Integrated computational prediction and experimental validation identifies promiscuous T cell epitopes in the proteome of Mycobacterium bovis.

The discovery of novel antigens is an essential requirement in devising new diagnostics or vaccines for use in control programmes against human tuberculosis (TB) and bovine tuberculosis (bTB). Identification of potential epitopes recognised by CD4+ T cells requires prediction of peptide binding to MHC class-II, an obligatory prerequisite for T cell recognition. To comprehensively prioritise potential MHC-II-binding epitopes from Mycobacterium bovis, the agent of bTB and zoonotic TB in humans, we integrated three binding prediction methods with the M. bovisproteome using a subset of human HLA alleles to approximate the binding of epitope-containing peptides to the bovine MHC class II molecule BoLA-DRB3. Two parallel strategies were then applied to filter the resulting set of binders: identification of the top-scoring binders or clusters of binders. Our approach was tested experimentally by assessing the capacity of predicted promiscuous peptides to drive interferon-γ secretion from T cells of M. bovis infected cattle. Thus, 376 20-mer peptides, were synthesised (270 predicted epitopes, 94 random peptides with low predictive scores and 12 positive controls of known epitopes). The results of this validation demonstrated significant enrichment (>24 %) of promiscuously recognised peptides predicted in our selection strategies, compared with randomly selected peptides with low prediction scores. Our strategy offers a general approach to the identification of promiscuous epitopes tailored to target populations where there is limited knowledge of MHC allelic diversity.

Gene expression profiling and antigen mining of the tuberculin production strain Mycobacterium bovis AN5.

Control of bovine tuberculosis (bTB) relies on regular testing of cattle with a crude preparation of mycobacterial antigens termed purified protein derivative (PPD). Worldwide production of bovine PPD uses the Mycobacterium bovis AN5, a strain that was originally isolated circa 1948 in Great Britain (GB). Despite its worldwide use, the AN5 strain is poorly characterised. AN5 was adapted to grow on glycerol in a process similar to that used for the derivation of the BCG vaccine strains; during this process, it is known that BCG deleted the genes for some potent antigens. Our previous analysis of the genome of M. bovis AN5 showed that it had not suffered extensive gene deletion events during in vitro adaptation. However, glycerol adaptation of AN5 strain may have caused differences in its global gene expression profile that could affect antigen expression. To assess this, we determined the transcriptome profile of AN5 and compared it to expression data for two endemic GB strains of M. bovis that account for approximately 61% of all GB bTB cases. Genes expressed at lower levels in AN5 compared to M. bovis field isolates were then screened for antigenicity in naturally infected animals. Using this approach a number of genes were found to be expressed at lower levels in AN5, including those for known antigens. Our results show that field strains of M. bovis show some significant differences in gene expression to AN5, and that this differential gene expression may impact on the antigen profiles expressed by AN5 during in vitro culture.