A transcriptomic and functional approach to investigation of antigen processing and adaptive immunity in Barramundi (Lates calcarifer)

Abstract

Due to a constantly growing world population, food security has become an increasingly important issue. Controlling diseases in aquaculture can significantly increase food production while minimising the need for new infrastructures. Vaccination has been a major success story in the global aquaculture industry, revolutionising disease control and almost eliminating antibiotic use in modern fish farms. However, there are some infections that remain difficult to control by vaccination including diseases caused by highly variable bacterial pathogens such as Streptococcus species. In Australia, for example, barramundi (Lates calcarifer, also known as Asian sea bass) are a significant aquaculture fish but the industry is hindered by the hypervariable bacterium Streptococcus iniae. Antigen presentation by the cellular immune system is essential for development of a protective adaptive immune response, therefore, understanding it is critical to improve vaccine efficacy. In mammals, antigen presentation is coordinated by dendritic cells (DCs), and the recent identification of dendritic-like cells in rainbow trout and zebrafish strongly suggests the existence of those cells in teleosts. However, some antigens such as lipopolysaccharides (LPS), a bacterial sugar often used to stimulate cells in mammalian systems, do not seem to trigger a strong primary inflammatory response in teleosts. Nevertheless, it has also been shown that vaccination with LPS in fish induced the production of specific antibodies, warranting further investigation into the kinetics of antibody production in teleosts. The main objective of my project was thus to gain a better understanding of the early adaptive immunity, such as pathogen recognition, and how it links to antibody production as well as the role of dendritic cells in teleosts, using both functional in vitro assays and new technologies such as Illumina sequencing and bioinformatics analysis.To do so, I used barramundi as an industry relevant study organism. Following the generation of an immune transcriptome for barramundi, pathogen recognition receptors (PRRs) were identified for a number of pathogen associated molecular patterns (PAMPs), including peptidoglycans and nucleic acids. However, the PRR mainly associated with LPS recognition, the Toll-Like Receptor (TLR)-4 was not found in the immune transcriptome, and neither were the molecules classically associated with LPS recognition through TLR-4. Potential LPS receptors were thus investigated further in barramundi. The C-type lectin receptor Mincle was identified as a partial LPS receptor in barramundi, as LPS stimulation did induce some cytokine transcription through the Mincle pathway (Chapter 2).Caspases and NOD-like receptor inflammasomes were also investigated but they did not seem to be conserved in barramundi, or were missing molecular domains crucial to pathogen binding, suggesting that they were not participating in LPS recognition in barramundi. Beta2-integrin, on the other hand, were well conserved in barramundi compared with mammalian and other teleosts. Moreover, barramundi cells adhered to substrates specific to those β2-integrins and were bound by specific anti-integrin antibodies, suggesting that the αMβ2 and αLβ2 integrin molecules identified in the transcriptome were folding correctly and displayed binding sites similar to those of their mammalian counterpart (Chapter 3).Cells that were morphologically and functionally similar to DCs were identified from barramundi primary cell cultures. Those cells were shown to migrate out of the spleen and head kidney of barramundi after injection of bacterial components in the peritoneum, before returning to the spleen, where they probably induce the adaptive immune response. Interestingly though, those cells did not seem to react strongly to LPS alone (Chapter 4).Finally, immunoglobulin was investigated further due to its role in pathogen clearance, and the genomic region coding for variable, junction and constant immunoglobulin genes was identified and annotated in barramundi. A vaccine trial with subsequent challenge was also performed to assess the kinetics and amplitude immunoglobulin production in barramundi, and different adjuvants were used to assess their potential role on the quality and quantity of immunoglobulin produced. Overall, all adjuvants increased the number of antibodies produced, and one adjuvant was identified to significantly improve the secondary production of antibodies (Chapter 5).Globally, the present work investigated pathogen recognition, in particular LPS recognition, in barramundi as well as the onset of immune memory with a focus on the role of DCs and the mechanisms underlying immunoglobulin production. The results highlighted in this thesis suggest that barramundi, and likely other teleost species, use a combination of non-classical molecular pathways to process LPS. Moreover, it was demonstrated that the quantity and quality of antibodies could be improved with the use of adjuvants in barramundi, providing invaluable information to improve vaccines for warm water species aquaculture in the future