Stephen Mutoloki

Alpha Interferon and Not Gamma Interferon Inhibits Salmonid Alphavirus Subtype 3 Replication In Vitro

ABSTRACT Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-α)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-γ in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-α [rIFN-α] and rIFN-γ) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-α and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-α induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-α is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2α) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-α results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-γ has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-α, while postinfection antiviral responses or postinfection treatment with rIFN-α is not able to limit viral replication.

Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L)

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 107.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.

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

Two strains of IPNV made by reverse genetics on the Norwegian Sp strain NVI-015 (GenBank AY379740) backbone encoding the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs were used to prepare inactivated whole virus (IWV), nanoparticle vaccines with whole virus, Escherichia coli subunit encoding truncated VP2-TA and VP2-PT, VP2-TA and VP2-PT fusion antigens with putative translocating domains of Pseudomonas aeruginosa exotoxin, and plasmid DNA encoding segment A of the TA strain. Post challenge survival percentages (PCSP) showed that IWV vaccines conferred highest protection (PCSP=42-53) while nanoparticle, sub-unit recombinant and DNA vaccines fell short of the IWV vaccines in Atlantic salmon (Salmo salar L.) postsmolts challenged with the highly virulent Sp strain NVI-015 (TA strain) of IPNV after 560 degree days post vaccination. Antibody levels induced by these vaccines did not show antigenic differences between the virulent and avirulent motifs for vaccines made with the same antigen dose and delivery system after 8 weeks post vaccination. Our findings show that fish vaccinated with less potent vaccines comprising of nanoparticle, DNA and recombinant vaccines got infected much earlier and yielded to higher infection rates than fish vaccinated with IWV vaccines that were highly potent. Ability of the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs to limit establishment of infection showed equal protection for vaccines made of the same antigen dose and delivery systems. Prevention of tissue damage linked to viral infection was eminent in the more potent vaccines than the less protective ones. Hence, there still remains the challenge of developing highly efficacious vaccines with the ability to eliminate the post challenge carrier state in IPNV vaccinology.

High gene expression of inflammatory markers and IL-17A correlates with severity of injection site reactions of Atlantic salmon vaccinated with oil-adjuvanted vaccines

BackgroundTwo decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but the underlying profile of inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices.Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscosa antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response.ResultsGranulomatous lesions were observed in all vaccinated fish. The presence of severe granulomas was associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, TGF-β (p = 0.001), IL-17A (p = 0.007) and its receptor (IL-17AR) (p = 0.009) representing TH17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of TH1 T cell lineage (IFN-γ, CD4) or TH2 (GATA-3) responses were differentially expressed.ConclusionsGranulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon have the profile of strong expression of genes related to innate immune responses. The expression of TGF-β, IL-17A and its receptor suggests an involvement of TH17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating also a TH2-profile. To what extent the expression of IL-17A and its receptor reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon when vaccinated with oil-based vaccines.

Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon.

Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.

Superior protection conferred by inactivated whole virus vaccine over subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon (Salmo salar L.)

Salmonid alphavirus 3 (SAV-3) is an emerging pathogen in Norwegian salmon farming and causes severe annual losses. We studied the immunogenicity and protective ability of subunit and DNA vaccines based on E1 and E2 spike proteins of salmonid alphavirus subtype 3 (SAV-3), and compared these to an experimental inactivated, whole virus (IWV) vaccine in Atlantic salmon. The antigens were delivered as water-in-oil emulsions for the subunit and inactivated vaccines and non-formulated for the DNA vaccines. The IWV and the E2 subunit prime-boost groups had circulating neutralizing antibodies at challenge, correlating with high protection against lethal challenge and 3-log(10) reduction of virus titer in heart for the IWV group. Prime-boost with E1 subunit vaccine also conferred significant protection against mortality, but did not correlate with neutralizing antibody levels. Protection against pathology in internal organs was only seen for the IWV group. Prime-boost with E1 and E2 DNA vaccines showed marginal protection in terms of reduction of viral replication in target organs and protection against mortality was not different from controls. The IWV group showed significant upregulation of IFNγ and IL2 mRNA expression at 4 weeks post challenge possibly indicating that other mechanisms in addition to antibody responses play a role in mediating protection against infection. This is the first report comparing the immunogenicity and protection against mortality for IWV vaccines and spike protein subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon. The IWV vaccine has superior immunogenicity over sub-unit and DNA vaccines.

