Time-course study of the protection induced by an interferon-inducible DNA vaccine against viral haemorrhagic septicaemia in rainbow trout.

The highly effective DNA vaccines against diseases caused by fish rhabdoviruses in farmed fish consist of a DNA plasmid vector encoding the viral glycoprotein under the control of a constitutive cytomegalovirus promoter (CMV). Among others, attempts to improve efficacy and safety of these DNA vaccines have focused on regulatory elements of plasmid vectors, which play a major role in controlling expression levels of vaccine antigens. Depending on the context, use of a fish-derived promoter with minimal activity in mammalian cells could be preferable. Another aspect related to the CMV promoter is that constitutive expression of the vaccine antigen may lead to rapid elimination of antigen expressing cells in the fish and thereby potentially reduce the long-term effects of the vaccine. In this study, we compared DNA vaccines with the interferon-inducible Mx promoter from rainbow trout and the CMV promoter, respectively. Plasmid constructs encoding the enhanced green fluorescent protein (EGFP) were used for the in vitro analysis, whereas DNA vaccines encoding the glycoprotein (G) of the viral haemorrhagic septicaemia virus (VHSV) were applied for the in vivo examination. The in vitro analysis showed that while the DNA vaccine with the CMV promoter constitutively drove the expression of EGFP in both fish and human cell lines, the DNA vaccine with the Mx promoter inducibly enhanced the expression of EGFP in the fish cell line. To address the impact on protection, a time-course model was followed as suggested by Kurath et al. (2006), where vaccinated fish were challenged with VHSV at 2, 8 and 78 weeks post-vaccination (wpv). The DNA vaccine with the CMV promoter protected at all times, while vaccination with the DNA vaccine containing the Mx promoter only protected the fish at 8 wpv. However, following induction with Poly (I:C) one week before the challenge, high protection was also evident at 2 wpv. In conclusion, the results revealed a more fish host dependent activity of the trout Mx promoter compared to the traditionally used cross species-active CMV promoter, but improvements will be needed for its application in DNA vaccines to ensure long term protection.

Sublethal concentrations of ichthyotoxic alga Prymnesium parvum affect rainbow trout susceptibility to viral haemorrhagic septicaemia virus.

Ichthyotoxic algal blooms are normally considered a threat to maricultured fish only when blooms reach lethal cell concentrations. The degree to which sublethal algal concentrations challenge the health of the fish during blooms is practically unknown. In this study, we analysed whether sublethal concentrations of the ichthyotoxic alga Prymnesium parvum affect the susceptibility of rainbow trout Oncorhynchus mykiss to viral haemorrhagic septicaemia virus (VHSV). During exposure to sublethal algal concentrations, the fish increased production of mucus on their gills. When fish were exposed to the algae for 12 h prior to the addition of virus, a marginal decrease in the susceptibility to VHSV was observed compared to fish exposed to VHSV without algae. If virus and algae were added simultaneously, inclusion of the algae increased mortality by 50% compared to fish exposed to virus only, depending on the experimental setup. We concluded that depending on the local exposure conditions, sublethal concentrations of P. parvum could affect susceptibility of fish to infectious agents such as VHSV.

Ichthyotoxicity of the microalga Pseudochattonella farcimen under laboratory and field conditions in Danish waters.

Blooms of the marine dictyochophyte Pseudochattonella farcimen have been associated with fish kills, but attempts to verify ichthyotoxicity of this microalga under experimental conditions have not been successful. In the early spring of 2009 and 2011, P. farcimen bloomed in the inner Danish waters. The blooms occurred at a seawater temperature of ~2°C and correlated with extensive kills of farmed salmonid fish (2009) and wild populations (2011). Several strains of P. farcimen were isolated from the 2009 bloom. However, exposure of rainbow trout Oncorhynchus mykiss to laboratory-grown P. farcimen cultures did not reveal any toxic effects. During the 2011 bloom, fish were exposed to bloom water under both laboratory and field conditions. While no clinical effect was observed on fish incubated in bloom water in the laboratory trial, a remarkable difference was seen in the field trial between rainbow trout kept in tanks supplied with a continuous flow of filtered versus non-filtered bloom water. Histological examination of the gill tissue revealed karyorrhexis and epithelial loosening in the affected fish. Microscopy analysis of algal cell morphology suggested that mucocysts detected on the cell surface only in freshly sampled bloom water might be associated with ichtyotoxicity.

DNA vaccination against a fish rhabdovirus promotes an early chemokine-related recruitment of B cells to the muscle.

