Targeting the innate immune system to develop novel prophylactic strategies: lessons from amphioxus (B. lanceolatum) and zebrafish (D. rerio)

Student thesis: Doctoral thesis


Immunization through vaccination is one of the most effective strategies to control infectious diseases. However, effective vaccines and alternative prophylactic tools for many fish diseases are still lacking. More studies on basic and applied immunology are required to improve the prevention and control of diseases in aquaculture. In this context, the thesis presents both basic and applied research. The Toll-like receptors (TLRs) are important for raising innate immune defense and their ligands are used as vaccine adjuvants to improve the immune responses. We studied the TLR system in the amphioxus B. lanceolatum. We identified 28 new putative TLR genes which consist in both non-vertebrate- and vertebrate-like TLRs. We cloned one of these genes, Bl_TLRj. The phylogenetic analysis together with functional analysis showed that it clusters with TLR11 family and particularly with subfamily 13. Moreover, Bl_TLRj responded against viral stimuli and showed high sequence identity with fish TLR13 and TLR22. Second, we developed two different infection models in zebrafish and we tested two potential nanoparticle adjuvants, IBsTNFα and NLc. The IBsTNFα are a highly stable, non-toxic, and low-cost protein-based biomaterial formed with nano-structured trout tumor necrosis factor alpha cytokine. Via oral intubation of adult zebrafish, combining flow cytometry, histology, and confocal microscopy, we show that IBsTNFα are able to cross the intestinal mucosal epithelial barriers, pass through the lamina propria, and reach the muscle layer. The expression of innate immune-related genes was significantly up-regulated in zebrafish intestine. Finally, IBsTNFα could protect zebrafish against a Mycobacterium marinum lethal infection when i.p. injected. The second particle tested, NLc, was previously developed in our lab and is composed by nanoliposomes encapsulating LPS and Poly I:C. The NLc was tested in our M. marinum bacterial infection model and it could protect zebrafish against a lethal infection when i.p. injected. Next, we explored the infective possibilities of two fish pathogens, M. marinum and Aeromonas hydrophila, in zebrafish larvae by immersion. The mortality of zebrafish larvae immersed with M. marinum showed no significant differences but zebrafish larvae infected with A. hydrophila by immersion showed significant differences compared to controls in a dose-dependent manner. NLc and IBsTNFα localized in the pharynx and intestine of zebrafish larvae at 3 and 5 dpf, respectively. The expression of immune-related genes such as IL-1β and IRF1α was significantly up-regulated after 48 h treatment with NLc in 2 dpf larvae. The 5 dpf larvae immersion in IBsTNFα could not significantly alter immune-related gene expression and IBsTNFα could not protect zebrafish larvae against A. hydrophila lethal infection.
Date of Award22 Dec 2017
Original languageSpanish
SupervisorNerea Roher Armentia (Director)


  • Immunology
  • Toll-like receptor
  • Adjuvants

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