Abacavir impairs an HLA-B*5701-restricted CD8+ T Cell response to an immunodominant Epstein-Barr virus epitope
Lucas, A., Meyer-Pannwitt, V., McKinnon, E., Burrows, S., Rist, M., Leary, S., Lucas, M., Mallal, S. and Phillips, E. (2013) Abacavir impairs an HLA-B*5701-restricted CD8+ T Cell response to an immunodominant Epstein-Barr virus epitope. In: 20th Conference on Retroviruses and Opportunistic Infections (CROI 2013), 3 - 6 March 2013, Atlanta, Georgia
Background: Structural and functional studies show that abacavir (ABC) binds non-covalently to HLA B*57:01 and alters the repertoire of peptide presented, some of which may re-stimulate memory CD8+ T cells to cause ABC hypersensitivity. We questioned whether the binding of ABC to HLA-B*5701 might also impair an HLA-B*5701 restricted CD8+ T cell antiviral response.
Methods: 52 putative B95-8 Epstein-Barr Virus (EBV) strain HLA-B*57:01 restricted epitopes were identified using NetMHC 3.2 (binding score <400 nM). 7 healthy adult HLA-B*57:01+ donors were screened with EBV peptide pools followed by confirmatory enzyme-linked immunosorbent assay (ELISpot) with individual EBV peptides and avidity testing to 0.001 g/mL peptide. In addition, 4 HLA-B*57:01+ and 31 negative healthy donors were screened with 20-mer peptides overlapping with 15 amino acids from EBNA3A, 3B, BZLF1, and BMLF1. 4 short-term lines were generated from 4 HLA-B*57:01+ ABC-naīve donors by pulsing peripheral blood mononuclear cells (PBMC) with 20 μg/mL VSFIEFVGW for 1 hour, washing and then 9 days culture in RPMI-1640 media containing 10% FCS and 10% T-Stim media and 100 U/mL IL-2. The resulting cell lines contained 5% to 35% peptide specific CD8+ T cells. Cell lines or PBMC taken from HLA-B*57:01+ donors were tested for peptide specific responses ąABC at a range of concentrations previously shown not to affect cell viability, by intra-cellular cytokine re-stimulation assays (ICS) or ELISpot. Peptide concentrations of 0.01, 0.1, 1, and 10 g/mL were used in the ICS assay and 1 g/mL in the ELISpot assay. Expression of interferon (IFN)-γ in the presence of increasing concentrations of ABC relative to baseline (no ABC) and the respective ratios were analyzed using a linear mixed effects model with nesting for multiple peptide concentrations per individual.
Results: 4 HLA-B*57:01-restricted epitopes were confirmed of which 2 were independently detected using overlapping peptides: VSFIEFVGW from EBNA3A and VAAHPEIGAW from BMLF1. Responses to VSFIEFVGW were of high avidity and detectable in all 11 donors confirming it as an immunodominant peptide. IFN-γ responses to VSFIEFVGW relative to baseline were reduced relative to baseline with increasing abacavir concentrations using both ICS (Fig a) and ELISpot assay (Fig b).
Conclusions: A new immunodominant HLA-B*57:01-restricted EBV epitope was identified. ABC was shown to impair the CD8+ T cell response to this epitope in a dose-dependent manner, suggesting that drugs that bind non-covalently to HLA molecules may modulate immune responses.
|Publication Type:||Conference Paper|
|Murdoch Affiliation:||Institute for Immunology and Infectious Diseases|
|Notes:||Figures missing from abstract|
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