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Tuberculosis (TB) is one of the leading causes of death worldwide, especially in developing countries. Neonatal BCG vaccination occurs in various regions, but the level of protection varies in different populations. Recently, Mycobacterium vaccae is found to be an immunomodulating therapeutic agent that could confer a significant level of protection against TB. It is the only vaccine in a phase III trial from WHO to assess its efficacy and safety in preventing TB disease in people with latent TB infection. However, the mechanism of immunotherapy of M. vaccae remains poorly understood. In this study, the full genome of M. vaccae was obtained by next-generation sequencing technology, and a proteogenomic approach was successfully applied to further perform genome annotation using high resolution and high accuracy MS data. A total of 3,387 proteins (22,508 unique peptides) were identified, and 581 proteins annotated as hypothetical proteins in the genome database were confirmed. Furthermore, 38 novel protein products not annotated at the genome level were detected and validated. Additionally, the translational start sites of 445 proteins were confirmed, and 98 proteins were validated through extension of their translational start sites based on N terminus-derived peptides. The physicochemical characteristics of the identified proteins were determined. Thirty-five immunogenic proteins of M. vaccae were identified by immunoproteomic analysis, and 20 of them were selected to be expressed and validated by Western blot for immunoreactivity to serum from patients infected with M. tuberculosis. The results revealed that eight of them showed strong specific reactive signals on the immunoblots. Furthermore, cellular immune response was further examined and one protein displayed a higher cellular immune level in pulmonary TB patients. Twelve identified immunogenic proteins have orthologous in H37Rv and BCG. This is the first study to obtain the full genome and annotation of M. vaccae using a proteogenomic approach, and some immunogenic proteins that were validated by immunoproteomic analysis could contribute to the understanding of the mechanism of M. vaccae immunotherapy. Tuberculosis (TB) is one of the leading causes of death worldwide, especially in developing countries. Neonatal BCG vaccination occurs in various regions, but the level of protection varies in different populations. Recently, Mycobacterium vaccae is found to be an immunomodulating therapeutic agent that could confer a significant level of protection against TB. It is the only vaccine in a phase III trial from WHO to assess its efficacy and safety in preventing TB disease in people with latent TB infection. However, the mechanism of immunotherapy of M. vaccae remains poorly understood. In this study, the full genome of M. vaccae was obtained by next-generation sequencing technology, and a proteogenomic approach was successfully applied to further perform genome annotation using high resolution and high accuracy MS data. A total of 3,387 proteins (22,508 unique peptides) were identified, and 581 proteins annotated as hypothetical proteins in the genome database were confirmed. Furthermore, 38 novel protein products not annotated at the genome level were detected and validated. Additionally, the translational start sites of 445 proteins were confirmed, and 98 proteins were validated through extension of their translational start sites based on N terminus-derived peptides. The physicochemical characteristics of the identified proteins were determined. Thirty-five immunogenic proteins of M. vaccae were identified by immunoproteomic analysis, and 20 of them were selected to be expressed and validated by Western blot for immunoreactivity to serum from patients infected with M. tuberculosis. The results revealed that eight of them showed strong specific reactive signals on the immunoblots. Furthermore, cellular immune response was further examined and one protein displayed a higher cellular immune level in pulmonary TB patients. Twelve identified immunogenic proteins have orthologous in H37Rv and BCG. This is the first study to obtain the full genome and annotation of M. vaccae using a proteogenomic approach, and some immunogenic proteins that were validated by immunoproteomic analysis could contribute to the understanding of the mechanism of M. vaccae immunotherapy. Tuberculosis (TB) 1The abbreviations used are: TB, Tuberculosis; BCG, Mycobacterium bovis bacillus Calmette and Guérin; CDS, protein-coding sequences; CELLO2GO, protein subCELlular LOcalization prediction with functional Gene Ontology annotation; FDR, false discovery rate; HIV, human immunodeficiency virus; IMP, integral membrane protein; PEP, posterior error probability; RFB, rifambutin; SAMS, S-adenosylmethionine synthase; TSS, translational start site. 1The abbreviations used are: TB, Tuberculosis; BCG, Mycobacterium bovis bacillus Calmette and Guérin; CDS, protein-coding sequences; CELLO2GO, protein subCELlular LOcalization prediction with functional Gene Ontology annotation; FDR, false discovery rate; HIV, human immunodeficiency virus; IMP, integral membrane protein; PEP, posterior error probability; RFB, rifambutin; SAMS, S-adenosylmethionine synthase; TSS, translational start site. is an infectious disease caused by the bacillus Mycobacterium tuberculosis, and it is one of the most lethal diseases worldwide. Although over 20 years after WHO declared TB a global public health emergency, the infection remains a major global health problem (1Gao L. Lu W. Bai L. Wang X. Xu J. Catanzaro A. Cardenas V. Li X. Yang Y. Du J. Sui H. Xia Y. Li M. Feng B. Li Z. Xin H. Zhao R. Liu J. Pan J. Yang H. Wang Y. Xu Z. Y. Xu W. H. Wang Z. Liu H. Zhao Y. infection in results of a In people were to have with TB, and people were WHO Furthermore, TB the human immunodeficiency as a leading of death M. J. R. B. M. M. A. in of and Although TB is and to and the of TB of the in the global against TB is vaccination with Mycobacterium bovis bacillus Calmette and was first used in In the of with BCG against TB and of TB in years of but the protection is in various The and of BCG for a novel It that the of protection to the of the vaccine the of in (1Gao L. Lu W. Bai L. Wang X. Xu J. Catanzaro A. Cardenas V. Li X. Yang Y. Du J. Sui H. Xia Y. Li M. Feng B. Li Z. Xin H. Zhao R. Liu J. Pan J. Yang H. Wang Y. Xu Z. Y. Xu W. 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Zheng et al. (Fri,) studied this question.