The PD1 inhibitory pathway and mature dendritic cells contribute to abacavir hypersensitivity in human leukocyte antigen transgenic PD1 knockout mice.

Based on recent genome-wide association studies, abacavir-induced hypersensitivity is highly associated with human leukocyte antigen (HLA)-B*57:01 allele. However, the underlying mechanism of this occurrence is unclear. To investigate the underlying mechanism, we developed HLA-B*57:01 transgenic mice and found that application of abacavir could cause CD8 T cell activation with elevation in PD1 expression; however, severe skin hypersensitivity was not observed. To eliminate the immunosuppressive effect of PD1, HLA-B*57:01 transgenic/PD1 knockout (01Tg/PD1) mice were generated by mating HLA-B*57:01 transgenic mice and PD1 knockout mice. Thereafter, 01Tg/PD1 mice were treated with abacavir. Similar to the above results, severe skin hypersensitivity was not observed. Therefore, we treated 01Tg/PD1 mice with an anti-CD4 antibody to deplete CD4 T cells, followed by abacavir topically and orally. Severe abacavir-induced skin hypersensitivity was observed in 01Tg/PD1 mice after depletion of CD4 T cells, in addition to significant CD8 T cell activation and dendritic cell maturation. Taken together, we succeeded in reproducing severe skin hypersensitivity in a mouse model. And we found that through the combined depletion of PD1 and CD4 T cells, CD8 T cells could be activated and could proceed to clonal proliferation, which is promoted by mature dendritic cells, thereby eventually inducing severe skin hypersensitivity.

[Importance of HLA in Determining Individual Differences in the Onset of Adverse Drug Reactions].

Individuals vary in their susceptibility to adverse reactions to medications, some of which can be potentially life-threatening. Idiosyncratic drug toxicity (IDT) has been shown to be strongly associated to specific polymorphisms in genes encoding human leukocyte antigens (HLAs) by recent genome-wide association studies. However, the pathogenic mechanisms governing such reactions remain unclarified, at least in part because of a lack of suitable experimental animal models to assess IDT. This review describes our work on the specific allele/drug combination of HLA-B*57:01 and abacavir, an antiretroviral drug targeting the human immunodeficiency virus. As abacavir is known to trigger an HLA-dependent immune response, we engineered a transgenic mouse model-HLA-Tg-by partially substituting the mouse HLA sequence for the corresponding human sequence. Local abacavir exposure was found to trigger a significant immune response in an HLA-dependent manner, and oral administration induced liver injury partially via concurrent activation of the innate immune system. Additionally, we developed a technique for evaluating structural alterations in HLA complexes resulting from drug exposure based on phage display to ensure specificity. Further scrutiny of the mechanism(s) underlying drug-induced immune reactions using the HLA-Tg model, as well as enhanced methods for predicting adverse event incidence, are anticipated to help resolve issues surrounding HLA-associated drug hypersensitivity.

Kinetics of Abacavir-Induced Remodelling of the Major Histocompatibility Complex Class I Peptide Repertoire.

Abacavir hypersensitivity syndrome can occur in individuals expressing the HLA-B*57:01 major histocompatibility complex class I allotype when utilising the drug abacavir as a part of their anti-retroviral regimen. The drug is known to bind within the HLA-B*57:01 antigen binding cleft, leading to the selection of novel self-peptide ligands, thus provoking life-threatening immune responses. However, the sub-cellular location of abacavir binding and the mechanics of altered peptide selection are not well understood. Here, we probed the impact of abacavir on the assembly of HLA-B*57:01 peptide complexes. We show that whilst abacavir had minimal impact on the maturation or average stability of HLA-B*57:01 molecules, abacavir was able to differentially enhance the formation, selectively decrease the dissociation, and alter tapasin loading dependency of certain HLA-B*57:01-peptide complexes. Our data reveals a spectrum of abacavir mediated effects on the immunopeptidome which reconciles the heterogeneous functional T cell data reported in the literature.

New genetic predictors for abacavir tolerance in HLA-B*57:01 positive individuals.

