Proteomic Analysis of Antigen 60 Complex of M. bovis Bacillus Calmette-Guérin Reveals Presence of Extracellular Vesicle Proteins and Predicted Functional Interactions

Tuberculosis (TB) is ranked among the top 10 causes of death worldwide. New biomarker-based serodiagnostics and vaccines are unmet needs stalling disease control. Antigen 60 (A60) is a thermostable mycobacterial complex typically purified from Bacillus Calmette-Guérin (BCG) vaccine. A60 was historically evaluated for TB serodiagnostic and vaccine potential with variable findings. Despite containing immunogenic proteins, A60 has yet to be proteomically characterized. Here, commercial A60 was (1) trypsin-digested in-solution, analyzed by LC-MS/MS, searched against H37Rv and BCG Uniprot databases; (2) analyzed using STRING to predict protein-protein interactions; and (3) probed with anti-TB monoclonal antibodies and patient immunoglobulin G (IgG) on Western blot to evaluate antigenicity. We detected 778 proteins in two A60 samples (440 proteins shared), including DnaK, LprG, LpqH, and GroEL1/2, reportedly present in mycobacterial extracellular vesicles (EV). Of these, 107 were also reported in EVs of , and 27 key proteins had significant protein-protein interaction, with clustering for chaperonins, ribosomal proteins, and proteins for ligand transport (LpqH and LprG). On Western blot, 7/8 TB and 1/8 non-TB sera samples had reactivity against 37-50 kDa proteins, while LpqH, GroEL2, and PstS1 were strongly detected. In conclusion, A60 comprises numerous proteins, including EV proteins, with predicted biological interactions, which may have implications on biomarker and vaccine development.