Towards an HCV vaccine

Ward, Scott Matthew. (2002). Towards an HCV vaccine PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

       
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Author Ward, Scott Matthew.
Thesis Title Towards an HCV vaccine
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Eric Gowans
Total pages 194
Collection year 2002
Language eng
Subjects L
320599 Pharmacology not elsewhere classified
780105 Biological sciences
Formatted abstract Hepatitis C virus (HCV) infects more than 1% of the world population and leads to persistent infection in ~80% of exposed individuals. To date, the only effective treatment is interferon therapy which leads to recovery in less than 40% of cases. Therefore, development of a prophylatic and/or therapeutic vaccine would be beneficial in preventing HCV-associated chronic disease.

Viral clearance is usually associated with a strong cytopathic T lymphocyte (CTL) response. Several studies have demonstrated that a strong, early and ongoing CTL response is important in resolving acute infection. Hence it is proposed that stimulation of the CTL response may be useful to prevent infection per se and terminate persistent infection. This thesis describes the development of two putative vaccines designed to elicit an HCV-specific CTL response.

Previous studies have shown that exogenously-administered hepatitis B surface antigen (HBsAg) virus-like particles (VLPs) are able to generate a CTL response via an alternate MHC class 1 processing pathway. It was proposed that any protein internal to the HBsAg VLPs would also elicit a CTL response. This led to the development of hepatitis delta virus VLPs to deliver HCV CTL epitopes (polyB-HDAg VLPs). The HCV CTL epitopes were designed in a polyepitope (polytope) manner. The polyBHDAg VLPs were constructed and characterised and were found to have similar biophysical properties to wild-type hepatitis delta VLPs as analysed by immunoblot. density gradient centrifugation and immunoprecipitation studies. The ability of the polyB-HDAg VLPs to elicit a CTL response in mice was tested in a standard chromium release assay to measure cytolytic activity, without success.

The second vaccine delivery system was based on the Kunjin virus replicon (self-replicating RNA). The replicon RNA expresses the virus polyprotein with the structural proteins replaced by a foreign protein. This allows self-replication of the RNA by the virus non-structural proteins and expression of the foreign protein without production of virions. The Kunjin replicon was used to incorporate a new polytope that consisted of 7 HCV CTL epitopes and one Influenza CTL epitope (Kun-Poly). The self-replicating nature of the RNA was confirmed in cell culture by Northern blot hybridisation using a poly tope-specific probe. Expression was confirmed, by detection of the Kunjin NS3 protein in immunofluorescence studies, up to 10 days post-transfection in BHK cells without an apparent cytopathic effect as determined by haemotoxylin and eosin staining.

The peptides of the corresponding epitopes within the polytope were tested in mice for their ability to elicit a CTL response. The Influenza peptide was strongly immunogenic but the HCV peptides did not elicit a detectable CTL response. Initial vaccinations with one dose of Kun-Poly RNA showed that an Influenza-specific CTL response was elicited more efficiently by intradermal compared to intramuscular delivery. Two µg of RNA delivered in the ear pinnae of mice was sufficient to elicit a detectable CTL response 10 days post-vaccination. Further vaccination studies showed that 4 of the 7 HCV CTL epitopes were able to elicit specific CTL responses following three doses of Kun-Poly RNA, although this was not sufficient to afford protection from recombinant vaccinia virus challenge. This work provides a basis for future HCV vaccine developments using the Kunjin replicon system.
Keyword Hepatitis C -- Vaccination

 
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Created: Fri, 24 Aug 2007, 17:52:37 EST