Abstract
Tuberculosis is a global infectious disease caused by Mycobacterium tuberculosis (Mtb). Although novel Mtb biomarkers from both the pathogen and host have been studied, more breakthroughs are still needed to meet different clinic requirements. In an effort to identify Mtb antigens, chaperone-peptide complexes were purified from TB infected lungs using free-solution isoelectric focusing combined with high resolution LTQ Orbitrap Velos mass spectrometry. Antigen specific cellular immune responses in vitro were then examined. Those efforts led to the identification of six Mtb peptides only identified in Tuberculosis lung samples and that were not found in the control samples. Additionally, antigen-specific IFN-γ secretion, T-cell proliferation, cytokine expression, and a cytotoxic assay were also evaluated. Among the peptides isolated, we identified a 34 amino acid peptide named PKAp belonging to a serine/threonine-protein kinase, as being able to generate Mtb-specific cellular immune responses as noted by elevated antigen-specific cytokine secretion levels, increased CD8(+) T-cell proliferation and a strong cytotoxic lymphocyte (CTL) response. Moreover, the immune stimulating abilities of PKAp were further validated in vivo, with target peptide immunized mice showing an increased cellular IFN-γ in both the lungs and spleen without causing immunopathogenesis. In conclusion, we identified novel functional Mtb antigens directly from the granulomatous lesions of Tuberculosis patients, inducing not only significant antigen-specific IFN-γ secretion but also a marked cytotoxic lymphocyte functional response. These findings indicated that PKAp has potential as a novel antigen biomarker for vaccine development.
Highlights
From the ‡MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100176, China; §Beijing Tuberculosis and Thoracic Tumor Research Institute, 101149, China; ¶Guangdong Key Laboratory for emerging infectious diseases, Shenzhen Key Laboratory of Infection and Immunity, Shenzhen Third People’s Hospital, Guangdong Medical College, Shenzhen, 518020, China; ʈDepartment of Neurosurgery, University of Colorado, Denver, Colorado 80045
Mycobacterium tuberculosis (Mtb) Specific Chaperone (Hsp70/Gp96)-associated Peptides Identified in Lung Tissues—Clinical pulmonary TB samples, along with control lung adenocarcinoma and pulmonary fungal infection samples (Table I), were lysed to verify the presence of Hsp70 and Gp96 (Fig. 1A)
A proper preventive or therapeutic vaccine is urgently needed for TB control, with BCG being the only vaccine in clinic use and its protective efficacy controversial
Summary
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]. No Mtb chaperone-associated peptides have been isolated directly from patients, the present study explores the possible existence of these complexes in TB lung tissue
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