Exploiting the retrograde transport of disarmed toxins for the delivery of exogenous antigens into MHC class 1 presentation pathway
The targeting of exogenous antigen into the MHC class 1-restricted presentation pathway is required for the induction of cytotoxic T lymphocytes (CTL) which have been shown to be an important component of the protective response to intracellular antigens and also induce immunity to tumour cells. Thus, induction of a CTL response is an important goal in vaccine research and a number of delivery systems are being investigated. Certain cytotoxic proteins that catalytically modify substrates in the cytosol of mammalian cells undergo retrograde vesicular transport from the cell surface to the endoplasmic reticulum before translocating into the cytosol. In this study, Shiga-like toxin 1 (SLT) from Escherichia coil 0157: H7 was used to deliver an antigenic peptide for presentation by major histocompatibility complex (MHC) class I molecules. The SLT A chain was genetically fused with a nonamer peptide derived from influenza virus Matrix protein, positioned at either the N- or C-terminus of the toxin (designated SLT N-Ma or C-Ma). The SLT coding sequence was also mutated to convert an active site glutamate into aspartate to significantly reduce ribosome-inactivating activity, and hence the inherent cytotoxicity of the toxin chimeras. Recombinant holotoxins carrying the viral peptide were expressed in E. coil and purified to homogeneity before use. HLA-A2-transfected HeLa cells were allowed to internalize the disarmed toxin-peptide chimeras and were used as targets of influenza Matrix-specific cytotoxic T lymphocytes (CTL). HLA-A2-matched cells, unable to internalize SLT, were used as negative controls. Results from this study show that SLT N-Ma but not SLT C-Ma is capable of sensitizing HeLa A2 cells for lysis by cytotoxic T -lymphocytes (CTL) whilst no killing of SLT-resistant cells has been observed. Treatment of cells with the Golgi stack-disrupting agent brefeldin A successfully blocked the presentation of the M58- 66 epitope at the cell surface, confirming that the antigenic peptide was liberated intracellularly. This strategy was repeated for ricin A-chain, placing a nonamer peptide derived from influenza virus nucleoprotein at either the N-terminus or within an external loop of the toxin A-chain (designated RTA N-NP or Clal-NP). Fusion proteins were expressed in E. coli and purified, followed by reassociation with ricin B-chain. Preliminary results have failed to show the delivery of peptide antigen to class-i molecules by the ricin chimeras. This work is ongoing. This thesis demonstrates that Shiga-like toxin-1, known to be endocytosed from the cell surface to the ER lumen and then transferred to the cytosol of eukaryotic cells, can intersect the MHC class 1 presentation pathway and effectively carry antigenic peptide to class 1 molecules in vitro. This approach opens up new possibilities for the generation of CD8+ T-cell vaccines for use against infectious agents, cancer and autoimmune disorders.