Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically... Show moreWhereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use. Show less
Improved tuberculosis diagnostics and tools for monitoring treatment response are urgently needed. We developed a robust and simple, PCR-based host-blood transcriptomic signature, RISK6, for... Show moreImproved tuberculosis diagnostics and tools for monitoring treatment response are urgently needed. We developed a robust and simple, PCR-based host-blood transcriptomic signature, RISK6, for multiple applications: identifying individuals at risk of incident disease, as a screening test for subclinical or clinical tuberculosis, and for monitoring tuberculosis treatment. RISK6 utility was validated by blind prediction using quantitative real-time (qRT) PCR in seven independent cohorts. Prognostic performance significantly exceeded that of previous signatures discovered in the same cohort. Performance for diagnosing subclinical and clinical disease in HIV-uninfected and HIV-infected persons, assessed by area under the receiver-operating characteristic curve, exceeded 85%. As a screening test for tuberculosis, the sensitivity at 90% specificity met or approached the benchmarks set out in World Health Organization target product profiles for non-sputum-based tests. RISK6 scores correlated with lung immunopathology activity, measured by positron emission tomography, and tracked treatment response, demonstrating utility as treatment response biomarker, while predicting treatment failure prior to treatment initiation. Performance of the test in capillary blood samples collected by finger-prick was noninferior to venous blood collected in PAXgene tubes. These results support incorporation of RISK6 into rapid, capillary blood-based point-of-care PCR devices for prospective assessment in field studies. Show less
