Structural Elements Recognized by Abacavir-Induced T Cells.

Adverse drug reactions are one of the leading causes of morbidity and mortality in health care worldwide. Human leukocyte antigen (HLA) alleles have been strongly associated with drug hypersensitivities, and the causative drugs have been shown to stimulate specific T cells at the sites of autoimmune destruction. The structural elements recognized by drug-specific T cell receptors (TCRs) in vivo are poorly defined. Drug-stimulated T cells express TCRs specific for peptide/HLA complexes, but the characteristics of peptides (sequence, or endogenous or exogenous origin) presented in the context of small molecule drugs are not well studied. Using HLA-B*57:01 mediated hypersensitivity to abacavir as a model system, this study examines structural similarities of HLA presented peptides recognized by drug-specific TCRs. Using the crystal structure of HLA-B*57:01 complexed with abacavir and an immunogenic self peptide, VTTDIQVKV SPT5a 976-984, peptide side chains exhibiting flexibility and solvent exposure were identified as potential drug-specific T cell recognition motifs. Viral sequences with structural motifs similar to the immunogenic self peptide were identified. Abacavir-specific T cell clones were used to determine if virus peptides presented in the context of abacavir stimulate T cell responsiveness. An abacavir-specific T cell clone was stimulated by VTQQAQVRL, corresponding to HSV1/2 230-238, in the context of HLA-B*57:01. These data suggest the T cell polyclonal response to abacavir consists of multiple subsets, including T cells that recognize self peptide/HLA-B*57:01 complexes and crossreact with viral peptide/HLA-B*57:01 complexes due to similarity in TCR contact residues.

Oxypurinol directly and immediately activates the drug-specific T cells via the preferential use of HLA-B*58:01.

Allopurinol (ALP) hypersensitivity is a major cause of severe cutaneous adverse reactions and is strongly associated with the HLA-B*58:01 allele. However, it can occur in the absence of this allele with identical clinical manifestations. The immune mechanism of ALP-induced severe cutaneous adverse reactions is poorly understood, and the T cell-reactivity pattern in patients with or without the HLA-B*58:01 allele is not known. To understand the interactions among the drug, HLA, and TCR, we generated T cell lines that react to ALP or its metabolite oxypurinol (OXP) from HLA-B*58:01(+) and HLA-B*58:01(-) donors and assessed their reactivity. ALP/OXP-specific T cells reacted immediately to the addition of the drugs and bypassed intracellular Ag processing, which is consistent with the "pharmacological interaction with immune receptors" (p-i) concept. This direct activation occurred regardless of HLA-B*58:01 status. Although most OXP-specific T cells from HLA-B*58:01(+) donors were restricted by the HLA-B*58:01 molecule for drug recognition, ALP-specific T cells also were restricted to other MHC class I molecules. This can be explained by in silico docking data that suggest that OXP binds to the peptide-binding groove of HLA-B*58:01 with higher affinity. The ensuing T cell responses elicited by ALP or OXP were not limited to particular TCR Vß repertoires. We conclude that the drug-specific T cells are activated by OXP bound to HLA-B*58:01 through the p-i mechanism.

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.

The ADP-ribose-1''-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus 229E mediate resistance to antiviral interferon responses.

Several plus-strand RNA viruses encode proteins containing macrodomains. These domains possess ADP-ribose-1″-phosphatase (ADRP) activity and/or bind poly(ADP-ribose), poly(A) or poly(G). The relevance of these activities in the viral life cycle has not yet been resolved. Here, we report that genetically engineered mutants of severe acute respiratory syndrome coronavirus (SARS-CoV) and human coronavirus 229E (HCoV-229E) expressing ADRP-deficient macrodomains displayed an increased sensitivity to the antiviral effect of alpha interferon compared with their wild-type counterparts. The data suggest that macrodomain-associated ADRP activities may have a role in viral escape from the innate immune responses of the host.

Abacavir induced T cell reactivity from drug naïve individuals shares features of allo-immune responses.

Abacavir hypersensitivity is a severe hypersensitivity reaction which occurs exclusively in carriers of the HLA-B*57∶01 allele. In vitro culture of PBMC with abacavir results in the outgrowth of abacavir-reacting CD8+ T cells, which release IFNγ and are cytotoxic. How this immune response is induced and what is recognized by these T cells is still a matter of debate. We analyzed the conditions required to develop an abacavir-dependent T cell response in vitro. The abacavir reactivity was independent of co-stimulatory signals, as neither DC maturation nor release of inflammatory cytokines were observed upon abacavir exposure. Abacavir induced T cells arose in the absence of professional APC and stemmed from naïve and memory compartments. These features are reminiscent of allo-reactivity. Screening for allo-reactivity revealed that about 5% of generated T cell clones (n = 136) from three donors were allo-reactive exclusively to the related HLA-B*58∶01. The addition of peptides which can bind to the HLA-B*57∶01-abacavir complex and to HLA-B*58∶01 during the induction phase increased the proportion of HLA-B*58∶01 allo-reactive T cell clones from 5% to 42%. In conclusion, abacavir can alter the HLA-B*57∶01-peptide complex in a way that mimics an allo-allele ('altered self-allele') and create the potential for robust T cell responses.

Towards a coronavirus-based HIV multigene vaccine.

Human immunodeficiency virus (HIV) infection represents one of the major health threats in the developing world. The costly treatment of infected individuals with multiple highly efficient anti-HIV drugs is only affordable in industrialized countries. Thus, an efficient vaccination strategy is required to prevent the further spread of the infection. The molecular biology of coronaviruses and particular features of the human coronavirus 229E (HCoV 229E) indicate that HCoV 229E-based vaccine vectors can become a new class of highly efficient vaccines. First, the receptor of HCoV 229E, human aminopeptidase N (hAPN or CD13) is expressed mainly on human dendritic cells (DCs) and macrophages indicating that targeting of HCoV 229E-based vectors to professional antigen presenting cells can be achieved by receptor-mediated transduction. Second, HCoV 229E structural genes can be replaced by multiple transcriptional units encoding various antigens. These virus-like particles (VLPs) containing HCoV 229E-based vector RNA have the ability to transduce human DCs and to mediate heterologous gene expression in these cells. Finally, coronavirus infections are associated with mainly respiratory and enteric diseases, and natural transmission of coronaviruses occurs via mucosal surfaces. In humans, HCoV 229E causes common cold by infecting the upper respiratory tract. HCoV 229E infections are mainly encountered in children and re-infection occurs frequently in adults. It is thus most likely that pre-existing immunity against HCoV 229E will not significantly impact on the vaccination efficiency if HCoV 229E-based vectors are used in humans.

EDEM contributes to maintenance of protein folding efficiency and secretory capacity.

A stringent quality control process selects misfolded polypeptides generated in the endoplasmic reticulum (ER) for ER-associated degradation (ERAD). Here we assessed the maintenance of efficient glycoprotein folding in cells with defective ERAD caused by lack of adaptation of the intralumenal level of ER degradation-enhancing alpha-mannosidase-like protein (EDEM) to an increase in the ER cargo load. When these cells were converted into factories for production of high levels of human beta-secretase, maturation of this N-glycosylated aspartic protease progressed as in wild-type cells initially to gradually become less efficient. Up-regulation of EDEM to strengthen the ERAD machinery (but not up-regulation of calnexin to reinforce the folding machinery) was instrumental in maintaining folding efficiency and secretory capacity. Our data underscore the important role that the degradation machinery plays in maintaining a functional folding environment in the ER.