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Exploiting the retrograde transport of disarmed toxins for the delivery of exogenous antigens into MHC class 1 presentation pathway
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Noakes, Karen (1999) Exploiting the retrograde transport of disarmed toxins for the delivery of exogenous antigens into MHC class 1 presentation pathway. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b1368729~S15
Abstract
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.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Antineoplastic agents, T cells, Escherichia coli, Major histocompatibility complex | ||||
Official Date: | March 1999 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Biological Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Roberts, L. M. (Lynne M.) | ||||
Extent: | xvi, 160 leaves | ||||
Language: | eng |
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