Roy A. Dalmo

What happens to the DNA vaccine in fish? A review of current knowledge.

The primary function of DNA vaccines, a bacterial plasmid DNA containing a construct for a given protective antigen, is to establish specific and long-lasting protective immunity against diseases where conventional vaccines fail to induce protection. It is acknowledged that less effort has been made to study the fate, in terms of cellular uptake, persistence and degradation, of DNA vaccines after in vivo administration. However, during the last year some papers have given new insights into the fate of DNA vaccines in fish. By comparing the newly acquired information in fish with similar knowledge from studies in mammals, similarities with regard to transport, blood clearance, cellular uptake and degradation of DNA vaccines have been found. But the amount of DNA vaccine redistributed from the administration site after intramuscular administration seems to differ between fish and mammals. This review presents up-to-date and in-depth knowledge concerning the fate of DNA vaccines with emphasis on tissue distribution, cellular uptake and uptake mechanism(s) before finally describing the intracellular hurdles that DNA vaccines need to overcome in order to produce their gene product.

Adjuvants and immunostimulants in fish vaccines: Current knowledge and future perspectives.

Vaccination is the most adequate method to control infectious diseases that threaten the aquaculture industry worldwide. Unfortunately, vaccines are usually not able to confer protection on their own; especially those vaccines based on recombinant antigens or inactivated pathogens. Therefore, the use of adjuvants or immunostimulants is often necessary to increase the vaccine efficacy. Traditional adjuvants such as mineral oils are routinely used in different commercial bacterial vaccines available for fish; however, important side effects may occur with this type of adjuvants. A search for alternative molecules or certain combinations of them as adjuvants is desirable in order to increase animal welfare without reducing protection levels. Especially, combinations that may target specific cell responses and thus a specific pathogen, with no or minor side effects, should be explored. Despite this, the oil adjuvants currently used are quite friendlier with respect to side effects compared with the oil adjuvants previously used. The great lack of fish antiviral vaccines also evidences the importance of identifying optimal combinations of a vaccination strategy with the use of a targeting adjuvant, especially for the promising fish antiviral DNA vaccines. In this review, we summarise previous studies performed with both traditional adjuvants as well as the most promising new generation adjuvants such as ligands for Toll receptors or different cytokines, focussing mostly on their protective efficacies, and also on what is known concerning their effects on the fish immune system when delivered in vivo.

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.

Bath immunostimulation of rainbow trout (Oncorhynchus mykiss) fry induces enhancement of inflammatory cytokine transcripts, while repeated bath induce no changes

The application of immunostimulants may be a cost-effective practice in production of delicate and fragile fish fry since it may confer disease resistance. Bath administration to fish fry is considered an ideal delivery route for mass manipulation since there is no need for individual handling. In the current study, rainbow trout fry were bathed with three different immunostimulants: Plasmid DNA, lactoferrin and beta-glucan at two different doses (0.1 microM and 1.0 microM) for 45 min, four times with an interval of 1 week. Ten fish per treatment group were sampled, and RNA of pooled tissues consisting of eye, tongue, skin, gill, thymus, spleen, head kidney, liver, small and large intestine were isolated from the fish obtained at first, second and fourth bathing (24 and 72 h post-bathing). Before cDNA transcription, two parallel samples were pooled giving a total of 5 parallels in each treatment group. Results showed that plasmid DNA and lactoferrin as well as beta-glucan treated fish possessed higher gene expression with regard to the pro-inflammatory cytokines IL-1beta, TNF-alpha, IL-6 and the anti-inflammatory cytokines IL-10 and TGF-beta after the first bathing, especially 24 h post-bathing in the high-dose groups. This indicates that bath delivery of immunostimulants can induce pro-inflammatory responses. No significant changes of the pro-inflammatory cytokine IL-17A transcripts, compared to the respective controls, were observed. Gene expression levels in the immunostimulated fish after the fourth bathing did not show significant differences compared to controls.

