatory activity against salmonid viruses

Viral infections in the aquaculture of salmonids can lead to high mortality and substantial economic losses. Thus, there is industrial interest in new molecules active against these viruses. Here we describe the production, purification, and the physicochemical and structural characterization of high molecular weight dextrans synthesized by Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10. The purified dextrans, and commercial dextrans with molecular weights ranging from 10 to 2000kDa, were assayed in infected BF-2 and EPC fish cell-line monolayers for antiviral activity. Only T2000 and dextrans from MN1 and RTF10 had significant antiviral activity. This was similar to results obtained against infectious pancreatic necrosis virus. However the dextran from MN1 showed ten-fold higher activity against hematopoietic necrosis virus than T2000. In vivo assays using the MN1 polymer confirmed the in vitro results and revealed immunomodulatory activity. These results together with the high levels of dextran production (2gL(-1)) by Lb. sakei MN1, indicate the compounds potential utility as an antiviral agent in aquaculture.

Trout oral VP2 DNA vaccination mimics transcriptional responses occurring after infection with infectious pancreatic necrosis virus (IPNV)

Time-course and organ transcriptional response profiles in rainbow trout Oncorhynchus mykiss were studied after oral DNA-vaccination with the VP2 gene of the infectious pancreatic necrosis virus (IPNV) encapsulated in alginates. The profiles were also compared with those obtained after infection with IPNV. A group of immune-related genes (stat1, ifn1, ifng, mx1, mx3, il8, il10, il11, il12b, tnf2, mhc1uda, igm and igt) previously selected from microarray analysis of successful oral vaccination of rainbow trout, were used for the RTqPCR analysis. The results showed that oral VP2-vaccination qualitatively mimicked both the time-course and organ (head kidney, spleen, intestine, pyloric ceca, and thymus) transcriptional profiles obtained after IPNV-infection. Highest transcriptional differential expression levels after oral vaccination were obtained in thymus, suggesting those might be important for subsequent protection against IPNV challenges. However, transcriptional differential expression levels of most of the genes mentioned above were lower in VP2-vaccinated than in IPNV-infected trout, except for ifn1 which were similar. Together all the results suggest that the oral-alginate VP2-vaccination procedure immunizes trout against IPNV in a similar way as IPNV-infection does while there is still room for additional improvements in the oral vaccination procedure. Some of the genes described here could be used as markers to further optimize the oral immunization method.