As the global population increases, estimated to reach 9 billion by the year 2050, global food security becomes a priority. A prominent disease implicated in financial loss to the poultry industry, on a global scale, is infectious bursal disease virus (IBDV). Vaccination against IBDV is sub-optimal and difficult to deliver. Therefore it has been highlighted as a key area for the development of an oral vaccine. A highly conserved capsid protein from IBDV (VP2) was identified, and sub-cloned into a bacterial expression cassette. This protein was fused to a potential carrier protein (cholera toxin B chain), previously shown to mediate the exit from the gut lumen into the lamina propria. However, to allow this antigen to reach the mucosal associated lymphoid tissue, the protein antigen must remain in its native conformation through the stomach. This work developed a delivery system to meet this end. By encapsulation within a fatty acid coated, protein adsorbed-solid core drug delivery system (SCDDS), it was shown that a model protein antigen (GST-GFP) could be protected from low pH (i.e. pH 2.0) and proteases. Protease protection was demonstrated against the exposure of myristic acid coated, GST-GFP adsorbed silica, to both protease K (100 μU, 1hour (100% protection)) as well as a simulated in vitro stomach environment (pepsin (0.2 mg) (100% protection)). Having demonstrated protection from proteases at pH 2.0 and pH7.4, it was then shown that GST-GFP could be released from the myristic acid coated silica at pH 8.8 (consistent with the small intestine). As much as ~15% (15 μg) (w/w) GST-GFP was released from the aforementioned system. The evidence supporting this conclusion was drawn from molar ellipticity calculations that showed the proportion of helical structure in relation to regions of beta sheet remained constant, pre-adsorption and post-release (16.9% α-helix, 20.8% β-sheet, 43.3% random coil). Finally, this work has shown that if a recombinant antigen was fused to cholera toxin B chain (but not shiga toxin B chain), it was capable of mediating transcytotic passage across, differentiated, polarised Caco-2 cells (1/1000th input (10 ng)). In conclusion and based upon the evidence provided above, this system warrants further optimisation and investigation to serve as an oral vaccine delivery system to treat IBDV.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:616531 |
| Date | January 2013 |
| Creators | Pettit, Wendy Marie |
| Contributors | Richardson, Simon; Mitchell, John |
| Publisher | University of Greenwich |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://gala.gre.ac.uk/11376/ |
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