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Title: An insight into the mechanisms of abacavir induced hypersensitivity reactions with a view to designing safer therapeutics
Author: Thomson, Paul J.
ISNI:       0000 0004 7659 0377
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Immune-mediated hypersensitivity reactions to the antiretroviral drug abacavir are observed exclusively in individuals carrying the HLA-B*57:01 risk allele. Abacavir is hypothesised to bind within the peptide binding groove of HLA-B*57:01 and alter the array of peptides presented on the cell surface. Indeed, presentation of these unique abacavir-self-peptide complexes is thought to initiate a CD8+mediated T-cell response to the drug in hypersensitive patients. Consistent with previous findings abacavir-specific T-cell clones from healthy donors expressing HLA-B*57:01 were found to be activated via 2 independent mechanisms, relying on and exclusive of antigen processing respectively. Furthermore, clones yielded a varied Tcell receptor expression and cytokine secretion properties when stimulated with abacavir, indicating the presence of multiple unique subsets of abacavir-responsive T-cells. Eighteen abacavir substituted analogues were synthesised with modifications to the cyclopropyl moiety of the compound, namely derivatives of the azetidine group. Eight of these analogues completely lacked any T-cell activity, while others strongly induced IFN-? secretion from the abacavir-responsive clones. Interestingly, activation of T-cells was demonstrated to be enantiomeric-specific around the cyclopropyl and cyclopentyl moiety. However, no analogue bereft of T-cell activity possessed sufficient antiviral potency to be considered a replacement for abacavir in the therapeutic setting. Molecular modelling revealed the unfavourable binding characteristics of non-cross-reacting analogues within the peptide binding groove of HLA-B*57:01, something not observed with T-cell activating analogues. Furthermore, peptide elution studies revealed a distinct shift towards small chain terminating peptides in the presence of cross-reacting abacavir analogues but not non-cross reactive analogues. Raising clones to the respective analogues of abacavir yielded a plethora of T-cells with reactivity towards the analogues including those that initially did not cross react with abacavir clones, indicating the importance of T-cell cloning assays in the analysis of the immunogenicityof HLA-binding drugs. While the role of altered self-peptides presented on the surface of the MHC in the presence of abacavir is thought to drive the CD8+ mediated T-cell reactions, the peptide sequences that activate T-cells in the presence of abacavir has not been fully defined. Further studies with a previously presumed immunogenic peptide (NTVELRVKI) demonstrated little to no reactivity towards abacavir clones in its natural form, whilst producing non-specific T-cell activity in the amidated form. Dissecting the contribution of the abacavir-induced-selfpeptides in T-cell responses to abacavir is difficult as these peptides are naturally loaded onto MHCs for presentation on the cell surface. However, using a panel of five abacavir inducedself peptides which had underwent kinetic studies previously to examine their dependence on abacavir for presentation on the surface of antigen presenting cells over time, peptide responsive T-cell clones were generated in the presence of abacavir. At low concentrations of the drug, these clones demonstrated an enhanced response to abacavir in the presence of the individual self-peptides. Finally, two clones raised solely to abacavir, displayed strong IFN-? secretion to two of the self-peptides in the absence of the drug. These findings suggest that the abacavir-dependent self-peptides do seem to contribute to the activation of T-cells. This work highlights the involvement of peptides in the CD8+ T-cell-mediated hypersensitivity reactions to abacavir in HLA-B*57:01+ individuals. Furthermore, it emphasises the importance of a holistic approach to the design and subsequent analysis of the safety of a new compound for integration into the therapeutic setting.
Supervisor: Naisbitt, Dean J. ; Park, B. K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral