Immune responses elicited in rainbow trout through the administration of infectious pancreatic necrosis virus-like particles

7 páginas, 5 figuras, 2 tablas -- PAGS nros. 378-384Virus like particles (VLPs) against viral pathogens not only constitute a novel approach for the development of antiviral vaccines for an specific virus, but also for the creation of multivalent vaccines in which antigens from other pathogens may be expressed on the surface of these VLPs. Despite positive results on protection for many of these VLPs in both fish and mammals, not many studies have focused on the immune response triggered by these particles; studies that may provide hints for the identification of immune mechanisms responsible for antiviral protection, which are mostly unknown in fish. In the current work, we have studied the levels of transcription of several immune genes in the spleen of rainbow trout (Oncorhynchus mykiss) intraperitoneally injected with VLPs from infectious pancreatic necrosis virus (IPNV) focusing on the chemokine response as well as the response of genes related to interferon (IFN) production. Surprisingly, the capacity of VLPs to induce chemokines differed from that of live IPNV, suggesting a direct effect of viral replication on the chemokine response in this organ. While VLPs up-regulated the transcription of CK3, CK10 and CXCd and down-modulated CK5B, CK6 and CK9 transcription, a previous study in which the transcription of γIP, CXCd, CK1, CK3, CK5B, CK6, CK7A, CK9 and CK12 had been studied demonstrated that IPNV only significantly up-regulated CK6 and down-modulated CK3 in the spleen. On the other hand, the administration of VLPs produced a strong mobilization to the peritoneum of CD4+, IgM+, IgT+ and CD83+ leukocytes similar to that induced by the live viral infection. In both cases, this leukocyte recruitment seemed to be greatly mediated through CK3, CK5B, CK9 and CK10 chemokine production. These results together with the fact that VLPs strongly induced non-specific lymphocyte proliferation and specific anti-IPNV antibody production point to VLPs as excellent candidates for vaccine developmentThis work was supported by projects AGL2008-03519-C04-02 and AGL2010-18454 of the Spanish Ministry of Science and Innovation (Plan Nacional)Peer reviewe

Evaluación del nivel de infectividad de cepas de virus de la necrosis pancreatica infecciosa (IPNV) y el nivel de inducción de la proteina Mx3 como mecanismo antiviral

Trabajo presentado en el XX Congreso Sociedad Mexicana de Patólogos Veterinarios, celebrado en México, en 201

Inmunohistochemical detection of trout IgM⁺ cells and IgT⁺ cells in different segments of the digestive tract of naïve fish.

<p>Detection of IgM⁺ cells in the foregut (A), pyloric caeca (C) and midgut (E) segments revealed that IgM⁺ cells are usually located in the LP, with some IEL IgM⁺ in the pyloric caeca. Arrows show examples of LP lymphocytes (LPL) and IELs. Detection of IgT⁺ cells in the foregut (B), pyloric caeca (D) and midgut (F) segments showed that most IgT⁺ cells where located between enterocytes, as IELs. The figures show a magnification image and an insert figure of the general location in which the detail was observed. Bar: 100 µm.</p

Correlation of VP2 transcription with transcription of immune genes.

<p>Pearson correlation between the levels of transcription of the VP2 gene and the transcription levels of the different immune genes studied in the different segments of the intestinal tract. N = 6.</p>*<p>p<0.05;</p>**<p>p<0.01.</p

Secreted IgM and membrane IgM modulation in response to oral DNA vaccination.

<p>Trout were orally vaccinated and sampled as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066118#pone-0066118-g004" target="_blank">Figure 4</a> and levels of secreted IgM (A) and membrane IgM (B) transcription in the different segments were studied through real time PCR. Data are shown as the mean relative gene expression normalized to the transcription of the house-keeping gene EF-1α ± SD (n = 6). The relative significance of differences between fish vaccinated with either the empty plasmid or empty microspheres and vaccinated fish at each segment of the digestive tract was determined through a one-way ANOVA and is shown above the bars as *.</p

IgM, IgT and IgD modulation in response to oral DNA vaccination.

<p>Trout were orally vaccinated with 10 µl of suspension of the vaccine microspheres each containing either 10 µg of pDNA-VP2 or 10 µg of the pDNA empty plasmid diluted in 10 µl of PBS. Finally, a third group received the same volume of an empty microsphere suspension. At day 10 post-vaccination, trout were sacrificed and the different segments of the digestive tract removed for RNA extraction and analysis of immune gene transcription through real time PCR. Levels of IgM (A), IgT (B) and IgD (C) transcription in the different segments were studied through real time PCR. Data are shown as the mean relative gene expression normalized to the transcription of the house-keeping gene EF−1α ± SD (n = 6). The relative significance of differences between fish vaccinated with either the empty plasmid or empty microspheres and vaccinated fish at each segment of the digestive tract was determined through a one-way ANOVA and is shown above the bars as *.</p

Gut segments used in this study.

<p>Schematic model illustrating the different segments in the trout digestive tract used in this study.</p