Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population

RESEARCH ARTICLE Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Richard Baguma1☯, Adam Penn-Nicholson1☯*, Erica Smit1, Mzwandile Erasmus1, Jonathan Day1, Lebohang Makhethe1, Marwou de Kock1, E. Jane Hughes1, Michele van Rooyen1, Bernadette Pienaar1†, Lynnett Stone1, Willem Hanekom1, Michael J. Brennan2, Robert S. Wallis3, Mark Hatherill1, Thomas J. Scriba1 1 South African Tuberculosis Vaccine Initiative (SATVI), Institute of Infectious Disease and Molecular Medicine and Division of Immunology, Department of Pathology, University of Cape Town, Cape Town, South Africa, 2 Aeras, Rockville, Maryland, United States of America, 3 The Aurum Institute, Johannesburg, South Africa ☯ These authors contributed equally to this work. † Deceased. * OPEN ACCESS Citation: Baguma R, Penn-Nicholson A, Smit E, Erasmus M, Day J, Makhethe L, et al. (2017) Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population. PLoS ONE 12(9): e0184563. https://doi.org/10.1371/journal.pone.0184563 Editor: Pere-Joan Cardona, Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, SPAIN Received: June 20, 2017 Accepted: August 25, 2017 Published: September 8, 2017 Copyright: © 2017 Baguma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract The determinants of immunological protection against Mycobacterium tuberculosis (M.tb) infection in humans are not known. Mycobacterial growth inhibition assays have potential utility as in vitro surrogates of in vivo immunological control of M.tb. We evaluated a whole blood growth inhibition assay in a setting with high burden of TB and aimed to identify immune responses that correlate with control of mycobacterial growth. We hypothesized that individuals with underlying M.tb infection will exhibit greater M.tb growth inhibition than uninfected individuals and that children aged 4 to 12 years, an age during which TB incidence is curiously low, will also exhibit greater M.tb growth inhibition than adolescents or adults. Neither M.tb infection status, age of the study participants, nor M.tb strain was associated with differential control of mycobacterial growth. Abundance and function of innate or T cell responses were also not associated with mycobacterial growth. Our data suggest that this assay does not provide a useful measure of age-associated differential host control of M.tb infection in a high TB burden setting. We propose that universally high levels of mycobacterial sensitization (through environmental non-tuberculous mycobacteria and/or universal BCG vaccination) in persons from high TB burden settings may impart broad inhibition of mycobacterial growth, irrespective of M.tb infection status. This sensitization may mask the augmentative effects of mycobacterial sensitization on M.tb growth inhibition that is typical in low burden settings. Funding: An author of this publication (Scriba) received funding from the European and Developing Countries Clinical Trials Partnership (EDCTP) through a project entitled “Inflammatory determinants of risk of tuberculosis disease” (grant PLOS ONE | https://doi.org/10.1371/journal.pone.0184563 September 8, 2017 1 / 19 Mycobacterial growth inhibition in a TB endemic setting #TA.2011.40200.010). However, EDCTP cannot accept any responsibility for information or views expressed herein. This work was also supported by a grant from the US FDA (FDA grant #IU18FD004012-01, PI Brennan). Aeras provided support in the form of salaries for author MJB, but did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: Even though one author of this manuscript (MJB) was employed by Aeras, a nonprofit biotechnology organization developing new tuberculosis (TB) vaccines, there are no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Introduction Almost a quarter of the world’s population is estimated to be infected with Mycobacterium tuberculosis (M.tb) [1], driving the most deadly epidemic due to an infectious agent. In 2015, approximately 10.4 million people developed active tuberculosis (TB) disease, resulting in 1.4 million deaths [2]. Several risk factors for developing TB disease following M.tb infection have been identified, including compromised immunity due to HIV co-infection, diabetes, gender and age [3]. Pre-adolescent children older than 4 years of age have much lower rates of progression to TB disease following infection than adolescents or adults [4–7]. Furthermore, in such children pulmonary TB typically manifests as a mild, pauci-bacillary disease, whereas adolescents and adults more commonly present with multi-bacillary disease, more pronounced lung infiltration with immunopathology and lung cavitation [6,7]. The low risk of TB in children within this “golden age of TB” presents an opportunity to study natural resistance and/or characteristics of successful immunity to M.tb in humans, which are not well understood. The need for a TB vaccine that is more efficacious than Bacillus Calmette-Guérin (BCG) is urgent. Efforts to develop TB vaccines are hampered by the lack of reliable immunological correlates of protection or biomarkers that predict vaccine efficacy [8]. The adaptive immune response mediated by T cells is necessary for host control of M.tb infection [9]. As a consequence, frequencies of antigen-specific IFN-γ-producing and polyfunctional CD4 T cells coexpressing IFN-γ, TNF-α and IL-2 have been commonly measured markers of vaccine immunogenicity in preclinical and clinical testing of TB vaccine candidates. However, antigen-specific Th1 cells are not sufficient for complete protection in mouse models of TB [10,11]. In human infants, frequencies and cytokine co-expression profiles of BCG-specific CD4 and CD8 T cells did not correlate with risk of TB [12,13]. Efforts to screen and select the most promising novel vaccine candidates ideally require a functional assay that can measure immune-mediated control or killing of intracellular mycobacteria. A number of mycobacterial growth inhibition assays (MGIA) have been developed to measure functional inhibition of mycobacterial replication by blood leukocytes [14–21]. Animal studies in mice [22,23] (...truncated)