Immunoproteomic analysis of cellular and adaptive responses in naïve and recovered TB patients following intra-lung re-challenge with live BCG

Tuberculosis disease (TB) remains a major global health challenge today, despite the availability of vaccine and an armoury of small molecule drugs. Notably, response rates to the century-old bacillus Calmette-Guerin (BCG) vaccine vary widely, for reasons that remain poorly understood. We have therefore developed a lung-oriented controlled human infection model of tuberculosis disease to better understand TB immunopathogenesis at the site of disease and across a spectrum of disease susceptibilities.

We recruited 99 healthy South African participants who had previously either single or multiple bouts of TB disease, a latent TB infection, or who appeared to have sterilising immunity. Live BCG, sterile PPD and saline were bronchoscopically instilled into separate lung segments (n = 65). A control group (n = 34) underwent a single bronchoscopy without challenge. Bronchoalveolar lavage (BAL) samples were collected at baseline and 3 days after re-challenge and analysed using SWATH-based proteomics, as well as using mycobacterial protein- and lipid-microarrays, in order to quantify cellular immune responses and anti-microbial antibody signatures as a function of challenge and disease susceptibility.

We quantified 4901 peptide IDs and 949 human protein IDs in the BAL samples through the SWATH-based analysis, with a data completeness of 79% across all samples. Differential expression was analysed in limma with Benjamin-Hochberg multiple testing correction, comparing baseline and 3 days post-rechallenge samples across the four disease susceptibility groups. We also quantified antigen-specific IgG and IgA titres in BAL and serum using custom mycobacterial protein NAPPA microarrays comprising 687 cell wall-associated antigens, as well as on home-made mycobacterial lipid microarrays.

We observed that immune responses were highly compartment specific (BAL vs. blood) and localized to the challenged lung segments. We also found that cellular and humoral responses increased in magnitude with increasing disease susceptibility, but that these were highly heterogeneous within a challenge- and susceptibility group. In particular, integration of our multi-omic proteomic and antibody data demonstrates the presence of previously unrecognised sub-groups within the clinically-defined latent TB infection group and points to significant mechanistic differences in the immune responses upon re-exposure to the pathogen between these sub-groups. More specifically, STRING analysis of our data identifies a co-ordinated antigen-specific IgA- and neutrophil-driven response at the site of disease in one LTBI sub-group that is likely to be protective but which is absent in in the other LTBI sub-group. Collectively, our integrated proteomic data therefore sheds important new light on the latent TB infection state, with implications amongst others for control of TB transmission, as well as for WHO modelling of elimination of TB disease by 2050.