Rajina ShakyaDepartment of Toxicology

• Nucleoside analogue• Potent anti-viral activity against HIV • Main side-effcet : Hypersensitivity in-

clude fever, rash, gastrointestinal symptoms (nausea, vomiting, diarrhea, or abdominal pain), and lethargy or malaise.

ABACAVIR

HYPERSENSITIVITY

Set of undesirable reactions produced by the normal immune system, including allergies and autoimmunity. These reactions may be damaging, uncomfortable, or oc-casionally fatal.

Approximately, 5–9% of patients treated with abacavir developed hyper-sensitivity reaction. Symptoms usually appear within the first 6 weeks of treatment and worsen with continued ther-

apy and improve within 72 h of abacavir discontinuation. Rechallenging, with abacavir after a hypersensitivity reaction typically results in recurrence of

symptoms within hours, with the potential to induce a more severe clinical syndrome.

ABACAVIR HYPERSENSITIVITY

• The susceptibility loci associated with abacavir hypersensitivity was further investigated by us-ing recombinant AH mapping, within the central non-human leukocyte antigen (HLA) region of the MHC.

• Combination of HLA-B*5701 and a haplotypic polymorphism of Hsp70- Hom is highly predic-tive of abacavir hypersensitivity, which provides the mechanistic basis of the hypersensitivity syndrome.

MATERIALS AND METHODS• Retrospective study N=200• Prospective study N=48

Demographic data:

Origin Total Patients (N=248)European or Asain Indian

Descent214

African 11

Austratians 15

Asian 8

Cases Observed patients

Abacavir Hypersensitive 18

Abacavir Hypersensitive not excluded

15

Abacavir tolerant individ-ual

215

CLASSIFICATION OF HYPERSENSITIVITY CASES

PATCH TEST

Cases Case ob-served

Patch test

Abacavir HypersensitiveW 57.1 AH 14 9

W/o 57.1 AH 4 3

Abacavir Hypersensitive not excluded15 3

Abacavir tolerant individual (W57.1 AH)HLA-B*5701 alleles 2

Full 57.1AH(HLA-B*5701, C4A6, AND HLA-DRB1*0701,HLA-DQ3)

2

• 24 and 48 h of application• Absence of an allergic response for positive results

Typing of MHC Markers and Assignment of Alleles Specific to the 57.1 AH

• Carried out by using standard genetic assays and sequencing. • Single-nucleotide polymorphisms (SNPs) within genes located in the C4A6–MEGT1 region

were examined by using the SNP database.• Primers were designed based on oligonucleotide sequences reported within the database. • A set of well characterized Epstein–Barr virus-transformed homozygous B cell lines represent-

ing commonly occurring European AHs was used to determine alleles carried on the 57.1 AH. • Full-length sequencing across the Hsp70 gene cluster was performed by using overlapping

primer pairs.

Measurement of the TNF Response to Abacavir

• Intracellular expression of TNF was assessed in cultured peripheral blood mononuclear cells (PBMCs) grown in the presence and absence of abacavir(Ziagen 300-mg).

• The PBMCs were stained with CD45-ECD, CD14-FITC, or IgG1- FITC antibodies and ana-lyzed on flow cytometer.

CD4 and CD8 Cell Depletion. • TNF levels were measured in supernatants of 1ml of whole blood cultured in the absence and

presence of abacavir (1 g/ml) for 24 h at 37°C. • Whole blood was depleted of either CD4 or CD8 T cells.

