Raafat, Nermin Abdel Fattah. Vaccinia virus expressing ICP47 : a novel platform for cancer vaccines highlighting tumor epitopes and hiding viral antigens. 2010, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_8968
Recombinant poxviruses expressing tumor associated antigens (TAAs) are evaluated since 20 years as immunogenic vaccine vector in clinical trials. Some caveats for using recombinant viral vector are due to either prior systemic immunity to poxviruses or immunodominance of viral antigens which may reduce the induction of immune response against weaker tumor antigens. To address this issue, we developed a recombinant vaccinia virus (VV) expressing herpes simplex virus (HSV) type I protein ICP47. This protein down-regulates MHC class-I antigen presentation by blocking the transporter associated with antigen processing (TAP), which translocates peptides, generated by proteasomal protein degradation, into the endoplasmic reticulum for loading onto MHC class I molecule.
HSV-US12 gene, coding for ICP47 was introduced into wild type vaccinia virus (VV) and into r.VV expressing MART-1/Melan-A27-35 HLA-A201 ER-targeted epitope. Following infection with non-replicating recombinant virus, effect of ICP47 expression on cell surface MHC-class-I, MHC class-II and co-stimulatory molecules was characterized by antibody staining and FACS analysis. Human T-lymphocytes were stimulated in vitro with autologous CD14+ cells infected with r.VV-US12, r.VV-Mart-US12 or control virus. Responsiveness of specific CD8+ and CD4+ T cells to viral proteins and to recombinant epitopes was monitored by MHC-multimer staining and cytokines (IFNγ & IL-2) expression analysis. In order to highlight in vitro the immunogenic advantage provided by ICP47 blockade of viral epitopes, T cells were pre-sensitized with WT-VV infected APC and then used as responders to r.VV-Mart-US12 or r.VV-Mart infected APC.
Cells infected with HSV-US12-r.VV, demonstrated a decreased ability of presenting MHC class-I antigens to CD8+ T cells whereas MHC-class-II restricted presentation to CD4+ T cells remained unaffected. Cell surface expression of CD80 as a costimulatory molecule and CD44 as cell adhesion molecule was not affected in cells infecied with VV expressing ICP47. Co-expression of ER-Melan-A/Mart-127-35 appeared to partially compensate for the ICP47 related surface MHC class-I down-regulation and preserve a strong capacity to induce CTL response against the TAA derived peptide. The differential prime-stimulation for Mart epitope, in conditions where clearance of infected APC by VV specific CTL could become a limiting factor, showed significantly enhanced CTL responses to Mart stimulation as characterized by IFN-γ and IL-2 gene expression in cultures primed with r.VV-Mart-US12 as compared to cultures primed with r.VV-Mart.
Thus, viral vectors expressing ICP47 confirmed a diminished TAP-dependant processing of endogenous class-I restricted epitopes while the immunogenic potential of recombinant epitopes directly targeted to the ER was enhanced. Such reagents could become of high relevance especially in multiple-boost vaccine protocol required in cancer immunotherapy.
|Advisors:||Spagnoli, Giulio C.|
|Committee Members:||Zajac, Paul and Hirsch, Hans H.|
|Faculties and Departments:||03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Oncology Surgery (Spagnoli/Heberer)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||148 Bl.|
|Last Modified:||30 Jun 2016 10:41|
|Deposited On:||07 May 2010 08:20|
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