Natalia A. Ballesteros

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.

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.

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.

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.

Establishment and characterization of a new cell line (SSP-9) derived from Atlantic salmon Salmo salar that expresses type I if n

In the present work, the establishment and biological characterization of a new cell line, SSP-9, derived from the pronephros of the Atlantic salmon Salmo salar, are reported. These cells grew well in Leibovitzs (L15) medium supplemented with 10% foetal calf serum at temperatures from 15 to 25° C, and they have been sub-cultured over 100 passages to produce a continuous cell line with an epithelial-like morphology. The SSP-9 cells attached and spread efficiently at different plating densities, retaining 80% of cell viability after storage in liquid nitrogen. When karyotyped, the cells had 40-52 chromosomes, with a modal number of 48. Viral susceptibility tests showed that SSP-9 cells were susceptible to infectious pancreatic necrosis virus and infectious haematopoietic necrosis virus, producing infectious virus and regular cytopathic effects. Moreover, these cells could be stimulated by poly I:C, showing significant up-regulation in the expression of the genes that regulate immune responses, such as ifn and mx-1. SSP-9 cells constitutively express genes characteristic of macrophages, such as major histocompatibility complex (mhc-II) and interleukin 12b (il-12b), and flow cytometry assays confirmed that SSP-9 cells can be permanently transfected with plasmids expressing a reporter gene. Accordingly, this new cell line is apparently suitable for transgenic manipulation, and to study host cell-virus interactions and immune processes.

Gene2Path: A Data Analysis Tool to Study Fish Gene Pathways by Automatic Search of Orthologous Genes

Most of the gene regulation pathways data from biochemical and molecular experiments are drawn from humans or from species commonly used as experimental animal models. Accordingly, the software packages to analyse these data on the basis of specific gene identification codes (IDs) or accession numbers (AN) are not easy to apply to other organisms that are less characterized at the genomic level. Here, we have developed the Gene2Path programme which automatically searches pathway databases to analyse microarray data in an independent, species-specific way. We have illustrated the method with data obtained from an immune targeted rainbow trout microarray to search for orthologous pathways defined for other well known biological species, such as zebrafish, although the software can be applied to any other case or species of interest. The scripts and programme are available and free at the “GENE2PATH” web site http://gene2path.no-ip.org/cgi-bin/gene2path/index.cgi. A user guide and examples are provided with the package. The Gene2Path software allows the automated searching of NCBI databases and the straightforward visualization of the data retrieved based on a graphic network environment.