Oral administration of lipopolysaccharide to Atlantic salmon (Salmo salar L.) fry. Uptake, distribution, influence on growth and immune stimulation

Abstract Atlantic salmon fry were fed Aeromonas salmonicida lipopolysaccharide (LPS)-coated feed for 62 days and then challenged with virulent A. salmonicida bacteria. The fry were fed LPS-coated feed also after challenge. Fry that were fed LPS feed (0.1% LPS) showed a higher mortality throughout the challenge period (accumulated mortality of 57% after 42 days) compared with fry fed control feed (accumulated mortality of 36.5% after 42 days). Fry receiving LPS-coated feed showed an increase in mean weight of 10.7% at day 62 compared with fry receiving control feed. The increase was, however, not statistically significant. Sixty days of feeding with LPS-coated feed did not result in measurable amounts of specific antibodies against A. salmonicida LPS in the homogenised fry. The total amount of immunoglobulins (Igs) was, however, slightly increased. Studies of immunohistochemical localisation and radioactive LPS distribution revealed high levels of LPS in the intestinal epithelial cells. Head kidney, liver and heart showed low levels of radioactivity and no immunohistochemical staining. In another set of experiments, Atlantic salmon fry were fed LPS-coated feed (0.03% and 0.01%, respectively) for 64 days and then challenged with A. salmonicida and Vibrio anguillarum . The accumulated mortality in fry challenged with A. salmonicida was 40.9%, 34.3% and 30.8% after feeding 0.03% and 0.01% LPS and control feed, respectively. The accumulated mortality in fry challenged with V. anguillarum was 47.0%, 55.0% and 55.1% after feeding 0.03% and 0.01% LPS and control feed, respectively. Fry receiving 0.03% and 0.01% LPS-coated feed showed a statistically significant increase in mean weight at day 64 compared with fry receiving control feed.

Strategies and hurdles using DNA vaccines to fish

DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen – and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish.

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.

Early immune responses in Atlantic salmon (Salmo salar L.) after immunization with PLGA nanoparticles loaded with a model antigen and β-glucan

Polymeric nanoparticles (NPs) of poly (lactic-co-glycolic) acid (PLGA) possess adjuvant properties. To date, there are few studies exploring their application as antigen carriers for vaccination of fish. This study presents a preclinical assessment of the early innate and adaptive immune responses in Atlantic salmon following immunization with PLGA NPs. A model antigen (TNP-LPH) and an immunostimulant (β-glucan) were entrapped in NPs of 300-400nm either alone or in combination. Both the antigen and the β-glucan were efficiently entrapped (>50%) in particles and an antigen release study indicated particle stability up to 50 days at 8°C. Spleen and head kidney were analyzed for pro-inflammatory markers (TNF-α, IL-1β, IL-8, C3a) and T cell cytokines, effector molecules and transcription factors (IFN-γ, T-bet, GATA-3, granzyme A, IL-10, Foxp3) at mRNA transcription levels 2, 4 and 8 days post i.p. immunization. NPs alone were able to moderately up-regulate pro-inflammatory immune responses. Addition of immunogenic cargo, either an antigen or β-glucan generally increased the gene expression of pro-inflammatory markers, while administering both resulted in the highest gene expression. These findings were also reflected by concurrently increased levels of IL-10. Comparing the treatment groups injected with antigen and β-glucan co-administered either in NPs or FCA demonstrated that the magnitude of the acute pro-inflammatory responses was equal between the treatments or highest in the NP injected group. Although elevated expression of granzyme A in the NP injected groups (carrying antigen and/or β-glucan) was observed, PLGA NPs were unable to induce T cell differentiation on mRNA gene expression levels, as increased levels of the indicating cytokines and transcriptions factors failed to occur. In conclusion, this study demonstrates that PLGA NPs have potential as an adjuvant in salmon vaccines as they enhance the early pro-inflammatory responses to immunization.

The spotted wolffish (Anarhichas minor Olafsen) complement component C3 : isolation, characterisation and tissue distribution

The complement component C3 was isolated from spotted wolffish (Anarhichas minor Olafsen) serum by polyethylene glycol precipitation, anion exchange chromatography and gel filtration. Silver staining in SDS-PAGE and rabbit anti-wolffish C3 antiserum used in Western blotting revealed that spotted wolffish C3 contains two polypeptide chains, M(r)65 and 115kDa, respectively. The high molecular weight alpha-chain of the C3 incorporated 14C-methylamine suggesting that it contained a reactive thioester group. The deduced amino acid sequence, after screening a liver cDNA expression library, showed that the wolffish C3 contained key amino acids for binding C3 convertase, factor H, I and properdin. Also, high degree of homology to other vertebrate C3 was found in the beta-alpha junction site. Phylogenetic tree analysis indicated that the Japanese flounder and spotted wolffish that belong to order pleuronectiformes and perciformes, respectively, are phylogenetically close species. Immunohistochemical experiments showed that liver hepatocytes and blood contained C3, and in situ hybridisation experiments revealed that liver hepatocytes expressed C3.