A novel tuberculosis antigen identified from human tuberculosis granulomas

Mycobacterium tuberculosis (Mtb),1 the infectious agent that causes tuberculosis, is associated with an estimated 1.4 million deaths per year and remains a major global health concern (1). Current research and diagnostics have focused on antigen screening and biomarker discovery, with most antigen screening methods focused on the bacterial pathogen itself, with less focus on the Mtb infected host (2). The pathogenic progression of TB occurs in the lungs, making the characterization of any functional antigens existing in the lungs during infection potentially useful for immunotherapy or vaccine development. The immune response to an Mtb infection results in the formation of a granuloma that initially contains bacterial expansion, but may fail to eliminate the pathogen (3, 4). This immune response brings with the possibility of identifying Mtb functional antigens in the lung tissue and to gain a clearer understanding of the immune mechanisms (5, 6). Although it has been well studied that a T-cell mediated adaptive immune response plays a central role during Mtb infection and is crucial in both protection and pathogenesis, a better understanding of the antigen induced immune response and correlations to pathogenicity is necessary (2, 7). It has been reported that heat shock proteins (such as the HSP70 family members) and others chaperones such as Gp96 can specifically bind many hydrophobic sequences, enabling them to bind foreign peptides associated with intracellular bacterial or viral challenge (8), such as Gp96 associating with a HBV-specific peptide (9). Previous studies have shown that chaperone-peptide complexes can induce a disease-specific immune response (10–12), with the gp96-peptide complex from H37Rv infected cells able to induce a protective antigen specific immune response (13). Currently, no Mtb chaperone-associated peptides have been isolated directly from patients, thus the present study explores the possible existence of these complexes in TB lung tissue. To achieve this objective, the free-solution isoelectric focusing (FS-IEF) technique, which has been reported to enrich chaperones in cell lysates or tissue samples, was combined with Linear Trap Quadrupole (LTQ) OrbitrapVelos mass spectrometry, which was used to identify the associated Mtb peptides. Using these techniques, we obtained chaperone-rich cell lysates from the granulomatous lung lesions of active TB patients and identified six Mtb-associated peptides not noted in the control samples. Among them, a peptide (PKAp) derived from Mtb Protein Kinase A not only contributed to significant antigen-specific IFN-γ secretion, but also contributed to CTL function and T-cell proliferation. Importantly, murine immunization with PKAp derived peptides elicited an antigen-specific cellular activation without the occurrence of immune pathogenesis.