Abacavir hypersensitivity syndrome (ABC HSS) is strongly associated with carriage of human leukocyte antigen (HLA)-B*57:01, which has a 100% negative predictive value for the development of ABC HSS. However, 45% of individuals who carry HLA-B*57:01 can tolerate ABC. We investigated immune and non-immune related genes in ABC HSS (n = 95) and ABC tolerant (n = 43) HLA-B*57:01 + patients to determine other factors required for the development of ABC HSS. Assignment of phenotype showed that ABC HSS subjects were significantly less likely than tolerants to carry only ERAP1 hypoactive trimming allotypes (p = 0.02). An altered self-peptide repertoire model by which abacavir activates T cells is in keeping with observation that endoplasmic reticulum aminopeptidase 1 (ERAP1) allotypes that favour efficient peptide trimming are more common in ABC HSS patients compared to patients who tolerate ABC. Independently, non-specific immune activation via soluble cluster of differentiation antigen 14 (sCD14) may also influence susceptibility to ABC HSS.

Drug hypersensitivity in HIV infection.

PURPOSE OF REVIEW: Immune-mediated adverse drug reactions (IM-ADRs) are many times more common in HIV-infected patients. Usual offending drugs include antiretroviral and antiinfectives, but the burden of specific drug IM-ADRs is population-specific; changing as new and fixed dose combinations enter the market, and drug-resistance patterns demand. This review considers recent literature on epidemiology, mechanisms, clinical management and prevention of IM-ADRs amongst persons living with HIV/AIDS. RECENT FINDINGS: Epidemiological studies continue to describe high rates of delayed hypersensitivity to known offenders, as well as similar reactions in preexposure prophylaxis. IM-ADRs to oral and injectable integrase strand transfer inhibitors are reported with expanding use. The clinical spectrum and management of IM-ADRs occurring in HIV-infected populations is similar to uninfected; with exceptions such as a recently described severe delayed efavirenz DILI with high mortality. Furthermore, the context can be unique, such as the lower than expected mortality in a Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) cohort from a HIV/TB high burden setting. Programmatic data showing the near complete elimination of Abacavir drug hypersensitivity syndrome following implementation of HLA-B57:01 screening is a stellar example of how prevention is possible with mechanistic insight. SUMMARY: IM-ADRs remain a challenge in persons living with HIV. The complexities posed by polypharmacy, overlapping drug toxicities, drug interactions, overlap of IM-ADRs with other diseases, limited alternative drugs, and vulnerable patients with advanced immunosuppression with high mortality, necessitate increased use of drug provocation testing, treat-through and desensitization strategies. There is an urgent need for improved diagnostics and predictive biomarkers for prevention, or to guide treat-through, rechallenge and desensitization approaches.

The contribution of pharmacogenetics to pharmacovigilance.

Since the beginning of this century, information on pharmacogenetics appears in the summary of product characteristics (SPC) of drugs. Pharmacogenetic tests particularly concern the enzymes involved in the metabolism of drugs, among which P450 cytochromes. Some patients known as poor metabolisers eliminate some drugs more slowly, causing overdoses and adverse drug reactions (ADRs). The best-known examples are AVK and VKORC1-CYP2C9 or clopidogrel and CYP2C19. In the USA, the tests are recommended before the introduction of these drugs to prevent the occurrence of ADRs. Other tests are also commonly performed to address the toxicity of certain anticancer drugs (DPYD-capecitabine, UGT1A1-irinotecan, TPMT 6-mercaptopurine). Pharmacogenetic testing is also available to identify HLA loci that are very strongly associated with the occurrence of immuno-allergic reactions to a specific drug. The best-known example is HLA-B*5701, strongly associated with hypersensitivity to abacavir, and this test is now always prescribed before the instatement of this drug.

The importance of hapten-protein complex formation in the development of drug allergy.

