Although the first successful rabies vaccine administration was in 1885, rabies continues to be the tenth most lethal infection worldwide causing significant human life loss. Endemic to under-developed and developing countries, economic restraints limit the widespread administration of either pre-exposure or post-exposure prophylaxis. This research project is a continuum to the objective of producing rabies vaccine, both its passive and active components, in plants. The main component of this research was the work done to reverse engineer the plant rhabdovirus, Lettuce Yellows Necrotic Virus (LNYV), as a viral-vector vaccine candidate. The strategy followed was that of a minigenome cassette where, using a reporter gene, the cis and the trans elements of the viral transcription cycle were demonstrated to function in planta. A number of molecular tools such as ribozymes, introns, and hybrid genomic sequences were utilised to achieve precise viral temini, to allow cloning of viral genes, and to raise anti-LNYV antiserum reagent. For the second component of this project, development of rabies immunoglobulins proceeded with further optimisation of the neutralising 62-71-3 monoclonal antibody. Previous research in our group has demonstrated the efficacy of chimaeric 62-71-3 mAb in neutralising a battery of lyssaviruses. Through an extended collaboration, two humanized variants of the murine light chain and three humanized variants of the heavy chains of the chimaeric 62-71-3 mAb (human constant and murine variable regions), were combined into six fully humanized antibodies, expressed in Nicotiana benthamiana and characterised. Their neutralization efficacy was demonstrated using a rabies pseudotype neutralization assay. In addition, to investigate their binding efficacy, at first the antigenic site I, proven to be the chimaeric 62-71-3 mAb binding site, was investigated through immunoblotting. Based on the results, the difficulty in obtaining rabies virus glycoprotein was circumvented via in vitro synthesis of the rabies glycoprotein, and a competition ELISA was developed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:719154 |
| Date | January 2017 |
| Creators | Ibrahim, Ahmad El Cheikh |
| Publisher | St George's, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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