Ali, Zainab; (2019) Development of novel delivery technologies for vaccination through oral administration of farmed salmon. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In current times, the aquaculture industry has been faced with the dramatic on-set of various viral diseases specifically infectious pancreatic necrosis virus (IPNV) in Atlantic salmon (Salmo salar). In order for the industry to persevere through and successfully meet a consistently growing demand, substantial measures need to be taken. The process of vaccination is known to reduce incidence of disease in addition to reducing intensity of infection as well. Even though vaccination has gradually become an essential part of disease control, there are still some challenges that need to be faced head on. Maintenance of antigen viability, incorporation of antigen in an appropriate delivery system and process of antigen delivery require further understanding as limited research has been reported on the development of oral delivery systems as far as vaccination in aquaculture is concerned. This thesis explored three fundamental elements of oral drug delivery development; optimisation of microbead fabrication techniques, characterisation of microbeads through their unique mechanical and dissolution properties and finally the analysis of immune response following consumption of orally delivered antigen. The first objective of this thesis was to demonstrate the potential of electrospraying (ES) and aerodynamically assisted jetting (AAJ) as fabrication methodologies for advanced orally deliverable systems. Micron-ranged delivery systems have previously been utilized for the long-term delivery of active biological compounds. However conventional methodologies pose significant challenges, particularly poor sample repeatability, lack of homogeneity and poor control, among other drawbacks. This thesis reports ES and AAJ as possessing the ability to produce highly monodisperse, porous and reproducible polymeric microbeads at low cost. Operational maps obtained through an extensive sizing study established that ES possessed the ability to produce microbeads ranging from 250 – 2500 µm while AAJ produced beads ranging from 25 – 60 µm. The sizes of microbeads were seen to be primarily controlled through the fine tuning of process and solution parameters. Since these polymeric microbeads offer protection of the active pharmaceutical ingredients (API), their own stability is crucial in order to achieve optimal performance. Effects of polymer concentration, type of cross-linking agent and fabrication methodologies on the mechanical stability of microbeads were studied. Polymeric microbeads also offer the imperative feature of safeguarding an API through proteolytic enzymes and harsh pH encountered in the digestive tract of fish. This prospect was studied by looking into the release characteristics of microbeads, using a model API and fabricated microbeads using various types of alginate. Microbead release kinetics were addressed through a custom tailored in vitro dissolution test representative of the environmental conditions faced by A. salmon. To achieve the final objective of this thesis, A. salmon was exposed to a dose of viral antigen by using orally administered microbeads with encapsulated antigen. ELISA analysis indicated that at least some exposed fish had higher level of IgM antibodies targeting the specific IPNV antigen used for immunization. These results were also complemented by qPCR profiling in the head kidney, also including those encoding important immune transcription factors. Both treatments, with and without viral antigen showed an effect on a number of genes in head kidney. The most distinct expression profiles in the group of fish exposed to the viral antigen were seen at day 21 post exposure while at day 57 gene expression in both groups looked more similar. STAT6, IL17A, Tbet and TCRy were found to be regulated at time points selected in this study. The work presented in this thesis involves several stages in the process of development and optimization of the oral vaccine delivery system for an IPNV antigen in A. salmon. The system allows easy incorporation in fish feeds, however, it is not clear if the observed activation of the immune response will end up being protective against the disease. The hope is that the activation of the immune response by the studied antigen delivery system can be further optimized and tested in a challenge trial with live IPNV.

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