The road to successful DNA vaccination of Atlantic salmon against virus diseases

Viral diseases cause large losses in salmon farming and are a continuous threat to the industry. Our research group studies DNA vaccines, which is a promising alternative to the traditional vaccines based on inactivated virus. A DNA vaccine is a plasmid, which contains the gene for the surface protein of the virus. DNA vaccine is known to provide high protection against the rhabdovirus IHNV in salmon. On the other hand, DNA vaccine against infectious salmon anemia virus (ISAV) based on a plasmid expressing the surface protein hemagglutinin esterase (HE), provides low protection. However, we have recently shown that vaccination of salmon with HE plasmid together with a plasmid containing a gene for type I interferon (IFN), provides strong protection and antibody production against ISAV (Chang et al.,Vaccine 2015, 33:2442-8). We have also shown that a DNA vaccine against salmonid alphavirus (SAV) causing pancreatic disease (PD) provides far better protection and antibody production against SAV than the vaccine that has been used for PD over the last 10 years (Chang et al., J Fish Dis. 2017, 40:1775-81). The DNA vaccine against SAV is based on a plasmid that expresses the entire structural polyprotein of SAV and had a good effect without the need to add IFN plasmid. In this project we have analyzed gene transcriptions in muscle at the injection site to understand why IFN enhances the protective effect of the DNA vaccine against ISAV and how the DNA vaccines against ISAV and SAV initiate the adaptive immune response. Gene transcripts were measured with quantitative reverse transcription PCR (RT-qPCR) and microarray analyzes. To understand the adjuvant effect of IFN in the DNA vaccine against ISAV, we analysed gene transcripts in muscle at weeks 1 and 2 after injection of IFN plasmid, HE plasmid, control plasmid and PBS, respectively. The results showed that the IFN plasmid as expected increased transcripts of typical IFN-induced genes (ISG), but also of certain chemokines as well as gene markers for B cells, T cells and antigen presenting cells (APC). This indicates that the IFN plasmid stimulates attraction of lymphocytes and APC through induction of chemokines. The control plasmid and the HE plasmid gave a similar pattern in gene expression as the IFN plasmid although they yielded lower up-regulation of genes than IFN plasmid. This suggests that plasmid DNA itself has adjuvant activity. However, the HE plasmid gave significantly lower up-regulation of immune genes than the control plasmid, suggesting that HE has an inhibitory effect on ISGs. Taken together, this study therefore suggests that IFN plasmid acts as an adjuvant in the DNA vaccine against ISAV by counteracting inhibitory effect of HE on plasmid-induced immune genes. We then analyzed gene transcripts in salmon muscle after vaccination with SAV-plasmid, control plasmid or PBS, respectively. The results showed that the SAV-plasmid and the control plasmid had similar capabilities to up-regulate ISGs. This shows that the structural polyprotein of SAV does not inhibit plasmid mediated upregulation of ISGs, which explains why the SAV plasmid does not require co-injection of IFN-plasmid to provide protection. The SAV plasmid also provided stronger up-regulation of IFN-gamma and IFN-gamma induced genes than the control plasmid. Compared to the control plasmid, the SAV plasmid also gave larger increase in transcripts of marker genes for B cells, T cells and APC, suggesting that the SAV plasmid provides increased attraction of cells important in initiating the immune response to SAV. The attraction of lymphocytes and APC to the injection site can be explained by the fact that SAV plasmid increased upregulation of the chemokine CXCL10 and the proinflammatory cytokines IL-1beta and TNFalpha in the muscle. We also wanted to investigate whether DNA vaccines against proteins in general could give antibody response in salmon and if cellular localization of the protein has something to say for the effect. For this purpose, we chose DNA vaccine against ovalbumin (OVA) as a model. Plasmid constructs were produced which gave expression of OVA intracellularly, on the cell surface and which gave secretion of OVA from the cells. Groups of salmon smolt were injected intramuscularly with these plasmids while fish in another group were injected intraperitoneally with OVA in oil adjuvant. Ten weeks later, blood was harvested from the groups and the IgM antibody response to OVA was measured with ELISA. The results showed that OVA in oil adjuvant gave a strong antibody response while none of the OVA plasmids gave antibody response. Based on this, we concluded that DNA vaccines against a soluble protein do not appear to give antibody response to salmon. In summary, only DNA vaccines against surface proteins of enveloped viruses have so far been shown to give a strong specific adaptive immune response in Atlantic salmon.

Farmed Atlantic salmon is attacked by several pathogenic viruses, which represent a continuous threat for the aquaculture industry. Vaccines based on inactivated virus or recombinant virus protein are available, but do not give satisfactory protection. Moreover, the viruses that cause heart and skeletal muscle inflammation and cardiomyopathy syndrome cannot yet be grown in cell culture. This calls for new approaches to develop more effective virus vaccines for salmon. Our group is working on DNA vaccines because they are safe, cheap and gives a very high level of protection of salmonids against rhabdovirus infection. While DNA vaccines against other salmonid viruses have previously shown rather modest protection, our group has recently developed a novel method for obtaining a high level of protection with a DNA vaccine against infectious salmon anemia virus (ISAV) using interferon (IFN) as an adjuvant. IFN-plasmids provided a strong increase in protection and antibody production against ISAV when injected together with a plasmid containing the virus antigen hemagglutininesterase (HE) compared to injection of HE plasmid alone. Our recent work has shown that the properties of the virus antigen construct are highly important for the outcome of DNA vaccination since a DNA vaccine construct made against salmonid alphavirus (SAV) gave much better protection than a commercial vaccine based on whole inactivated virus while addition of an IFN-plasmid inhibited the protective immune response. In this project we are going to study the molecular signature of immune genes induced by the SAV DNA vaccine construct, study why IFN plasmid inhibits the immune response of the SAV construct and investigate how we can obtain adjuvant activity of IFN plasmid also with the SAV construct. We are also going to study how subcellular location of protein antigens influences the immune response to DNA vaccines.