Implicación de la respuesta inmunitaria de la gónada de peces durante una infección con nodavirus y desarrollo de vacunas | Immune response in fish gonad upon nodavirus infection and vaccines development

Gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) are the most important fish species for Mediterranean aquaculture and also for Spain. Among the problems to improve their culture are the high economic losses produced by pathologies. Thus, nodavirus (NNV), which actually is spreading worldwide, produces very high mortalities in European sea bass, a very susceptible species, and uses seabream as a resistant reservoir. In addition, this virus shows vertical transmission through gametes and gonadal fluids and no clearly effective preventive methods are available yet. Thus, in this Doctoral Thesis, we aimed to evaluate the interaction between European sea bass and gilthead seabream gonad and NNV (Part 1) and how to prevent its pathology and dissemination (Part 2).The Part 1 of this Doctoral Thesis contains four chapters (III.1.1 to III.1.4). In Chapter III.1.1., we studied whether NNV is able to colonize and replicate in the gonad of European sea bass and gilthead seabream males, and demonstrate whether the virus is able to replicate into the tissue. We firstly demonstrated that NNV infects and replicates into the testis of both species since we localized viral RNA in somatic cells of testis of both species and in germ cells of gilthead seabream by in situ PCR (isPCR) whether capsid protein (CP) and B2 peptide of the virus were only detected in gilthead seabream testis by immunocytochemistry. Furthermore, infective viral particles were rescued from the gonad of both species using cell culture techniques. Regarding reproductive functions, NNV infection altered sex hormones 17β-estradiol and 11-ketotestosterone synthesis and their serum levels and the sensitivity of brain and testis to these hormones, whereas there was no disruption of testicular functions according to the results related to gonadosomatic index (GSI), the gene expression level of dmrt1 and the morphology of the testis.In Chapters III.1.1 to III.1.4, we also determined how the immune responses are affected in gonad and brain, the main target tissue of the virus. Our results (Chapter III.1.1) showed a higher inflammatory response in gonad and brain of European sea bass, showing the up-regulation of all the pro-inflammatory genes analysed (tnfa, il6 and il1b), compared to gilthead seabream specimens, in which only il6 gene was slightly up-regulated in brain. These results are in concordance with the fact that European sea bass specimens reached mortality rates up to 55% after only 15 days of infection, whilst gilthead seabream was able to overcome the infection. Type I interferon (IFN) response (Chapter III.1.2) is one of the most powerful innate immune responses against virus. In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes but the viral RNA sensors and mediators leading to the IFN production are scarcely known. Thus, we have searched for the presence of these genes in both fish models used in this Doctoral Thesis and evaluated their expression upon NNV infection in brain and gonad, usually used by certain pathogens to be transmitted vertically. We succeeded in identifying the sequences coding for MDA5, TBK1, IRF3, IFN, Mx and PKR proteins in both species, and, in addition, for LGP2, MAVS, TRAF3, TANK and IRF7 in European sea bass. In order to characterize the IFN pathway, we studied their expression in two different cell lines, SAF-1 from gilthead seabream and DLB-1 from European sea bass brain, in which most of the genes were up-regulated after the in vitro treatment with NNV or polyinosinic-polycytidylic acid (pI:C). After the in vivo infection with NNV in gilthead seabream, most of studied genes were up-regulated in brain and unaltered in gonad, probably favouring the dissemination through gonad fluids or gametes. Strikingly, in European sea bass, all the genes analysed were up-regulated in the gonad but only mda5, lgp2, irf3, mx and pkr did in brain. In both species, the tbk1 gene was not stimulated by any treatment; neither in vivo nor in vitro, suggesting that it could not be relevant in this route. These findings support the idea that the innate immune response in European sea bass brain is unable to clear the virus and points to the importance of the gonad immunity to control the dissemination of NNV, an aspect that is worthy to investigate in aquatic animals.Antimicrobial peptides (AMPs) have a crucial role in fish innate immune response, being considered an essential component of the first line of defence against pathogens. Moreover, the importance of AMPs in fish gonad is based on the needed to develop an efficient immune response without damage the germ cells to avoid the used of the tissue by some pathogens as a vehicle or a reservoir to be transmitted to the progeny, as occurs with NNV, but no study has looked into the gonad of infected fish. Regarding this, we have characterized the antimicrobial response triggered by NNV in the testis of European sea bass and in the gilthead seabream, both in vivo and in vitro, and compared with that present in the serum and brain (Chapter III.1.3.). First, our data show a great antiviral response in the brain of gilthead seabream and in the gonad of European sea bass since mx gene expression was greatly up-regulated. In addition, for the first time, our results demonstrate that the antimicrobial activities (complement, lysozyme and total bactericidal) and the expression of several AMP genes (c3, lyz, hamp, dic, pis or bdef) in the gonad and brain of both species are very different, but generally activated in the European sea bass, probably related with the differences of susceptibility upon NNV infection. Furthermore, our in vitro results suggest that some AMPs (lyz, hamp and dic) are locally regulated playing a local immune response in the gonad, while others (c3) are more dependent of the systemic immune system.Histones (H1 to H4) are the primary proteins which mediate the folding of DNA into chromatin, however, and in addition to this function, histones have also been related to antimicrobial activity in vertebrates, in fact, mammalian H1 is mobilized as part as the anti-viral immune response. In fish, histones with AMP activity have been isolated and characterized