Acyclovir Has Low but Detectable Influence on HLA-B*57:01 Specificity without Inducing Hypersensitivity

Abstract

Immune mediated adverse drug reactions (IM-ADRs) remain a significant source of patient morbidity that have more recently been shown to be associated with specific class I and/or II human leukocyte antigen (HLA) alleles. Abacavir-induced hypersensitivity syndrome is a CD8+ T cell dependent IM-ADR that is exclusively mediated by HLA-B*57:01. We and others have previously shown that abacavir can occupy the floor of the peptide binding groove of HLA-B*57:01 molecules, increasing the affinity of certain self peptides resulting in an altered peptide-binding repertoire. Here, we have identified another drug, acyclovir, which appears to act in a similar fashion. As with abacavir, acyclovir showed a dose dependent increase in affinity for peptides with valine and isoleucine at their C-terminus. In agreement with the binding studies, HLA-B*57:01 peptide-elution studies performed in the presence of acyclovir revealed an increased number of endogenously bound peptides with a C-terminal isoleucine. Accordingly, we have hypothesized that acyclovir acts by the same mechanism as abacavir, although our data also suggest the overall effect is much smaller: the largest changes of peptide affinity for acyclovir were 2-5 fold, whereas for abacavir this effect was as much as 1000-fold. Unlike abacavir, acyclovir is not known to cause IM-ADRs. We conclude that the modest effect of acyclovir on HLA binding affinity in contrast to the large effect of abacavir is insufficient to trigger a hypersensitivity syndrome. We further support this by functional in vitro studies where acyclovir, unlike abacavir, was unable to produce an increase in IFN-γ upon expansion of HLA-B*57:01+ PBMCs from healthy donors. Using abacavir and acyclovir as examples we therefore propose an in vitro pre-clinical screening strategy, whereby thresholds can be applied to MHC-peptide binding assays to determine the likelihood that a drug could cause a clinically relevant IM-ADR.

Fig 1. The workflow of the virtual screening protocol for screening of similar drugs to abacavir.

Fig 2. Chemical structures for abacavir and the other seven drugs that were predicted to bind in the F-pocket of HLA-B*57:01.

Fig 3. Effects of abacavir and acyclovir on HLA-B*57:01 binding specificity.

Specific peptides with a terminal valine that showed an increased affinity for HLA-B*57:01 in the presence of abacavir were tested. Values are represented as geometric mean with 95% CI of two independent runs in triplicates, analyzed for statistical significance by Mann-Whitney U test comparing log IC₅₀ values vs. vehicle; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig 4. Effects of acyclovir (2 mg/mL) on the affinity of C-terminal residues for HLA-B*57:01.

Values are represented as geometric mean with 95% CI of the fold difference between vehicle/acyclovir treatment. The experiment was run 6 times with each run performed in triplicates. Analyzed for statistical significance by column statistics; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001). The most pronounced affinity increases for HLA-B*57:01 in the presence of 2 mg/mL of acyclovir were found for peptides with a cysteine, isoleucine and valine at the C-terminus.

Fig 5. Effects of abacavir and acyclovir on HLA-B*57:01 binding specificity.

Specific peptides with a terminal isoleucine that showed an increased affinity for HLA-B*57:01 in the presence of abacavir were tested. Values are represented as geometric mean with 95% CI of two independent runs in triplicates, analyzed for statistical significance by Mann-Whitney U test comparing log IC₅₀ values vs. vehicle; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig 6. Acyclovir and abacavir alter the binding specificity of HLA-B*57:01.

The peptide KAAKYRVSV was radiolabeled and tested for binding to HLA-B*57:01 in increasing doses of acyclovir and abacavir. Values are represented as geometric mean with 95% CI of four experimental runs in triplicates. Analysed by statistical significance by one-sided Mann Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig 7. PBMC from a healthy HLA-B*57:01 positive donor (Donor 1) where primed with abacavir at day 0, cultured for 14 days and then restimulated 1:10 with (A) HLA-B*57:01 single antigen line (C1R.B57), (B) with O/N abacavir treated C1R.B57 (C1R.B57.ABC) or (C) with O/N acyclovir treated C1R.B57 (C1R.B57.ACY).

Antigen activated cells were detected by ICS for IFN-γ production and CD8+/ IFN-γ T-cells quantitated using flow cytometry. (D) PBMC from two healthy HLA-B*57:01 positive donors were either primed with abacavir (ABC primed), primed with acyclovir (ACY primed) or had no treatment (Control. PBMC were cultured for 14 days and then stimulated 1:10 with treated and untreated single antigen line, C1R.B57, as indicated.