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Title: Engineering immunoglobulin genes for novel Tuberculosis vaccine production in plants
Author: Webster, Gina
ISNI:       0000 0004 7427 0564
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2017
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Tuberculosis is ranked in the top ten leading causes of death from an infectious disease. In 2015, an estimated 10.4 million people developed TB and 1.8 million died from the disease. There is a long-standing vaccine against TB, called BCG, but its ineffectiveness highlights the need for a new novel vaccine that can help prevent pulmonary TB. Ag85B, the most abundantly expressed protein in mycobacterial culture fluids, is a leading vaccine candidate currently used in various TB subunit vaccines in clinical trials. In this project Ag85B was used to form a novel polymeric immunoglobulin G scaffold (PIGS) vaccine candidate by fusion to truncated IgG y-chain. Moreover, IgM ptp was fused to the y-chain to facilitate polymeric structure formation. The predicted immune-complex-like structures should bind complement and FcRs with increased avidity. PIGS were cloned and expressed in Nicotiana benthamiana, purified using protein G affinity chromatography, and polymeric structures were observed by Native PAGE, dynamic light scattering and size exclusion chromatography. These PIGS were shown to be biologically active as they bind to C lq component of the complement cascade as well as FcyRs. More importantly, it was shown that PIGS had greater avidity to low affinity FcyRs, as would be expected for polymeric structures. PIGS were shown to bind to monocytic cells and be internalised. Immunisation and challenge studies in BALB/c and CD64 transgenic mice showed that PIGS are immunogenic but did not improve protection of mice from challenge with Mycobacterium tuberculosis compared to BCG. Adding polylC adjuvant to human PIGS increased immunogenicity, resulting in spleen CFU counts of M. tuberculosis that were statistically reduced compared to CFU counts from BCG immunised mice. One of the major drawbacks to plant molecular pharming is that often relatively low levels of recombinant protein expression are achieved. As plants were used to express the vaccine candidate, a codon optimization algorithm was tested using synonymous codon variants of the native human 2G12 heavy chain which could be applied to increase the expression levels of any plant-made antibody, including the tuberculosis vaccine candidate. However, preliminary results indicated no improvement in 2G12 monoclonal antibody yield using any of the codon optimized variants, compared to native sequence. In conclusion, this study demonstrated that plant-produced PIGS contained polymeric structures and were capable of eliciting an immune response in vivo. Addition of adjuvant, polyIC, resulted in increased immunogenicity of the human PIGS candidate and improved protection of mice from Mycobacterium tuberculosis, measured by the lower CFU counts, in the spleen, compared to BCG immunized mice.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available