Idiosyncratic adverse drug reactions are unpredictable, dose-independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. ...Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkages between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8 ⁺ T cells that required HLA-B*57:01 molecules for their function; however, the mechanism by which abacavir induces this pathologic T-cell response remains unclear. Here we show that abacavir can bind within the F pocket of the peptide-binding groove of HLA-B*57:01, thereby altering its specificity. This provides an explanation for HLA-linked idiosyncratic adverse drug reactions, namely that drugs can alter the repertoire of self-peptides presented to T cells, thus causing the equivalent of an alloreactive T-cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir and that were recognized by T cells of hypersensitive patients. The assays that we have established can be applied to test additional compounds with suspected HLA-linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA-linked hypersensitivities, and guide the development of safer drugs.
T cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals ...that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result.
To address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at http://epitope.liai.org:8080/tools/population.
We have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine), and also minimizing the variability of coverage obtained or projected in different ethnic groups.
This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC ...is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands, and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size-exclusion gel-filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained.
Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of ...interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.
We have analyzed by ex vivo ELISPOT the anti-vaccinia cytotoxic T lymphocyte responses of peripheral blood mononuclear cells from humans vaccinated with Dryvax vaccine. More than 6,000 peptides from ...258 putative vaccinia ORFs predicted to bind the common molecules of the HLA A1, A2, A3, A24, B7, and B44 supertypes were screened with peripheral blood mononuclear cells of 31 vaccinees. A total of 48 epitopes derived from 35 different vaccinia antigens were identified, some of which (B8R, D1R, D5R, C10L, C19L, C7L, F12, and O1L) were recognized by multiple donors and contain multiple epitopes recognized in the context of different HLA types. The antigens recognized tend to be > 100 residues in length and are expressed predominantly in the early phases of infection, although some late antigens were also recognized. Viral genome regulation and virulence factor were recognized most frequently, whereas few structural proteins were immunogenic. Finally, most epitopes were highly conserved among vaccinia virus Western Reserve, variola major and modified vaccinia Ankara, supporting their potential use in vaccine and diagnostic applications.
The recent explosion in genomic sequencing has made available a wealth of data that can now be analyzed to identify protein antigens, potential targets for vaccine development. Here we present, in ...the context of Plasmodium falciparum, a strategy that rapidly identifies target antigens from large and complex genomes. Sixteen antigenic proteins recognized by volunteers immunized with radiation-attenuated P. falciparum sporozoites, but not by mock immunized controls, were identified. Several of these were more antigenic than previously identified and well characterized P. falciparum-derived protein antigens. The data suggest that immune responses to Plasmodium are dispersed on a relatively large number of parasite antigens. These studies have implications for our understanding of immunodominance and breadth of responses to complex pathogens.
The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. ...The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.
Abstract Influenza virus remains a significant health concern, with current circulating strains that affect millions each year plus the threat of newly emerging strains, such as swine-origin H1N1 and ...avian H5N1. Our hypothesis is that influenza-derived HLA-class I–restricted epitopes can be identified for use as a reagent to monitor and quantitate human CD8+ T-cell responses and for vaccine development to induce protective cellular immunity. Protein sequences from influenza A virus strains currently in circulation, agents of past pandemics and zoonotic infections of man were evaluated for sequences predicted to bind to alleles representative of the most frequent HLA-A and -B (class I) types worldwide. Peptides that bound several different HLA molecules and were conserved among diverse influenza subtypes were tested for their capacity to recall influenza-specific immune responses using human donor PBMC. Accordingly, 28 different epitopes antigenic for human donor PBMC were identified and 25 were 100% conserved in the newly emerged swine-origin H1N1 strain. The epitope set defined herein should provide a reagent applicable to quantitate CD8+ T cell human responses irrespective of influenza subtype and HLA composition of the responding population. In addition, these epitopes may be suitable for vaccine applications directed at the induction of cellular immunity.
The purpose of this study was to immunize patients with HER-2/neu-overexpressing cancer with a multipeptide vaccine comprised of four class II HER-2/neu peptides that had been identified as the most ...immunogenic in a previous clinical trial. Furthermore, we questioned whether MHC binding affinity could predict the in vivo immunogenicity of the HER-2/neu helper peptides.
Four putative class II HER-2/neu peptides, which were found to generate detectable specific T-cell responses (stimulation index > 2) in a majority of patients in a previous study, were used to formulate a single vaccine. The multipeptide vaccine was administered intradermally with granulocyte macrophage colony-stimulating factor as an adjuvant. Ten patients with HER-2/neu overexpressing breast or lung cancer were enrolled. HER-2/neu peptide-and protein-specific T cell and antibody immune responses were measured. Competitive inhibition assays were used to analyze the class II HER-2/neu peptides for their binding affinity to 14 common HLA-DR alleles.
Twenty-five percent of patients developed HER-2/neu peptide-specific T-cell immunity, and 50% developed HER-2/neu peptide-specific antibody immunity. No patient developed HER-2/neu protein-specific T cell or antibody immunity. The majority of peptides exhibited high binding affinity, in vitro, to >/==" BORDER="0">3 of the 14 DR alleles analyzed.
The group of peptides used in this study demonstrated high binding affinity to multiple DR alleles suggesting that in vitro binding affinity may be able to predict the in vivo immunogenicity of class II peptides. However, only a minority of patients immunized with the multipeptide vaccine developed HER-2/neu peptide-specific T cell or antibody immunity, and none developed HER-2/neu protein-specific immunity.
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