PURPOSE OF REVIEW: Drug allergy is an adverse drug reaction that is immune-mediated. Immune activation can occur when drugs or haptens bind covalently to proteins and then act as antigens. The purpose of this review is to summarize the recent data on the formation of hapten-protein complexes and to assess the importance of these complexes in the generation of drug allergy. RECENT FINDINGS: The formation of hapten-protein complexes by drugs and their reactive metabolites has largely been investigated using model proteins such as human serum albumin. Precise identification of the structure of the hapten and the resulting modified residue(s) in the protein has been undertaken for a small number of drugs, such as p-phenylenediamine, nevirapine, carbamazepine, ß-lactams and abacavir. Some progress has also been made in identifying hapten-protein complexes in the serum of patients with allergy. SUMMARY: Drug-specific T cells have been isolated from different patients with allergy. Formation of hapten-protein complexes, their processing and antigen presentation have been implicated in the development of drug allergy to p-phenylenediamine, sulfonamides and ß-lactams. However, evidence also supports the pi mechanism of immune activation wherein drugs interact directly with immune receptors. Thus, multiple mechanisms of immune activation may occur for the same drug.

External quality assessment of patient HLA-B*57:01 testing prior to abacavir prescription.

Hypersensitivity reactions to the drug abacavir are strongly associated with possession of HLA-B*57:01. Hence, patients with HIV/AIDS who may be prescribed abacavir should be tested for this HLA allele and the drug withheld from those that possess B*57:01. The UK National External Quality Assessment Service for Histocompatibility and Immunogenetics has operated a scheme for B*57:01 testing since 2008 which, in 2013, involved 47 participants from 12 countries. A total of 24 B*57:01-positive, 2 B*57:03-positive and 22 B*57-negative blood samples (including 2 B*58 samples) were distributed to between 28 and 47 laboratories each year over 6 years. Participants, who were unaware of the samples' HLA types, tested and reported on their B*57/B*57:01 status. A total of 1868 reports were assessed over the 6 years. Of the 880 reports on B*57:01 samples, 93.4% were correctly assigned as B*57:01, 2.8% were assigned as groups of B*57 alleles including B*57:01, and 3.3% were reported as B*57 positive only. Over the 6 years, there were four (0.46%) false B*57:01 negative reports. All the B*57:03-positive and B*57-negative samples, involving 72 and 916 assignments, respectively, were essentially reported as B*57:01 negative. Thus, there were no false B57:01 positive assignments. The reporting of B*57:01 status over the last 3 years of the scheme was 99.8% sensitive and 100% specific. Over the last year, it was 100% sensitive and 100% specific.

Avidity determines T-cell reactivity in abacavir hypersensitivity.

The antiretroviral drug abacavir (abc) elicits severe drug hypersensitivity reactions in HLA-B*5701(+) individuals. To understand the abc-specific activation of CD8(+) T cells, we generated abc-specific T-cell clones (abc-TCCs). Abc reactivity could not be linked to the metabolism and/or processing of the drug, since abc metabolizing enzymes were not expressed in immune cells and inhibition of the proteasome in APCs did not affect TCC reactivity. Ca(2+) influx assays revealed different reactivity patterns of abc-TCCs. While all TCCs reacted to abc presented on HLA-B*5701 molecules, a minority also reacted immediately to abc in solution. Titration experiments showed that the ability to react immediately to abc correlated significantly with the TCR avidity of the T cells. Modifications of soluble abc concentrations revealed that the reactivity patterns of abc-TCCs were not fixed but dynamic. When TCCs with an intermediate TCR avidity were stimulated with increasing abc concentrations, they showed an accelerated activation kinetic. Thus, they reacted immediately to the drug, similar to the reaction of TCCs of high avidity. The observed immediate activation and the noninvolvement of the proteasome suggest that, in contrast to haptens, abc-specific T-cell stimulation does not require the formation of covalent bonds to produce a neo-antigenic determinant.

HLA alleles and drug hypersensitivity reactions.

The human leucocyte antigen (HLA) system is well known for its association with certain diseases such as ankylosing spondylitis, celiac disease and many others. More recently, severe and even fatal drug hypersensitivity reactions linked to particular HLA alleles have been discovered. The significance of these discoveries has led the European Medicines Agency (EMA) and its member state agencies to recommend HLA gene testing before initiation of drug treatment. To date, the following drugs have been identified as causing significant drug hypersensitivity reactions in patients who have the following HLA alleles: abacavir and HLA-B*57:01, carbamazepine and HLA-B*15:02/A*31:01 and finally allopurinol and HLA-B*58:01. This review will outline and discuss these three drugs and their associated HLA alleles as well as examine the pathogenesis of the drug hypersensitivity reactions.

