Immune profiling in M. tuberculosis infection enables stratification of patients with active disease

Abstract

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) infection and is a major public health problem with an estimated 1.7 billion persons infected worldwide. Clinical challenges in TB include the lack of a blood-based test for active disease, and the absence of prognostic biomarkers for early treatment response. Current blood based tests, such as QuantiFERON-TB Gold (QFT), are based on an IFNγ readout following Mtb antigen stimulation. However, they do not distinguish active TB disease from asymptomatic Mtb infection. We hypothesized that the use of TruCulture, an improved immunomonitoring method for whole blood collection and immune stimulation, could improve the discrimination of active disease from latent Mtb infection. To test our hypothesis, we stimulated whole blood from active TB patients (before and after successful treatment), comparing them to asymptomatic latently infected individuals. Mtb-specific antigens (ESAT-6, CFP-10, TB7.7) and live bacillus Calmette-Guerin (BCG) were used for TruCulture stimulation conditions, with direct comparison to QFT. Protein analyses were performed on the culture supernatants using ELISA and Luminex multi-analyte profiling. TruCulture showed an ability to discriminate active TB cases from latent controls (p < 0.0001, AUC = 0.81, 95% CI: 0.69-0.93) as compared to QFT (p = 0.47 AUC = 0.56, 95% CI: 0.40-0.72), based on an IFNγ readout after Mtb antigen stimulation. The stratification of the two groups could be further improved by using the Mtb Ag/BCG IFNγ ratio response (p < 0.0001, AUC = 0.918, 95% CI: 0.84-0.98). We also identified additional cytokines that distinguished latent infection from TB disease; and show that the primary differences between the TruCulture and QFT systems were a result of higher levels of non-specific innate immune activation in QFT tubes, due to the lack of a buffering solution in the latter. We conclude that TruCulture offers a next-generation solution for whole blood stimulation and immunomonitoring with the possibility to discriminate active and latently infected persons.

Tuberculosis alters immune-metabolic pathways resulting in perturbed IL-1 responses

“…We and others have reported distinct secretion of IL-1 agonists and antagonists upon M.tb infection 8,10,62 , however, whether IL-1 signalling itself is impaired in TB disease remains poorly explored. Here, we showed perturbed IL-1β signalling in TB patients, with higher expression of most IL-1β-induced genes in TB compared to LTBI.…”

“…We and others have reported distinct secretion of IL-1 agonists and antagonists upon M.tb infection 8,10,62 , however, whether IL-1 signalling itself is impaired in TB preprint (which was not certified by peer review) is the author/funder. All rights reserved.…”

“…Adult patients with sputum Xpert MTB/RIF-positive TB disease who tested HIV-negative were identified and recruited at clinics in Worcester, South Africa 62 . Healthy QuantiFERON-TB Gold (QFT) positive ( M.tb- infected) adults were recruited from communities living in or around Worcester and those who matched TB cases by age and gender were enrolled as LTBI controls (Table 1).…”

“…TruCulture tubes were prepared in batch as previously described 14,15,62 with the following stimuli: QFT antigens (ESAT-6, CFP-10, TB7.7), Bacillus Calmette– Guérin (BCG; Sanofi Pasteur, 10 5 units/ml), and IL-1β (Peprotec, 25 ng/ml). They were resuspended in 2 ml of buffered media and maintained at −20°C until use.…”