Trained Innate Immunity of Fish Is a Viable Approach in Larval Aquaculture

The general understanding has been that only adaptive immunity is capable of immunological memory, but this concept has been challenged in recent years by studies showing that innate immune systems can mount resistance to reinfection—as the innate immune system can adapt its function following an insult. Innate immune training offers an attractive approach in intensive fish larval rearing, especially since the adaptive immune system is not fully developed. Trained innate immunity will potentially favor robust fish in terms of resistance to viral and bacterial diseases. So-called immunostimulants such as ß-glucans have for decades been used both in laboratories and in intensive fish aquaculture. Treatment of fish by ß-glucans (and by other substances with pathogen-associated molecular patterns) often induces activation of non-specific/innate immune mechanisms and induces higher disease resistance. The reported effects of e.g., ß-glucans fit nicely into the concept “trained innate immunity,” but the research on fish does not yet include analysis of epigenetic changes that may be a prerequisite for long-lasting trained innate immunity. In this “perspective,” we will discuss how in practical terms and based on prior knowledge one can introduce innate immune training in brood stock fish, and their offspring, and whether innate immune training by ß-glucans is a viable approach in larval aquaculture

Strategies and hurdles using DNA vaccines to fish

International audienceAbstractDNA 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

List of primers and their designated applications.

<p>Note: Restriction endonuclease site are underlined.</p

Immunofluorescence

<p><b>study following </b><b><i>in vitro</i></b><b> modulation of Eomes in Atlantic salmon.</b> (<b>A</b>) siRNA knockdown of Eomes expression (shown in pink) in spleen lymphocytes after 48 h. (<b>B</b>) Myc-labelled Eomes ectopic expression was detected with fluorescence labelling (pink) using alexafluor 594. Nuclear staining was performed with DAPI (blue).</p

Regulators of Eomes expression in salmon lymphocytes.

<p>Spleen leukocytes were stimulated for 24 h, 48 h, and 72 h with ConA+PHA+huIL-2, IFN-α (0.5 µg/ml, and 5 ng/ml), and the mRNA levels of Eomes (A), and IFN-γ (B) were determined by real-time PCR. Gene expression is normalized against EF-1α and is shown relative to the mean of the non-stimulated cells. Each bar represents the mean ± SE of triplicate samples. Different letters denote statistically significant differences between the groups.</p

<i>In vivo</i> regulation of Eomes expression in Atlantic salmon post infection.

<p>(A) Tissue specific expression of granzyme A, IFN-γ, CD8α, and Eomes at different time-points after <i>A. salmonicida</i> challenge, and the overall correlation between them shown from top to bottom respectively, in the left panel. (B) Tissue specific expression of granzyme A, IFN-γ, CD8α, at different time-points after IPN virus challenge, and the overall correlation between them including Eomes shown from top to bottom respectively, in the right panel (Eomes expression not shown since there was no significant differences). Data were normalized to EF-1α expression at each time-points and presented as mean ± S.E.M (n = 6). Statistical differences (<i>P</i><0.05, <i>P</i><0.01, and <i>P</i><0.001) between different time-points compared to control are indicated by asterisk (*, **, and ***) respectively, above the bars.</p

Tissue distribution of Eomes expression in Atlantic salmon.

<p>Expression of salmon Eomes in different organs as detected by real-time PCR. Gene expression data were normalized to EF-1α expression using skin as a calibrator. Bar represents the mean ± S.E.M (n = 6). Asterisk (*) above the bar shows significant difference (<i>P</i><0.05) compared with the organ that showed the lowest expression (skin). The value above the bars shows average real-time CT values of six fish.</p

Atlantic salmon Eomes promoter sequence

<p><b>with consensus transcription factor binding sites.</b> The consensus transcription binding sites were predicted by MatInspector and TRANSFAC. Putative transcription factor binding sites are underlined, while (−) sign indicates the binding sites identified on the negative strand. TATA box and transcription start site (+1) are shaded.</p

Structural and functional analysis of the 5′-upstream region of the salmon Eomes gene.

<p>HeLa cells were transiently transfected with 0.3 µg of Eomes promoter/luciferase plasmid and 0.1 µg of Eomes promoter/SEAP plasmid (internal control). HeLa cells were stimulated with LPS and after 24 h luciferase activities were measured. Units of luciferase activity were normalized to activity of cotransfected pSEAP (relative luciferase activity). The error bars represent S.E.M values (n = 3). Asterisk (*) above the bars shows significant difference (<i>P</i><0.05) compared to respective control and the different letters (a and b) on the bars denote significant difference compared to the smallest construct. The bent arrow represents the salmon Eomes transcriptional start site.</p