mainly from skin and gonads. Taking all this into account, we studied the histones immune related role in European sea bass and gilthead seabream (Chapter III.1.4.). Firstly, we identified the complete H1 and H2B coding sequences in both fish species and studied their pattern of expression under naïve conditions and upon NNV in vivo infection. Our results showed the highest expression of h1 gene in peripheral blood of both species and in thymus of European sea bass whilst in the case of h2b gene expression, the tissues where the h2b expression reached highest levels were European sea bass thymus and gilthead seabream blood and gonads. Upon in vivo infection with NNV, we found that h1 gene expression was stimulated in the gonad or brain of European sea bass or gilthead seabream, respectively, but never in head-kidney. However, h2b gene expression was exclusively up-regulated in the head-kidney of both species. These genes seemed to be systemically regulated since they were not altered in the male gonad of both species after an in vitro infection with NNV. All these data obtained prompted us to study their role on the immune response of head-kidney leucocytes upon viral (NNV), bacterial (Vibrio anguillarum or Photobacterium damselae) or chemical stimulation. Although further studies are needed, our data suggest that H1 has a role in the immune response against NNV in brain, while H2B seems to be more important in head-kidney. Moreover, the potential role of histones as anti-viral agents is suggested and further characterization is in progress.The Part 2 of this Thesis contains two chapters (III.2.1 and III.2.2) and is focused on the development of preventive tools to improve the survival of European sea bass larval and juveniles upon NNV infection. NNV is extremely harmful during larval development of European sea bass specimens, and at these early stages fish are very difficult to be vaccinated due to their small size or their immature immune system. Among preventive tools to avoid fish viral infections, vaccines need to be designed and created to improve aquaculture sector production, being the DNA vaccines the most promising. Moreover, oral administration must be investigated for mass vaccination of early stages due to their no stress-associated problems. Taking into account the difficulty of injecting a vaccine to a very small immunocompetent juvenile fish, we have created an oral chitosan-encapsulated DNA vaccine specifically against NNV (ChP-CP-pNNV), and administered orally to early juveniles of European sea bass during two days (Chapter III.2.1). After 90 days of vaccination, we studied the total and the specific anti-NNV IgM levels in serum, and also the gene expression of different sequences coding for proteins involved in cell-mediated cytotoxicity (CMC: tcrb and cd8a), the antigen presentation (mhc1a and mhc2b), antibodies (igmh and igth) and IFN (ifn, mx and ifng) responses after 7, 30 and 90 post-vaccination in the posterior gut. Our study shows that the oral ChP-CP-pNNV vaccine failed to show circulating specific antibodies after 90 days of vaccination and differences on immunoglobulin (igmh or igth) gene expression levels in the posterior gut of vaccinated and non-vaccinated fish from 30 days after vaccination onwards. However, the vaccine up-regulated the expression of genes related to CMC (tcrb and cd8a) and IFN response (ifn, mx and ifng). In addition, three months after vaccination the resistance to an experimental NNV infection was significantly improved by retarding the onset of the fish death and the accumulative mortality, with a relative percent survival (RPS) of 45% in ChP-CP-pNNV vaccinated sea bass. In conclusion, we created a chitosan-encapsulated DNA vaccine against NNV that is partly protective to European sea bass juveniles and this protection seems to be mediated by CMC and IFN pathways, although further studies are needed to improve the anti-NNV vaccine and to understand their mechanisms of action.The continuous transport of eggs and larvae between fish farms worldwide favours the spread of viral infections, especially, in the case of NNV. Although the first larval stages lack a mature immune system, the exposition to environmental pathogens is not a threshold thanks to the maternal transfer of immune factors from broodfish to their progeny. Thus vaccination of broodstock seems to be a feasible method to enhance the survival of the progeny eliciting the transfer of passive immunity. As the studies performed until now have mainly focussed on maternal transfer of specific immune factors and the innate immune response is also essential to understand fish immunity, we have studied the role of innate immune effectors on the maternal transfer of immunity (Chapter III.2.2). In this work, we used the previously constructed and partially protective DNA vaccine against NNV (CP-pNN) to inject mature fluent females of European sea bass. As DNA vaccines elicit few specific antibodies in vaccinated specimens, we focussed on different antimicrobial activities (peroxidase, protease, anti-protease, lysozyme and total bactericidal) in the serum of vaccinated or sham-vaccinated females and in eggs or larval homogenates of their progeny, from eggs to 69 days post-fertilization. We have also analysed the pattern of expression of granulocytes and macrophages gene markers (mpx and csf1a, respectively) and several AMP genes (hamp, dic, c3, lyz and nk-lys). Our results showed that the vaccine elicits the maternal transference of some bactericidal activity effectors since eggs from vaccinated group showed increased activities at 0 days post-fertilization. Indeed, the maternal transfer described in our work seemed to be restricted to protein factors as transcript levels of mRNA of several leucocyte markers or AMPs were undetected in eggs. Interestingly, the immunisation of the broodstock females promoted a higher and earlier innate immune response in their progeny than in controls.In conclusion, our study demonstrate for the first time the localization of NNV into the testis cells in fish and how the immune-reproductive interactions are regulated by NNV, which is hiding in the testis and likely using it to spread to the progeny. Furthermore, we have designed a DNA vaccine that shows promising properties as preventive tools against NNV disease when administered to juveniles or broodstocks females.