Recombinant Virus-Like Particles Presenting Epitopes for Antibody Generation
Virus-like particles (VLP) have been shown to be effective vessels for drug or gene delivery, and vaccination. Much research has focused on the generation of VLP carrying heterologous tumour antigens to initiate cell-mediated immunity against tumours, where the VLP enhances the immunogenicity of the attached antigen. However, VLP can also be modified to incorporate an antigen on the surface to facilitate antibody generation. Purification tags or specific coupling sites could also be incorporated onto the surface of the particles, improving their functionality. This study aimed to utilise the immune stimulatory properties of VLP to generate antibodies against a heterologous model antigen, producing a scaffold for delivery and enhancement of antibody mediated subunit vaccines. More specifically, Human Norovirus (HuNV) and Rabbit hemorrhagic disease virus (RHDV) capsid proteins were engineered to express the YG1 epitope from human tumour necrosis factor-alpha (hTNF-α) at positions predicted to be displayed on the surface of the VLP. Amino acids positions 368 of HuNV and 306 of RHDV capsid proteins were identified in the literature as confirmed sites of peptide insertion, with each locating to the exposed loops of the capsid protein P domain. Recombinant gene constructs expressing the YG1 peptide at these sites were produced by PCR. VLP were expressed in a baculovirus expression system and purified using differential centrifugation and cesium chloride gradient separation. Following expression, recombinant VLP assembly was confirmed by electron microscopy, and while the RHDV VLP was less stable than HuNV, they were both able to form particles. Rats immunised with these recombinant VLP generated IgA and IgG antibodies specific for both the native VLP carrier and the YG1 epitope. Thus initial data indicates that HuNV and RHDV VLP can be genetically engineered to act as vaccine delivery systems, leading to enhanced peptide based subunit vaccines. The confirmed insertion sites could also be used to incorporate purification tags, specific coupling sites, or allow multi-epitope capability, where peptides can be included at both the N-terminus and on the surface of the same VLP.
Advisor: Ward, Vernon
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: virus like particles; VLP; recombinant VLP; antibody generation
Research Type: Thesis