Title page for ETD etd-05062005-124623


Document Type Master's Dissertation
Author Van Rensburg, Ruan
URN etd-05062005-124623
Document Title Construction and structural evaluation of Viral Protein 7 of African horse sickness virus as a particulate, multiple peptide vaccine delivery system
Degree MSc(Genetics)
Department Genetics
Supervisor
Advisor Name Title
Prof H Huismans Committee Chair
Keywords
  • African horse sickness virus
  • vaccines development
Date 2004-04-24
Availability unrestricted
Abstract
The highly hydrophobic viral protein (VP) 7 of African horse sickness virus (AHSV) folds into a trimeric structure that aggregates to form flat, hexagonal crystals (Chuma et al., 1992). These crystals are composed of flat sheets of hexameric rings, similar to the rings of trimers seen in the outer core surface layer. The crystals have been shown to be highly immunogenic when used as a subunit vaccine and are able to elicit a strong immune response against subsequent viral infections (Wade-Evans et al., 1997). The aim of this study is to investigate the structural constraints of using these structures as a particulate, multiple peptide vaccine delivery system.

Three hydrophilic regions at amino acid position 144, 177°and 200 on the VP7 surface of this trimeric structure were targeted for insertion of peptides and a new vector was constructed in this study with a multiple cloning site at each one of the three top domain sites. The newly constructed three-site VP7 mutant gene was expressed in the Bac- To-Bac expression system and the recombinant proteins were investigated for its solubility and crystal formation by sucrose density gradient centrifugation. The structure and stability of the modified, trimeric VP7 was confirmed and further analyzed. Scanning electron microscopy showed the formation of large structures by the trimeric modified VP7 protein units. These' structures differed from the hexagonal crystals formed by unmodified VP7, resulting in rough-looking, flat circular structures attached by protein cables. The high yield of protein expression and the ease, with which these particles can be purified, makes this vector ideal for vaccine use. These protein structures also seemed to remain stable after being stored under different conditions. Studies were also conducted on the stability of these structures after sonication, enabling a range of different size particles to be presented to the immune system.

The purpose for the creation of multiple cloning sites was for the vaccine to be able to accommodate and efficiently present multiple epitopes to the immune system. An investigation was launched into the effect of peptide insertion at one or more of the multiple cloning sites. The initial study included the insertion of two small peptides from AHSV VP2 at amino acid sites 144 and 177 respectively. The size of the peptides that can be inserted is also very important in the use of virus-like particles as antigen carriers. In order to utilize the full potential of the VP7 particles as an antigen presentation system, it must be possible to accommodate large epitope-containing insertions. At the extreme, a stretch of 250 amino acids from AHSV VP2 was inserted into the 177 amino acid multiple cloning site of the three-site VP7. Structural evaluation of all these expressed proteins indicated that the structure of the VP7 subunit vaccine is stable and still retains the ability to form large aggregated structures from the trimeric units. Scanning electron microscope revealed that all these peptide-containing constructs retain approximately the same structural shape as the structures formed by the three-site VP7 mutant.

© 2003 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.

Please cite as follows:

Van Rensburg, R 2003, Construction and structural evaluation of Viral Protein 7 of African horse sickness virus as a particulate, multiple peptide vaccine delivery system, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-05062005-124623/ >

H1197/ag

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