The impact of pharmacogenetics in the treatment of allergic disease and asthma.

Personalized medicine includes the application of genomic information in predicting disease and therapeutic response to ultimately individualize patient care. Pharmacogenetics is key in achieving true personalized care. However, the clinical applicability of genetic testing to "everyday medicine" is yet to be realized. This paper will discuss areas in allergic/inflammatory disease that have been impacted by pharmacogenetic research and how this application may be brought from the "bench to the bedside."

[HLA-B*5701 and abacavir hypersensitivity reaction]. / Génotype HLA-B*5701 et hypersensibilité à l'abacavir.

A potentially life-threatening hypersensitive reaction occurs in association with initiation of HIV nucleoside analogue abacavir therapy in 4 to 8% of patients. Preliminary studies appear to confirm the role of the immune system in abacavir hypersensitivity. The reaction is possibly the result of presentation of drug peptides onto HLA, that may induce a pathogenic T-cell response. Hypersensitivity reaction to abacavir is strongly associated with the presence of the HLA-B*5701 allele and prospective HLA-B*5701 genetic screening has now been instituted in clinical practice to reduce the risk of hypersensitivity reaction.

HLA-B*5701 genetic screening prior to abacavir prescription in Georgia.

A hypersensitivity reaction to abacavir develops in approximately 2-8% of HIV patients receiving this drug and is strongly associated with presence of the human leukocyte antigen (HLA)-B*5701. Screening for HLA-B*5701 reduces the risk of developing an abacavir hypersensitivity reaction. The carriage rate of HLA-B*5701 has not been studied in Georgia before 2009. Objective of the study was to determine HLA-B*5701 prevalence in HIV-infected patients in Georgia. One hundred and sixty HIV positive patients attending Georgian Infectious Diseases, AIDS and Clinical Immunology Research Center in 2009 were recruited for the study. None of the patients had previously been treated with abacavir. Blood samples were collected and screened for HLA-B*5701 prior to abacavir prescription. Of 160 patients recruited 9 tested HLA B*5701 positive - 5.6% (95% CI: 2.6-10.4%). Of these nine patients 7 were males (male prevalence: 6.5%, 95% CI: 2.6-12.9 %) and 2 females (female prevalence: 4.8%, 95% CI: 0.6-16.2%). The first prospective study of HLA-B*5701 prevalence in Georgia show similar results to the results of other studies. Abacavir still remains one of the key drugs of antiretroviral regimens in Georgia and other countries. Therefore, prospective HLA-B*5701 screening should be implemented in all settings where abacavir is widely used to guide selection of ART regimens and to reduce the risk of potentially life threatening hypersensitivity reaction.

Human leukocyte antigen class I-restricted activation of CD8+ T cells provides the immunogenetic basis of a systemic drug hypersensitivity.

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

Abacavir: pretreatment screening: warning. Encouraging method but uncertainties remain.

HIV-infected patients carrying the HLA-B*5701 allele are at a significantly increased risk of developing hypersensitivity reactions to abacavir. It is therefore a good idea to identify these patients before initiating treatment. But even when patients test negative, hypersensitivity reactions are possible.

Immune responses to abacavir in antigen-presenting cells from hypersensitive patients.

