Tuberculous infections caused by mycobacteria, especially tuberculosis of humans and cattle, are important both clinically and economically. Human populations can be vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), and control measures for cattle involving vaccination are now being actively considered. However, diagnostic tests based on tuberculin cannot distinguish between genuine infection and vaccination with BCG. Therefore, identification of differential diagnostic antigens capable of making this distinction is required, and until now sequence-based approaches have been predominant. Here we explored the link between antigenicity and mRNA expression level, as well as the possibility that we may be able to detect differential antigens by analyzing quantified global transcriptional profiles. We generated a list of 14 candidate antigens that are highly expressed in Mycobacterium tuberculosis and M. bovis under a variety of growth conditions. These candidates were screened in M. bovis-infected and naïve cattle for the ability to stimulate a gamma interferon (IFN-␥) response. We identified one antigen, Rv3615c, which stimulated IFN-␥ responses in a significant proportion of M. bovis-infected cattle (11 of 30 cattle [37%] [P < 0.01]) but not in naïve or BCG-vaccinated animals. Importantly, the same antigen stimulated IFN-␥ responses in a significant proportion of infected cattle that did not respond to the well-characterized mycobacterial antigens ESAT-6 and CFP-10. Therefore, use of the Rv3615c epitope in combination with previously described differential tests based on ESAT-6 and CFP-10 has the potential to significantly increase diagnostic sensitivity without reducing specificity in BCG-vaccinated populations.

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In order to identify cytokines that may be useful as candidates for inclusion in diagnostic tests for Mycobacterium bovis infection in cattle, we compared the levels of gamma interferon (IFN-␥), interleukin 1␤ (IL-1␤), IL-4, IL-10, IL-12, macrophage inflammatory protein 1␤ (MIP-1␤), and tumor necrosis factor alpha (TNF-␣) in whole-blood cultures from tuberculosis (TB) reactor animals or TB-free controls following stimulation with M. bovis-specific antigens (purified protein derivative from M. bovis [PPD-B] or ESAT-6/CFP-10). In addition to IFN-␥ responses, the production of IL-1␤ and TNF-␣ was also statistically significantly elevated in TB reactor cattle over that in uninfected controls following stimulation with PPD-B or ESAT-6/CFP-10 peptides. Thus, we evaluated whether the use of these two additional readouts could disclose further animals not detected by measuring IFN-␥ alone. To this end, receiver operating characteristic (ROC) analyses were performed to define diagnostic cutoffs for positivity for TNF-␣ and IL-1␤. These results revealed that for ESAT-6/CFP-10-induced responses, the use of all three readouts (IFN-␥, TNF-␣, and IL-1␤) in parallel increased the sensitivity of detection of M. bovis-infected animals by 11% but also resulted in a specificity decrease of 14%. However, applying only IFN-␥ and IL-1␤ in parallel resulted in a 5% increase in sensitivity without the corresponding loss of specificity. The results for PPD-B-induced responses were similar, although the loss of specificity was more pronounced, even when only IFN-␥ and IL-1␤ were used as readout systems.In conclusion, we have demonstrated that the use of an additional readout system, such as IL-1␤, can potentially complement IFN-␥ by increasing overall test sensitivity for the detection of M. bovis infection in cattle.

Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM) and trehalose monomycolate (TMM), the apolar phthiocerol dimycocersates (PDIMs), triacyl glycerol (TAG), pentacyl trehalose (PAT), phenolic glycolipid (PGL), and mono-mycolyl glycerol (MMG). Polar lipids identified included glucose monomycolate (GMM), diphosphatidyl glycerol (DPG), phenylethanolamine (PE) and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs). These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1β, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.

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.