Rajakumar, PD; (2016) The chloroplast of Chlamydomonas reinhardtii as a platform for recombinant vaccine production. Doctoral thesis , UCL (University College London).

Text PriscillaDorothyRajakumar-Copyright Material Removed-Dec2016.pdf - Accepted Version Access restricted to UCL open access staff until 1 January 2025. Download (62MB)

Abstract

Infectious diseases represent an on-going problem adversely affecting the poultry industries and fish farming. Protein-base vaccines that are currently used for the prevention and spread of diseases have several drawbacks including high cost of recombinant production and delivery; and the needs to eliminate endotoxins when produced in bacterial platforms such E. coli. Therefore, there is an urgent need for effective, cheaper and safer vaccines. The microalga Chlamydomonas reinhardtii could be an ideal candidate for the production of recombinant protein such as vaccines. This is because: i) it has high growth rates; ii) it is generally regarded as safe (GRAS) to eat and therefore does not present a problem of endogenous toxin; iii) it has a genetically tractable chloroplast genome that allows a good expression of recombinant proteins that undergo correct folding to form an active product. Hence, in this research two C. reinhardtii transgenic chloroplast lines expressing the infectious bronchitis virus (IBV) muti-epitope genes (Spike Protein S540-564, Nulceocapsid Protein N67-83 and 120 residues of C terminus of the Nucleocapsid Protein) fused with the adjuvant, CTB and full length nervous necrosis virus (NNV) capsid gene were created. IBV causes respiratory disease in chickens. On the other hand, NNV causes cellular vacoulation and neuronal shape changes in fish. The IBV and NNV antigens were successfully detected anti-HA antibodies by western analysis. The lyophilised C. reinhardtii CTB-IBV that were fed to day 0 chicks, successfully triggered a mucosal and systemic immune response giving rise to antibodies against CTB, IBV and HA epitopes. The NNV fish vaccine trial is currently ongoing in Kasetart University, Thailand. Serum and mucus of NNV vaccinated fish will be analysed in the Purton Lab, UCL. The chloroplast of C. reinhardtii is an attractive organelle for the production of recombinant protein such as vaccines. However, the current level of recombinant protein production in the chloroplast of C. reinhardtii is normally less than 1% of total soluble protein. Therefore, in this study four C. reinhardtii mutant strains (UVM 2, 4, 10 and 11) were created through forward genetics. These mutants exhibit superior expression of recombinant genes in the chloroplast through forward genetics. These strains will be further analysed, so that they can be used to express recombinant genes such as peptide vaccine in the chloroplast of C. reinhardtii in the future.

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