The use of Toll-like receptor ligands as adjuvants in fish vaccines

• Published in: Developmental & Comparative Immunology
• Main Author: Arnemo, Marianne
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: 2016
• Subjects: Atlantic salmon
• Online Access: http://hdl.handle.net/10852/49842; http://urn.nb.no/URN:NBN:no-53560

Non-living antigens are often poorly immunogenic and require addition of adjuvants to elicit protective immunity. Due to the immunostimulatory potential of Toll-like receptor (TLR) ligands, they are explored as vaccine adjuvants. The development of efficient and cheap vaccines against aquatic viruses is important for a sustainable aquaculture industry and the adjuvants for fish vaccines need to be improved. However, increased knowledge of fish TLR function is required before their ligands can find their way into fish vaccines. The major aim if this thesis has been to contribute to a more detailed understanding of fish TLRs. First, the tissue distribution of all known Atlantic salmon TLRs, the immunostimulatory potential of a panel of TLR ligands in primary head kidney leucocytes, and the impact of viral infection on TLR expression in head kidney were investigated. Head kidney and spleen were the main TLR expressing organs in Atlantic salmon. Several TLR ligands induced expression of inflammatory cytokines in salmon head kidney leucocytes. TLR3, TLR7, and TLR8a1 were induced in vivo after viral infection. In order to functionally validate ligand-specific activation of fish TLRs, we established an in vitro reporter assay in a salmon cell line. However, classical TLR2 ligands failed to activate rainbow trout TLR2 signalling when using NF-?B activation as measure of activation. To test the in vivo immunostimulatory potential of a TLR ligand alone and in vaccine formulations, a cold-water zebrafish challenge model was used. The TLR3 ligand poly I:C induced expression of antiviral transcripts in zebrafish head kidney and pre-treatment with poly I:C delayed VHSV (viral haemorrhagic septicaemia virus)-induced mortality. Chitosan encapsulated poly I:C was demonstrated to provide protection against VHSV when co-injected with two different non-living antigens (inactivated whole VHSV and VHSV glycoprotein G). Due to decreasing levels of the dietary n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in Atlantic salmon feed, we investigated how minimal levels of these fatty acids affect TLR signalling in Atlantic salmon leucocytes. The ability of leucocytes to respond to TLR ligand stimuli was reduced with low dietary- and head kidney levels of EPA and DHA, indicating the importance of n- 3 fatty acids in resistance to infection and response to vaccines. Our results provide new knowledge in the fish TLR field and lend support to poly I:C as a promising adjuvant candidate in viral vaccines.