Oral vaccination of fish - antigen preparations, uptake, and immune induction

The oral route offers the most attractive approach of immunization of fish for a number of reasons: the ease of administration of antigens, it is less stressful than parenteral delivery and in principle, it is applicable to small and large sized fish; it also provides a procedure for oral boosting during grow-out periods in cages or ponds. There are, however, not many commercial vaccines available at the moment due to lack of efficacy and challenges associated with production of large quantities of antigens. These are required to stimulate an effective immune response locally and systemically, and need to be protected against degradation before they reach the sites where immune induction occurs. The hostile stomach environment is believed to be particularly important with regard to degradation of antigens in certain species. There is also a poor understanding about the requirements for proper immune induction following oral administration on one side, and the potential for induction of tolerance on the other. To what extent primary immunization via the oral route will elicit both local and systemic responses is not understood in detail. Furthermore, to what extent parenteral delivery will protect mucosal/gut surfaces and vice-versa is also not fully understood. We review the work that has been done on the subject and discuss it in light of recent advances that include mass production of antigens, including the use of plant systems. Different encapsulation techniques that have been developed in the quest to protect antigens against digestive degradation, as well as to target them for appropriate immune induction are also highlighted.

The kinetics of CD4+ and CD8+ T-cell gene expression correlate with protection in Atlantic salmon (Salmo salar L) vaccinated against infectious pancreatic necrosis

Infectious pancreatic necrosis virus (IPNV) is a highly contagious disease causing high mortalities in juvenile salmonids. Lack of correlation between neutralizing antibodies and infecting virus suggests a likelihood of involvement of the cellular mediated immune response in vaccine protection. To elucidate the kinetics of CD4 and CD8 T-cells responses in vaccine protection, Atlantic salmon (Salmo salar L) were vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine and challenged by cohabitation using a highly virulent Norwegian Sp strain. Analysis of T-cell gene expression in lymphoid organs (headkidney and spleen) showed that GATA-3 was positively correlated with increase in antibody levels when T-bet was low. Conversely, T-bet and FoxP3 were positively correlated with viral infection and negatively correlated with increase in antibody levels. Among the CD8+ T cell genes, expression of eomes and CD8α were positively correlated with increase in viral copy numbers and negatively correlated with increase in antibody levels. Up-regulation of granzyme A was highly correlated with increase in viral copy numbers in the LoAg and control groups indicating that this gene could save as a diagnostic marker of acute infection for IPNV during acute infection. In contrast, its down regulation in the HiAg which had low viral copy numbers corresponded with high antibody levels. Overall, these data show that the kinetics of CD4 and CD8 T-cell genes expression follow the same pattern as that observed in higher vertebrates. These findings suggest that functional signatures of the cellular mediated immune response could be evolutionary conserved across the vertebrate taxa and that they can effectively be used to monitor vaccine protection and infection progression of IPNV in Atlantic salmon.

Sequence similarities of the capsid gene of Chilean and European isolates of infectious pancreatic necrosis virus point towards a common origin

The Chilean salmonid industry was developed by importing breeding materials, a practice still in effect due to deficits in the national supply of roe. Importation of breeding materials is often associated with the transmission of pathogens. The objectives of this study were to compare the infectious pancreatic necrosis virus (IPNV) isolates from Chile to those of European origin and to determine the diversity of the Chilean IPNV. The VP2 genes of IPNV from Chilean fish (whose eggs originated from Scotland, Iceland and Norway) were compared to isolates from fish in Norway and Ireland. The results show that the isolates are identical (97-99%) and cluster into one genogroup. Our findings support previous reports of association between the trade-in breeding materials and transmission of pathogens. Furthermore, our results demonstrate the genotypic diversity of Chilean IPNV isolates. These findings have important implications for IPNV disease diagnosis and control in Chile.

Acquired immunity and vaccination against infectious pancreatic necrosis virus of salmon

Acquired immunity plays an important role in the protection of salmonids vaccinated against infectious pancreatic necrosis virus (IPNV) infections. In recent years, vaccine research has taken a functional approach to find the correlates of protective immunity against IPNV infections. Accumulating evidence suggests that the humoral response, specifically IgM is a correlate of vaccine protection against IPNV infections. The role of IgT on the other hand, especially at the sites of virus entry into the host is yet to be established. The kinetics of CD4+ and CD8+ T-cell gene expression have also been shown to correlate with protection in salmonids, suggesting that other arms of the adaptive immune response e.g. cytotoxic T cell responses and Th1 may also be important. Overall, the mechanisms of vaccine protection observed in salmonids are comparable to those seen in other vertebrates suggesting that the immunological basis of vaccine protection has been conserved across vertebrate taxa.