OBJECTIVES: A potentially life-threatening hypersensitive reaction accompanies the use of HIV nucleoside analogue abacavir (ABC) in 4-8% of Caucasian individuals. HLA-B*5701 and Hsp70 493T alleles have been shown to predict susceptibility to this hypersensitivity. DESIGN AND METHODS: This study was undertaken to provide a mechanistic understanding of the highly significant genetic association of HLA Class I and Hsp70 alleles with ABC hypersensitivity. RESULTS: In this study an ABC-induced localization of intracellular HSP70 to endosomal vesicles of antigen-presenting cells was demonstrated. This ABC-stimulated redistribution of endogenous HSP70 was substantially higher in the genetically homogenous HLA-B*5701, Hsp70 493T ABC-hypersensitive individuals and ABC-naive individuals in comparison with the heterogeneous tolerant patients (P = 0.023). Increased expression of HSP70 was also detected in the hypersensitive group as measured by flow cytometry (P = 0.032). Blocking of HSP70 and HSP70 cell surface receptors CD14 and TLR2 abrogated ABC-stimulated HSP70 redistribution in sensitized individuals to basal levels (P < 0.004). In addition, the use of TcRalphabeta and HLA-B57/58 antibodies also ablated the expression of HSP70. Cells expressing the activation markers CD40 were increased after ABC stimulation in the hypersensitive patients (P = 0.006). ABC-stimulated interferon-gamma levels were higher in hypersensitive patients in comparison with ABC-tolerant individuals with a mean of 123.54 versus 0 pg/ml (P = 0.001). CONCLUSION: The present data indicates that ABC stimulates an innate immune response and activates antigen-presenting cells via the endogenous HSP70-mediated Toll-like receptor pathway in genetically susceptible individuals potentially initiating the immuno-pathological hypersensitive response.

Pharmacogenetic testing: proofs of principle and pharmacoeconomic implications.

Several proofs of principle have established that pharmacogenetic testing for mutations altering expression and functions of genes associated with drug disposition and response can decrease the "trial-and-error" dosing and reduce the risk of adverse drug reactions. These proofs of principle include thiopurine methyltransferase and thiopurine therapy, dihydropyrimidine dehydrogenase/thymidylate synthase and 5-fluorouracil therapy, folate enzyme MTHFR and methotrexate therapy, UGT1A1 and irinotecan therapy and CYP450 2C9 and S-warfarin therapy. These evidences advocate for the prospective identification of mutations associated with drug response, serious adverse reactions and treatment failure. More recent evidence with the HLA basis of hypersensitivity to the retroviral agent abacavir demonstrates the potential of pharmacogenetic testing and its pharmacoeconomic implications. With the convergence of rising drug costs and evidence supporting the clinical benefits of pharmacogenetic testing, it will be important to demonstrate the improved net health outcomes attributed to the additional costs for this testing.

Predisposition to abacavir hypersensitivity conferred by HLA-B*5701 and a haplotypic Hsp70-Hom variant.

Susceptibility to a clinically significant drug hypersensitivity syndrome associated with abacavir use seems to have a strong genetic component. We have previously shown that the presence of HLA-B*5701 strongly predicts abacavir hypersensitivity and have identified a potential susceptibility locus within a 300-kb region between the MEGT1 and C4A6 loci in the central MHC. We now report the results of fine recombinant genetic mapping in an expanded patient population of 248 consecutive, fully ascertained, abacavir-exposed individuals in the Western Australian HIV Cohort Study, in which 18 cases of definite abacavir hypersensitivity (7.3%) and 230 tolerant controls were identified. Haplotype mapping within patients with allelic markers of the 57.1 ancestral haplotype suggests a susceptibility locus within the 14-kb Hsp70 gene cluster. HLA-B*5701 was present in 94.4% of hypersensitive cases compared with 1.7% of controls (odds ratio, 960; P < 0.00001). A haplotypic nonsynonymous polymorphism of Hsp70-Hom (HspA1L, resulting from the substitution of residue M493T in the peptide-binding subunit) was found in combination with HLA-B*5701 in 94.4% of hypersensitive cases and 0.4% of controls (odds ratio, 3,893; P < 0.00001). Individuals with abacavir hypersensitivity demonstrated increased monocyte tumor necrosis factor expression in response to ex vivo abacavir stimulation, which was abrogated with CD8(+) T cell depletion. These data indicate that the concurrence of HLA-B*5701 and Hsp70-Hom M493T alleles is necessary for the development of abacavir hypersensitivity, which is likely to be mediated by an HLA-B*5701-restricted immune response to abacavir.