In fish, intramuscular (i.m) injection of plasmid DNA encoding viral proteins has proved a highly effective vaccination strategy against some viral pathogens. The efficacy of DNA vaccination in teleost fish is based on the high level of viral antigen expression in muscle cells inducing a strong and long-lasting protection. However, the mechanisms through which this protection is established and effectuated in fish are still not fully understood. Moreover, similarities to mammalian models cannot be established since DNA vaccination in mammals usually induces much weaker responses. In this work, we have focused on the characterization of the immune cells that infiltrate the muscle at the site of DNA injection in vaccinated fish and the chemokines and chemokine receptors that may be involved in their infiltration. We have demonstrated through diverse techniques that B lymphocytes, both IgM⁺ and IgT⁺ cells, represented a major infiltrating cell type in fish vaccinated with a viral haemorrhagic septicaemia virus (VHSV) glycoprotein-encoding DNA vaccine, whereas in control fish injected with an oil adjuvant mainly granulocyte/monocyte-type cells were attracted. Among twelve chemokine genes studied, only CXCL11_L1, CK5B and CK6 mRNA levels were up-regulated in DNA vaccinated fish compared to fish injected with the corresponding vector backbone. Furthermore, the transcription of CXCR3B, a possible receptor for CXCL11_L1 was also significantly up-regulated in vaccinated fish. Finally, experiments performed with recombinant trout CK5B and CK6 and chemokine expression plasmids revealed that these chemokines have chemotactic capacities which might explain the recruitment of B cells to the site of DNA injection. Altogether, our results reveal that there is an early chemokine-related B cell recruitment triggered by i.m. DNA vaccination against VHSV which might play an important role in the initial phase of the immune response.

Dual DNA vaccination of rainbow trout (Oncorhynchus mykiss) against two different rhabdoviruses, VHSV and IHNV, induces specific divalent protection.

DNA vaccines encoding the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV are very efficient in eliciting protective immune responses against their respective diseases in rainbow trout (Oncorhynchus mykiss). The early anti-viral response (EAVR) provides protection by 4 days post vaccination and is non-specific and transient while the specific anti-viral response (SAVR) is long lasting and highly specific. Since both VHSV and IHNV are endemic in rainbow trout in several geographical regions of Europe and Atlantic salmon (Salmo salar) on the Pacific coast of North America, co-vaccination against the two diseases would be a preferable option. In the present study we demonstrated that a single injection of mixed DNA vaccines induced long-lasting protection against both individual and a simultaneous virus challenge 80 days post vaccination. Transfected muscle cells at the injection site expressed both G proteins. This study confirms the applied potential of using a combined DNA vaccination for protection of fish against two different rhabdoviral diseases.

Time course study of in situ expression of antigens following DNA-vaccination against VHS in rainbow trout (Oncorhynchus mykiss Walbaum) fry.

The present study was performed as a time course study of fish vaccinated with 20 microg plasmid DNA vaccine encoding either the VHSV G-protein or the VHSV N-protein. Samples of the injection site were collected sequentially over a 7-week period. The study revealed an intense positive staining by immunohistochemistry for the viral G-protein mainly in the membrane of intact myocytes, most prominent by days 10-27, and with concomitant infiltration of inflammatory cells by days 13-38 that subsequently lead to a marked reduction in the number of myocytes expressing the G-protein. By immunofluorescence, infiltrating cells positive for MHC II, IgM, and C3 were demonstrated. By contrast, in fish vaccinated with the VHSV-N construct, fewer, diffusely positive myocytes were found, most prominent by days 13-38, these having a positive reaction for the N-protein mainly in the cytoplasm and variably in the membrane. N-protein positive myocytes did not attract infiltrating cells to the same degree. Positive reaction for the N-protein almost ceased by day 48 post-vaccination.

A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot.

A DNA vaccine encoding the envelope glycoprotein from a fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), has previously been shown to induce both early and long time protection against the virus in rainbow trout. Challenge experiments have revealed that the immunity established shortly after vaccination is cross-protective against heterologous fish rhabdoviruses. In this study, we show that the DNA vaccine encoding the VHSV glycoprotein also induces early protection against a non-enveloped, positive-sense RNA virus belonging to the Nodavirus family, the Atlantic halibut nodavirus (AHNV). In a vaccine efficacy test using juvenile turbot as model fish, the fish injected with the VHSV vaccine were completely protected against a nodavirus challenge performed 8 days post vaccination, while the cumulative mortality in the control group reached 54%. A DNA vaccine carrying the gene encoding the capsid protein of AHNV revealed no protective properties against the nodavirus challenge. Histological examination of muscle tissue sections from the vaccine injection site showed that the DNA vaccine against VHSV triggered a pronounced inflammatory response in turbot similar to what has earlier been observed in rainbow trout.

Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens.

It was recently reported that DNA vaccination of rainbow trout fingerlings against viral hemorrhagic septicaemia virus (VHSV) induced protection within 8 days after intramuscular injection of plasmid DNA. In order to analyse the specificity of this early immunity, fish were vaccinated with plasmid DNA encoding the VHSV or the infectious haematopoietic necrosis virus (IHNV) glycoprotein genes and later challenged with homologous or heterologous pathogens. Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the two viral pathogens whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high. The early immunity induced by the DNA vaccines thus appeared to involve short-lived non-specific anti-viral defence mechanisms.