African horsesickness virus (AHSV) belongs to the Orbivirus genus in the family Reoviridae. This non-enveloped virus consists of an outer capsid formed by two structural proteins, VP2 and VP5, and an inner core formed by structural proteins VP7 and VP3. Three additional structural proteins associated with viral replication, as well as ten dsRNA molecules responsible for replication, are found inside the core. VP7 is the smallest of the structural proteins and each monomer consist of two domains, a hydrophilic top and hydrophobic bottom domain. Upon expression of VP7, the protein spontaneously assembles into trimers. Recombinant expression of the core protein VP7 results in large hexagonal structures formed by a double layer of these VP7 trimers with the hydrophobic bottom domains on the inside and hydrophilic top domains on the outside. The use of these crystal structures as a general display system for the display of foreign peptides/epitopes is being investigated in our group. In this regard, sites for the insertion of foreign peptides/epitopes were constructed at amino acid positions 177, 144 and 200 of the top domain of the VP7 protein and the resultant proteins named vectors AHSV-9 VP7-177, AHSV-9 VP7-144 and AHSV-9 VP7-200. Various inserts ranging from the HIV-1 ELDKWA epitope and FMDV VP1 epitopes to the eGFP peptide were inserted and subsequently analysed for immunogenicity. Results showed that a significant immune response was only elicited if the soluble trimer component of a chimeric VP7 protein was used for inoculation purposes. The crystal particles initially investigated as a display system did not result in any immune response. These results emphasized the importance of protein solubility for eliciting a significant immune response. The importance of solubility prompted an investigation into the use of the Bluetongue virus (BTV) VP7 protein as a vaccine display system. This protein is inherently more soluble than AHSV VP7 and does not result in crystal hexagonal structures if recombinantly expressed. An insertion site analogous to that of the AHSV-9 VP7-177 vector, located at amino acid 177 within an RGD loop in the top domain of VP7 was constructed. This new BTV VP7 vector, BTV-10 VP7-169, was characterised with regard to solubility and the ability to form trimers. In order to investigate the effect on solubility and trimerisation, FMDV VP1 epitope and eGFP were inserted into the BTV-10 VP7-169 vector. Results showed that following the construction of the insertion site, the vector was largely insoluble compared to the AHSV VP7 vectors and that insertion of the abovementioned peptides/epitopes did not have a significant effect on solubility. Although trimers were present, the yield was low compared to that of the AHSV chimeric VP7 proteins. Methods of improving the solubility of the chimeric VP7 proteins were investigated by treatment with solubilisation agents, sarkosyl and L-arginine. The results indicated that a strong denaturant such as sarkosyl can solubilise the particulate component of all chimeric VP7 proteins whereas Larginine had limited effect. The effect of these agents on the folding of the proteins were evaluated using fluorescence, since the ability to fluoresce is regarded as an indicator of correct folding. A comparison of the different VP7-eGFP proteins treated with these solubilisation agents showed that the sarkosyl solubilised proteins were not necessarily correctly folded. These results combined with the previously performed solubility assays indicated that a large proportion of correctly folded chimeric VP7 proteins associate with the particulate fraction. Investigation showed that expression of a large amount of correctly folded chimeric proteins results in the aggregation of these proteins within the expressing host cell. Once harvested these proteins remain associated with the insoluble fraction but can be solubilised by arginine treatment, or in some cases mere resuspension in a low-salt buffer, and used for vaccination purposes. In conclusion, the comparative analyses of solubility and trimerisation for the display vectors indicated that AHSV-9 VP7-144 vector may be most suitable for the display of foreign epitopes/peptides as it consistently yielded the largest component of correctly folded proteins. Furthermore, considering that large amounts of correctly folded chimeric VP7 proteins occurred in the insoluble component of each the VP7 display proteins, this study emphasize that use of solubility assays alone does not provide adequate information regarding the potential of a display vector for vaccination purposes. / Dissertation (MSc)--University of Pretoria, 2013. / Genetics / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/28837 |
| Date | 08 January 2013 |
| Creators | Bolton, Debora |
| Contributors | Prof H Huismans, Dr W Fick, dbolton77@gmail.com |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2013 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria |
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