Immune responses to oral pcDNA-VP2 vaccine in relation to infectious pancreatic necrosis virus carrier state in rainbow trout Oncorhynchus mykiss.

The VP2 gene of infectious pancreatic necrosis virus, encoded in an expression plasmid and encapsulated in alginate microspheres, was used for oral DNA vaccination of fish to better understand the carrier state and the action of the vaccine. The efficacy of the vaccine was evaluated by measuring the prevention of virus persistence in the vaccinated fish that survived after waterborne virus challenge. A real-time RT-qPCR analysis revealed lower levels of IPNV-VP4 transcripts in rainbow trout survivors among vaccinated and challenged fish compared with the control virus group at 45 days post-infection. The infective virus was recovered from asymptomatic virus control fish, but not from the vaccinated survivor fish, suggesting an active role of the vaccine in the control of IPNV infection. Moreover, the levels of IPNV and immune-related gene expression were quantified in fish showing clinical infection as well as in asymptomatic rainbow trout survivors. The vaccine mimicked the action of the virus, although stronger expression of immune-related genes, except for IFN-1 and IL12, was detected in survivors from the virus control (carrier) group than in those from the vaccinated group. The transcriptional levels of the examined genes also showed significant differences in the virus control fish at 10 and 45 days post-challenge.

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.

Food pellets as an effective delivery method for a DNA vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss, Walbaum).

A DNA vaccine based on the VP2 gene of infectious pancreatic necrosis virus (IPNV) was incorporated into feed to evaluate the effectiveness of this oral delivery method in rainbow trout. Lyophilized alginate-plasmid complexes were added to feed dissolved in water and the mixture was then lyophilized again. We compared rainbow trout that were fed for 3 consecutive days with vaccine pellets with fish that received the empty plasmid or a commercial pellet. VP2 gene expression could be detected in tissues of different organs in the rainbow trout that received the pcDNA-VP2 coated feed (kidney, spleen, gut and gill) throughout the 15 day time-course of the experiments. This pcDNA-VP2 vaccine clearly induced an innate and specific immune-response, significantly up-regulating IFN-1, IFN-γ, Mx-1, IL8, IL12, IgM and IgT expression. Strong protection, with relative survival rates of 78%-85.9% were recorded in the vaccinated trout, which produced detectable levels of anti-IPNV neutralizing antibodies during 90 days at least. Indeed, IPNV replication was significantly down-regulated in the vaccinated fish 45 days pi.

Modulation of genes related to the recruitment of immune cells in the digestive tract of trout experimentally infected with infectious pancreatic necrosis virus (IPNV) or orally vaccinated.

There are still many details of how intestinal immunity is regulated that remain unsolved in teleost. Although leukocytes are present all along the digestive tract, most immunological studies have focused on the posterior segments and the importance of each gut segment in terms of immunity has barely been addressed. In the current work, we have studied the regulation of several immune genes along five segments of the rainbow trout (Oncorhynchus mykiss) digestive tract, comparing the effects observed in response to an infectious pancreatic necrosis virus (IPNV) infection to those elicited by oral vaccination with a plasmid coding for viral VP2. We have focused on the regulation of several mucosal chemokines, chemokine receptors, the major histocompatibility complex II (MHC-II) and tumor necrosis factor α (TNF-α). Furthermore, the recruitment of IgM(+) cells and CD3(+) cells was evaluated along the different segments in response to IPNV by immunohistochemical techniques. Our results provide evidences that there is a differential regulation of these immune genes in response to both stimuli along the gut segments. Along with this chemokine and chemokine receptor induction, IPNV provoked a mobilization of IgM(+) and IgT(+) cells to the foregut and pyloric caeca region, and CD3(+) cells to the pyloric caeca and midgut/hindgut regions. Our results will contribute to a better understanding of how mucosal immunity is orchestrated in the different gut segments of teleost.

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.

The persistence of infectious pancreatic necrosis virus and its influence on the early immune response.

Persistent infection by IPNV was induced in RTG-2 and RTG-P1 cells in vitro and the influence of this phenomenon on viral infectivity, viral antigen expression and interference with homologous and heterologous viruses was characterized over successive passages. The induction of IFN was also assessed, as was the sequence of the VP2 viral capsid protein, the region believed to be responsible for virulence, attenuation or persistence. Viral antigen expression was recorded in cells with no evidence of cytopathic effects and in these conditions, flow cytometry was more sensitive than RT-PCR to demonstrate the presence of a non-lytic virus. Interference of homologous viral infection could be detected in cross-infection experiments and in RTG-P1 cells persistently infected with IPNV, the Mx1 promoter could still be activated for at least 5 successive passages. Indeed, although over-induction of luciferase was not observed by re-infection with homologous or heterologous viruses, a significant increase in luciferase was induced by poly I:C. IFN transcripts could be quantified by qRT-PCR in the persistent cells at several passages, suggesting that IFN plays a role in maintaining IPNV persistence. In addition, we observed the same determinants in the VP2 sequences from the persistent virus as those described previously for IPNV adaptation and persistence in cell culture.

In vitro and in vivo immune responses induced by a DNA vaccine encoding the VP2 gene of the infectious pancreatic necrosis virus.

The work presented here describes the construction of a plasmid encoding the VP2 gene of the infectious pancreatic necrosis virus (IPNV), its expression in BF-2 cells and an evaluation of its activity in brown trout (Salmo trutta L) soon after injection. Preliminary experiments to evaluate the potential of the plasmid to induce neutralizing antibodies were also performed. We established a BF-2 cell line that expresses VP2 constitutively and we examined the infection of these VP2-transfected BF-2 cells with homologous and heterologous viruses. The expression kinetics of IFN, and of the IFN-induced genes Mx and ISG15, was also evaluated in brown trout over a 15 day interval, and quantified by real-time or semi-quantitative PCR. Type I IFN and Mx are markers of the non-specific innate immune response to viruses and they are involved in antiviral defences. Our results demonstrate that expression of the IPNV VP2 protein in BF-2 cells induces an antiviral state against IPNV and against the infectious haematopoietic necrosis virus (IHNV). In BF-2 infected cells, VP2 inhibited both the IPNV and IHNV-induced cytopathic effect to some extent, as well as the virus yield. In vivo, VP2 was expressed in haematopoietic tissues such as the head kidney of 7 month-old trout. In addition, it induced early immune responses and specific immunity 30 days after injection. IFN mRNA expression increased sharply on the 1st and 15th day post-injection and expression of other IFN-induced genes as Mx and ISG15 was also detected soon after vaccination of brown trout. Moreover, specific antibodies were detected 30 days after vaccination. These results suggest that the VP2 gene is a good candidate for the design of IPNV-DNA vaccines and to investigate the use of cytokines as co-stimulatory molecules.