RESULTS200 participants from retrospec-

tive study

Clinically Abacavir Hypersensi-tivity case N=18

N=3 (receiving nevirapine therapy) : negative re-sponse to patch test after 48 hrs of application

N=2 rechallenged successfully with abacavir w/o developing ABC hypersensitivity

Meanwhile, third patient developed similar hyper-sensitivity reaction to efavirenz

RESULTSAnother retrospective cohort case “abacavir hypersensitivity

not excluded”

Typical and severe symptoms

Received clinical diagnosis within a few days of exposure to abacavir and received intensive supportive inpatient

care at a hospital

No symptoms were identified

Also, Individual more likely to be desensitized by the contin-ued administration of abacavir,Negative response to epicu-

taneous patch testing

Mapping of Putative Susceptibility Loci Within the MHC

• Markers of 57.1 AH, within the central MHC region, between C4A6 and MEGT1were identified.• Hsp70 cluster between MEGT1 and snRNP was compared with the sequence of 8.1, 7.1, and 18.2 AHs but

nonsynonymous substitutions was not identified.• The only nucleotide substitution in the Hsp70- Hom gene identified in most of the abacavir-hypersensitive

cases involved a T to C transition, which results in a change from methionine to threonine at amino acid residue.

• The Hsp70-Hom M493T allele on 57.1 AH, was detected to be 94.4% of the hypersensitive group compared with 22.2% of tolerant controls (17 of 18 individuals vs. 51 of 230 individuals, OR 59.7, Pc 0.00001).

• In combination with HLA-B*5701, the Hsp70-Hom M493T allele was strongly associated with abacavir hy-persensitivity.

Expression of Inflammatory Cytokine TNF in Abacavir-Hypersensitive Individuals

• Cytokine response was examined by measuring intracellular TNF levels in cultures of PBMCs from abacavir-hypersensitive, -tolerant, and unexposed HIV-positive patients.

• The proportion of TNF-positive cells was higher in patients with abacavir hypersensitivity (n =8) than in abacavir-tolerant controls (n=9) compared with decrease in tolerant controls.

• The three individuals classified as abacavir-tolerant on the basis of a negative epicutaneous patch test also had no increase in TNF-positive cells in response to abacavir stimulation.

Phenotype of T Cells Expressing TNF After Abacavir Stimulation• Further phenotypic characterization of the T cell response to abacavir as carried out on whole

blood cultures of two patients who were selected for analysis because they both carried the pre-dictive HLA-B*5701 and Hsp70-Hom M493T alleles yet were divergent in their clinical re-sponses to abacavir.

• Definite abacavir hypersensitivity occurred in case 17 within 1 week of drug exposure, whereas case 21 tolerated this drug without any clinical symptoms suggestive of hypersensitivity.

• Involvement of CD4 and CD8 T cells in the secretion of TNF in culture supernatants was deter-mined after stimulation with abacavir.

• Extracellular levels of TNF were higher in abacavir-stimulated whole blood cultures of the aba-cavir-hypersensitive individual compared with those of the abacavir-tolerant control.

• TNF levels were attenuated when CD8 T cells were depleted, compared with undepleted or CD4 T cell-depleted cultures.

DISCUSSION

• Recombinant haplotype mapping was used to confirm a strong genetic association between the 57.1 AH and definite abacavir hypersensitivity, with further evidence that the concurrence of the HLA-B*5701 allele and a haplotypic variant of the Hsp70-Hom allele represents a highly predictive marker of susceptibility.

• Patients with a higher CD8 T cell count (850 cells) at the time of exposure to aba-cavir have an increased risk of developing hypersensitivity.

• Lack of sufficient sensitivity when immunological response was measured for rechal-lenging a patient with abacavir in cases where a previous hypersensitivity reaction is suspected.

Discussion

• Abacavir or its metabolites may be involved in the haptenation of endogenous pep-tides and subsequent presentation of ‘‘altered self’’ in the context of HLA-B*5701, thus inducing vigorous T cell responses.

• Hsp70 plays a direct role in the selection of HLA-B*5701-restricted peptide sub-strates that are potentially haptenated through an abacavir-dependent mechanism.

Conclusion

• The HLA-B*5701 and Hsp70- Hom M493T alleles as highly predictive genetic mark-ers of susceptibility to abacavir hypersensitivity.

• These findings have significant implications in the clinical management of abacavir exposed HIV-infected patients.

• Also, it elucidate basic pathophysiological mechanisms underlying this and other idiosyncratic drug hypersensitivity reactions.