AbstractsBiology & Animal Science

Detection andcharacterization of lipopeptide-specific T cells in guinea pigssensitized with bacteria of the Mycobacterium tuberculosiscomplex

by Eva Kaufmann

Institution: Universität Giessen
Year: 2016
Posted: 02/05/2017
Record ID: 2124427
Full text PDF: http://geb.uni-giessen.de/geb/volltexte/2016/12103


The only available vaccine against TB – the Bacille Calmette-Guérin (BCG) vaccine – provides reliable protection against severe manifestations of childhood TB, but not against pulmonary tuberculosis in adults. Despite many attempts, no improved alternative TB vaccine has been fully licensed so far. It may be crucial for future vaccine development to gain further knowledge on the stimulatory antigens of this vaccine strain and their elicited immune responses. The aim of the current study was to investigate the BCG-mediated adaptive immune responses directed against lipid preparations (chloroform-methanol extracts, CMEs) of bacteria of the Mycobacterium tuberculosis complex (MTC). To this end, the BCG-sensitized guinea pig was employed as a model for human BCG vaccination. In particular, the present study addressed whether lipopeptides in the CME preparations constitute relevant stimulatory antigens for adaptive cellular immune responses upon BCG sensitization and whether the expression of stimulatory lipid antigens varies across the genus Mycobacterium. It was further attempted to identify the molecular nature of the stimulatory antigens present in CME. Outbred Dunkin-Hartley guinea pigs were sensitized with BCG or, for comparison purposes, with heat-inactivated wet mass of the virulent M. bovis strain AN5. Ex vivo, the proliferation of peripheral blood mononuclear cells (PBMCs) was investigated in a CFSE-based proliferation assay which was subsequently analyzed by flow cytometry. In this assay, CMEs of M. bovis BCG and M. tuberculosis H37Rv and, in addition, a Koch’s “Old Tuberculin” standard were used as antigenic stimuli. The molecular nature of the relevant stimulatory antigens within these preparations was investigated by means of delipidation and protease treatment. Furthermore, the stimulatory potentials of a lipopeptide-enriched subfraction of CME(H37Rv) (LppEL), of a defined mycobacterial protein antigen (AG85A), and of different BCG preparations (live or heat-inactivated bacteria, bacterial lysates derived from BCG cultures in the exponential as well as in the static growth phase, and culture supernatant) were tested for their stimulatory potentials in the ex vivo lymphocyte proliferation assay. A restimulation assay based on the antigen-specific expansion of ex vivo lymphocytes, the sorting for expanded lymphocytes and the restimulation of these cells, was established in this study and served subsequently for determination of the antigen-specificity of the stimulated lymphocytes as well as for investigating the distribution of the stimulatory antigens among the tested preparations (Tuberculin, CME(BCG), CME(H37Rv), LppEL). The distribution of the stimulatory antigens was further analyzed among the genus Mycobacterium using CMEs of several MTC strains, including clinical ones, (M. bovis AN5, M. tuberculosis CDC 1551, Haarlem 2336, HN 878, Beijing 1934, East African-Indian strains 1797 and 91/0079, M. canetti) and of non-tuberculous mycobacteria (NTM) (M. marinum, M. avium ssp. avium and M. avium ssp.…