Abstract
Identification of CD8+ T cell antigens/epitopes expressed by human pathogens with large genomes is especially challenging, yet necessary for vaccine development. Immunity to tuberculosis, a leading cause of mortality worldwide, requires CD8+ T cell immunity, yet the repertoire of CD8 antigens/epitopes remains undefined. We used integrated computational and proteomic approaches to screen 10% of the Mycobacterium tuberculosis (Mtb) proteome for CD8 Mtb antigens. We designed a weighting schema based upon a Multiple Attribute Decision Making:framework to select 10% of the Mtb proteome with a high probability of containing CD8+ T cell epitopes. We created a synthetic peptide library consisting of 15-mers overlapping by 11 aa. Using the interferon-γ ELISPOT assay and Mtb-infected dendritic cells as antigen presenting cells, we screened Mtb-specific CD8+ T cell clones restricted by classical MHC class I molecules (MHC class Ia molecules), that were isolated from Mtb-infected humans, against this library. Three novel CD8 antigens were unambiguously identified: the EsxJ family (Rv1038c, Rv1197, Rv3620c, Rv2347c, Rv1792), PE9 (Rv1088), and PE_PGRS42 (Rv2487c). The epitopes are B5701-restricted EsxJ24–34, B3905-restricted PE953–67, and B3514-restricted PE_PGRS4248–56, respectively. The utility of peptide libraries in identifying unknown epitopes recognized by classically restricted CD8+ T cells was confirmed, which can be applied to other intracellular pathogens with large size genomes. In addition, we identified three novel Mtb epitopes/antigens that may be evaluated for inclusion in vaccines and/or diagnostics for tuberculosis.
Highlights
Effective vaccines for many important human pathogens have not yet been developed, and rational design of effective vaccines requires detailed knowledge of correlates of protective immunity
Summary of T cell clone screening results Classically restricted CD8+ T cell clones isolated from ten donors (LTBI, n = 4; active TB, n = 6) had been previously screened against peptide pools representing known CD4 antigens which represented the following proteins: 1) Esat-6, Rv3875; 2) CFP-10, Rv3874; 3) Ag85B, Rv1886c; 4) Mtb9.8, EsxG, Rv0287; 5) 19 kd lipoprotein, Rv3763; 6) TB8.4,Mtb8.4, Rv1174c; 7) Mtb39a, Rv1196; and 8) Mtb9.9, family of ESAT-6 like proteins comprised of EsxN, Rv1793; EsxO, Rv2346c; EsxI, Rv1037c; EsxV, Rv3619c; and EsxL, Rv1198
Mycobacterium tuberculosis (Mtb)-specific, classically restricted T cell clones that did not recognize any of these eight antigens (n = 14) isolated from donors with latent tuberculosis infection (LTBI) (D504) or active TB (D432, D466), as well as non-classically restricted T cell clones from these same donors (n = 33) were screened against the peptide library
Summary
Effective vaccines for many important human pathogens have not yet been developed, and rational design of effective vaccines requires detailed knowledge of correlates of protective immunity. Except perhaps for HIV [2], we do not have a comprehensive understanding of human T cell antigens. This is due in part to the large genome size of many human pathogens making production and screening of peptide libraries prohibitively expensive and impractical to perform. Elimination of TB worldwide will require a more effective vaccine, and this, in turn, will require a detailed knowledge of protective TB immunity
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