Document Type Master's Dissertation Author Kretzmann, Heidi URN etd-05152007-103844 Document Title The characterization of African horsesickness virus VP7 particles with foreign peptides inserted into site 200 of the VP7 protein top domein Degree MSc (Genetics) Department Genetics Supervisor
Advisor Name Title Dr W Fick Prof H Huismans Keywords
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Date 2006-08-02 Availability unrestricted AbstractAfrican horsesickness virus (AHSV) VP7 development as a particulate vaccine system, for human and veterinary use, is based on the observation that VP7 forms hexagonal crystalline structures which can be exploited to present epitopes to the immune system. VP7 can be produced in large amounts by means of the baculovirus expression system and is easily purified. In order to display an epitope or small peptides in the VP7 top domain several VP7 display vectors have been constructed that will allow the insertion of peptides into at least three different amino acid sites. These vectors are VP7mu144, VP7mu177 and VP7mu200. Vector VP7mu177 in which different peptides were inserted into site 177 has been investigated by Rutkowska (2002). The aim of this investigation is to insert the same peptides into site 200 and compare the constructs with regard to assembly into VP7 particles, structure, stability and antigenicity. These VP7 vectors have to be characterized to determine which of these mutant sites will elicit the best immune response.
Insertion mutants were made of the major core protein of AHSV VP7 serotype 9 at specific amino acid positions for the possible use as a subunit vaccine by F. Maree (2000). In this study HIV-1 subtype C ALDSWK and RVLAIERYLKD epitopes with flanking regions were cloned using restriction enzyme tagged PCR products. The recombinants were expressed using the baculovirus expression system.
The insertion of HIV epitopes in the vectors did not significantly alter the protein expression levels. However, the chimeric VP7mu200 and VP7mu177 proteins did not show the characteristic crystalline structures observed with VP7mu200, VP7mu177 and unmodified VP7 in insect cells. Scanning Electron Microscopy revealed flat, rounded particulate structures with a rough to layered surface. The insertion of 50, 53 and 101 amino acids thus abolishes the smooth hexagonal crystalline formation characteristic of the VP7 protein. This is partly due to steric interference which most likely disrupted the hydrophobic bonds. The structural disruption of the VP7 particle observed with the 50 aa insertion is not further distorted in the 101 aa insert.
Comparative studies show VP7mu200 to be more sensitive to the hydrophobic nature of an insert with regards to particle assembly than VP7mu177. VP7mu177 with and without HIV epitopes assemble into large particles predominantly found in fractions 1-4 at the bottom of a sucrose density gradient. VP7mu200 with a double HIV insert predominantly assembles into large particles (fractions 1-4) and the single HIV inserts assemble into predominantly smaller particles (fractions 5-8). Mechanical lysis of the infected cells containing the chimeric VP7mu200 particles compared to detergent treatment of similarly infected samples reveal possible lipid association suggesting that VP7mu200 may form inclusion bodies. Despite differences in particle assembly between the vectors, both chimeric VP7mu177 and chimeric VP7mu200 particles were shown to be antigenic. Future prospects include conformation and immunology studies pertaining to immunizations with the chimeric proteins, analysis of sera to ascertain if antibodies have been raised as well as neutralization assays to distinguish between the capabilities of the different insertion sites.
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