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.ancestral haplotype ͉ microsatellite ͉ human leukocyte antigen ͉ single-nucleotide polymorphism

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 therapy is associated with significant drug hypersensitivity in approximately 8% of recipients, with retrospective studies indicating a strong genetic association with the HLA-B*5701 allele. In this prospective study, involving 260 abacavir-naive individuals (7.7% of whom were positive for HLA-B*5701), we confirm the usefulness of genetic risk stratification, with no cases of abacavir hypersensitivity among 148 HLA-B*5701-negative recipients.

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Background: Vitamin E isomers may protect against atherosclerosis. The aim of this study was to compare the effects of supplementation with either ␣-tocopherol (␣T) or mixed tocopherols rich in ␥-tocopherol (␥T) on markers of oxidative stress and inflammation in patients with type 2 diabetes. Methods: In a double-blind, placebo-controlled trial, 55 patients with type 2 diabetes were randomly assigned to receive (500 mg/day) (a) ␣T, (b) mixed tocopherols, or (c) placebo for 6 weeks. Cellular tocopherols, plasma and urine F 2 -isoprostanes, erythrocyte antioxidant enzyme activities, plasma inflammatory markers, and ex vivo assessment of eicosanoid synthesis were analyzed preand postsupplementation. Results: Neutrophil ␣T and ␥T increased (both P <0.001) with mixed tocopherol supplementation, whereas ␣T (P <0.001) increased and ␥T decreased (P <0.005) after ␣T supplementation. Both ␣T and mixed tocopherol supplementation resulted in reduced plasma F 2 -isoprostanes (P <0.001 and P ‫؍‬ 0.001, respectively) but did not affect 24-h urinary F 2 -isoprostanes or erythrocyte antioxidant enzyme activities. Neither ␣T nor mixed tocopherol supplementation affected plasma C-reactive protein, interleukin 6, tumor necrosis factor-␣, or monocyte chemoattractant protein-1. Stimulated neutrophil leukotriene B 4 production decreased significantly in the mixed tocoph-

IL-10 inhibits the production of many pro-inflammatory cytokines. Polymorphisms in the IL10 gene promoter at positions -1082G-->A, -819C-->T and -592C-->A occur as three haplotypes, ATA, GCC and ACC. These influence several infectious and inflammatory diseases including community-acquired pneumonia, where a role for IL-10 is suggested by fluctuations in plasma levels of the cytokine. However, the effects of the haplotypes on IL-10 production are unclear. We stimulated peripheral blood mononuclear cells (PBMC) from at least five individuals homozygous for each of the three haplotypes with lipopolysaccharide (LPS, 10 microg/ml) or heat-killed Streptococcus pneumoniae (10(7)cfu/ml) and measured IL-10 mRNA by RT-PCR. Following S. pneumoniae stimulation, PBMC with the ATA haplotype had higher IL-10 mRNA levels than those with the GCC haplotype at 4 h (independent t-test; P=0.024), or the ACC haplotype at 4 h ( P<0.0001) and 8 h ( P=0.007). Following LPS stimulation, IL-10 mRNA levels were not significantly influenced by the IL10 haplotype, but similar trends were observed, consistent with the variable outcome of published studies. The results suggest that the -819 and/or -592 alleles affect transcription.

Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic “hypotheses” arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFNγ responses in 290 individuals from the same cohort which gave rise to 874 HLA-HIV associations in genetic analyses, taking into account autologous viral sequences and individual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this “reverse genomics” approach. Many HLA adapted epitopes elicited equivalent or higher magnitude IFNγ responses than the non-adapted epitopes, particularly in Nef. At a population level, inclusion of all the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We conclude that HLA-HIV associations do mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations as well as test, train and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA adapted variant responses may have negative effects on natural and vaccine immunity against HIV, and therefore has implications for diversity coverage approaches in HIV vaccine design.

HIV-1 mutations which reduce or abolish cytotoxic T lymphocyte responses against virus-infected cells are frequently selected in acute and chronic HIV-infection. Among population HIV-1 sequences, immune selection is evident as HLA allele-associated substitutions of amino acids within or near CD8 T cell epitopes. In these cases, the non-adapted epitope is susceptible to immune recognition until an escape mutation renders the epitope less immunogenic. However, several population-based studies have independently identified HLA-associated viral changes which lead to formation of a new T cell epitope, suggesting that the immune responses which these variants or “neo-epitopes” elicit provide an evolutionary advantage to the virus rather than the host. Here, we examined functional characteristics of eight CD8 T cell responses that result from viral adaptation in 125 HLA-genotyped individuals with chronic HIV-1 infection. Neo-epitopes included well-characterised immunodominant epitopes restricted by common HLA alleles and in most cases, the T cell responses against the neo-epitope exhibited significantly greater functional avidity and higher IFNγ production than T cells for non-adapted epitopes but were not more cytotoxic. Neo-epitope formation and emergence of the cognate T cell response co-incident with a rise in viral load was then observed in-vivo in an acutely infected individual. These findings demonstrate that HIV-1 adaptation not only abrogates immune recognition of early targeted epitopes, but may also increase immune recognition to other epitopes, which elicit immunodominant but non-protective T cell responses. These data have implications for immunodominance associated with polyvalent vaccines based on the diversity of chronic HIV-1 sequences.