Dissecting mechanisms of immunodominance to the common tuberculosis antigens ESAT-6, CFP10, Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ)
Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.The Journal of Immunology (Impact Factor: 4.92). 04/2012; 188(10):5020-31. DOI: 10.4049/jimmunol.1103556
Diagnosis of tuberculosis often relies on the ex vivo IFN-γ release assays QuantiFERON-TB Gold In-Tube and T-SPOT.TB. However, understanding of the immunological mechanisms underlying their diagnostic use is still incomplete. Accordingly, we investigated T cell responses for the TB Ags included in the these assays and other commonly studied Ags: early secreted antigenic target 6 kDa, culture filtrate protein 10 kDa, Rv2031c, Rv2654c, and Rv1038c. PBMC from latently infected individuals were tested in ex vivo ELISPOT assays with overlapping peptides spanning the entirety of these Ags. We found striking variations in prevalence and magnitude of ex vivo reactivity, with culture filtrate protein 10 kDa being most dominant, followed by early secreted antigenic target 6 kDa and Rv2654c being virtually inactive. Rv2031c and Rv1038c were associated with intermediate patterns of reactivity. Further studies showed that low reactivity was not due to lack of HLA binding peptides, and high reactivity was associated with recognition of a few discrete dominant antigenic regions. Different donors recognized the same core sequence in a given epitope. In some cases, the identified epitopes were restricted by a single specific common HLA molecule (selective restriction), whereas in other cases, promiscuous restriction of the same epitope by multiple HLA molecules was apparent. Definition of the specific restricting HLA allowed to produce tetrameric reagents and showed that epitope-specific T cells recognizing either selectively or promiscuously restricted epitopes were predominantly T effector memory. In conclusion, these results highlight the feasibility of more clearly defined TB diagnostic reagent.
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- "Sets of overlapping 15 or 16-mer peptides spanning various allergen and bacterial antigens were screened for immune reactivity as previously described (Oseroff et al., 2010; Arlehamn et al., 2012; Oseroff et al., 2012). Antigen specific cytokine production in donor peripheral-blood mononuclear cells (PBMC) was measured in dual or single ELISPOT assays. "
ABSTRACT: Computational prediction of HLA class II restricted T cell epitopes has great significance in many immunological studies including vaccine discovery. In recent years, prediction of HLA class II binding has improved significantly but a strategy to globally predict the most dominant epitopes has not been rigorously defined. Using human immunogenicity data associated with sets of 15-mer peptides overlapping by 10 residues spanning over 30 different allergens and bacterial antigens, and HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB), we optimized a strategy to predict the top epitopes recognized by human populations. The most effective strategy was to select peptides based on predicted median binding percentiles for a set of seven DRB1 and DRB3/4/5 alleles. These results were validated with predictions on a blind set of 15 new allergens and bacterial antigens. We found that the top 21% predicted peptides (based on the predicted binding to seven DRB1 and DRB3/4/5 alleles) were required to capture 50% of the immune response. This corresponded to an IEDB consensus percentile rank of 20.0, which could be used as a universal prediction threshold. Utilizing actual binding data (as opposed to predicted binding data) did not appreciably change the efficacy of global predictions, suggesting that the imperfect predictive capacity is not due to poor algorithm performance, but intrinsic limitations of HLA class II epitope prediction schema based on HLA binding in genetically diverse human populations. Copyright © 2015. Published by Elsevier B.V.
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- "Sets of overlapping 15 or 16mer peptides spanning various allergen and bacterial antigens were screened for immune reactivity as previously described    (Supplemental Table 1). Antigen specific cytokine production induced in donor PBMC was measured in dual ELISPOT assays. "
ABSTRACT: Computational prediction of HLA class II restricted T cell epitopes has great significance in many immunological studies including vaccine discovery. With the development of novel bioinformatics approaches, prediction of HLA class II binding has improved significantly but a strategy to predict the most dominant HLA class II epitopes has not been defined. Using different sets of peptides from various allergen and bacterial antigens and HLA class II binding prediction tools from the IEDB, we have designed a strategy to predict the top epitopes from any antigen. We found that the top 21% of 15-mer peptides overlapping by 10 residues (based on the predicted binding to seven DRB1 and DRB3/4/5 alleles) capture 50% of the immune response. This corresponded to an IEDB consensus percentile rank of 19.82 which could be used as a universal prediction threshold.
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- "The work described above developed reagents and approaches to broadly characterize human T-cell epitopes in the general human population (32, 33, 39, 41, 68), and characterized in detail the T-cell epitopes recognized in a panel of model TB antigens (50). Most importantly, using LTBI donor PBMCs, we performed the first truly genome-wide screen of ex vivo human CD4+ MTB T-cell reactivity. "
ABSTRACT: We have recently described the first true genome-wide screen for CD4(+) T-cell reactivity directed against Mycobacterium tuberculosis (MTB) in latent TB-infected individuals. The approach relied on predictions of HLA-binding capacity for a panel of DR, DP, and DQ alleles representative of those most commonly expressed in the general population, coupled with high throughput ELISPOT assays. The results identified hundreds of novel epitopes and antigens, and documented the novel observation that T cells in latent MTB infection are confined to the CXCR3(+)CCR6(+) phenotype and largely directed against three antigenic "islands" within the MTB genome. In parallel, we have made generally available to the scientific community the technical approaches and reagents developed in the process, such as motifs, algorithms, and binding assays for several common HLA class II alleles, and a panel of single allele HLA class II transfected cell lines representative of the most frequent specificities in the general population. Recent efforts have been focused on characterization of epitopes and antigens recognized by patients with active TB and individuals vaccinated with BCG, with the aim of providing the first systematic evaluation of the overlap between latent, active, and BCG cohorts. The definition of a broad range of epitopes restricted by common HLA molecules, will facilitate development of diagnostic reagents, allow a rigorous evaluation of T-cell responses associated with TB infection in humans, and enable the evaluation of the immunogenicity of different vaccine candidates. Furthermore, it might suggest new candidates for vaccine and diagnostic development.