C57BL/6 mice were vaccinated with plasmid DNA encoding Ag85 from Mycobacterium tuberculosis, with Ag85 protein in adjuvant, or with a combined DNA prime-protein boost regimen. While DNA immunization, as previously described, induced robust Th1-type cytokine responses, protein-in-adjuvant vaccination elicited very poor cytokine responses, which were 10-fold lower than those observed with DNA immunization alone. Injection of Ag85 DNA-primed mice with 30 to 100 g of purified Ag85 protein in adjuvant increased the interleukin-2 and gamma interferon (IFN-␥) response in spleen two-to fourfold. Further, intracellular cytokine analysis by flow cytometry also showed an increase in IFN-␥-producing CD4 ؉ T cells in DNA-primedprotein-boosted animals, compared to those that received only the DNA vaccination. Moreover, these responses appeared to be better sustained over time. Antibodies were readily produced by all three methods of immunization but were exclusively of the immunoglobulin G1 (IgG1) isotype following protein immunization in adjuvant and preferentially of the IgG2a isotype following DNA and DNA prime-protein boost vaccination. Finally, protein boosting increased the protective efficacy of the DNA vaccine against an intravenous M. tuberculosis H37Rv challenge infection, as measured by CFU or relative light unit counts in lungs 1 and 2 months after infection. The capacity of exogenously given protein to boost the DNA-primed vaccination effect underlines the dominant role of Th1-type CD4؉ helper T cells in mediating protection.Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. The only TB vaccine presently available is an attenuated strain of Mycobacterium bovis termed M. bovis BCG. The efficacy of BCG remains controversial, particularly against pulmonary TB in young adults (5), and development of a better vaccine is urgently needed to counter the global threat of this disease (22).Secreted and surface-exposed cell wall proteins are major antigens recognized by the protective immune response against TB and immunization with whole-culture filtrate, a rich source of these extracellular proteins, can protect mice and guinea pigs to some extent against subsequent challenge with the tubercle bacillus (1, 14, 15). A major portion of the secreted proteins in Mycobacterium tuberculosis and BCG culture filtrate is formed by the Ag85 complex, a 30-to 32-kDa family of three proteins (Ag85A, Ag85B, and Ag85C) (38) which all possess a mycoloyltransferase enzyme activity required for the biogenesis of cord factor (4), a dominant structure necessary for maintaining cell wall integrity (19,29). Ag85 complex induces strong T-cell proliferation and gamma interferon (IFN-␥) production in most healthy individuals infected with M. tuberculosis and/or Mycobacterium leprae (24) and in BCGvaccinated mice (16), making it a promising candidate as a protective antigen. Vaccination with naked plasmid DNA encoding Ag85A and Ag85B can stimulate strong humoral and cell-mediated immune responses and confer si...
The continuous flow of new research articles on MDR-TB diagnosis, treatment, prevention and rehabilitation requires frequent update of existing guidelines. This review is aimed at providing clinicians and public health staff with an updated and easy-to-consult document arising from consensus of Global Tuberculosis Network (GTN) experts.The core published documents and guidelines have been reviewed, including the recently published MDR-TB WHO rapid advice and ATS/CDC/ERS/IDSA guidelines.After a rapid review of epidemiology and risk factors, the clinical priorities on MDR-TB diagnosis (including whole genome sequencing and drug-susceptibility testing interpretations) and treatment (treatment design and management, TB in children) are discussed. Furthermore, the review comprehensively describes the latest information on contact tracing and LTBI management in MDR-TB contacts, while providing guidance on post-treatment functional evaluation and rehabilitation of TB sequelae, infection control and other public health priorities.
Diagnosis of leprosy is a major obstacle to disease control and has been compromised in the past due to the lack of specific reagents. We have used comparative genome analysis to identify genes that are specific to Mycobacterium leprae and tested both recombinant proteins and synthetic peptides from a subset of these for immunological reactivity. Four unique recombinant proteins (ML0008, ML0126, ML1057, and ML2567) and a panel of 58 peptides (15 and 9 mer) were tested for IFN-γ responses in PBMC from leprosy patients and contacts, tuberculosis patients, and endemic and nonendemic controls. The responses to the four recombinant proteins gave higher levels of IFN-γ production, but less specificity, than the peptides. Thirty-five peptides showed IFN-γ responses only in the paucibacillary leprosy and household contact groups, with no responses in the tuberculosis or endemic control groups. High frequencies of IFN-γ-producing CD4+ and CD8+ T cells specific for the 15- and 9-mer peptides were observed in the blood of a paucibacillary leprosy patient. 9-mer peptides preferentially activated CD8+ T cells, while the 15-mer peptides were efficient in inducing responses in both the CD4+ and CD8+ T cell subsets. Four of the six 9-mer peptides tested showed promising specificity, indicating that CD8+ T cell epitopes may also have diagnostic potential. Those peptides that provide specific responses in leprosy patients from an endemic setting could potentially be developed into a rapid diagnostic test for the early detection of M. leprae infection and epidemiological surveys of the incidence of leprosy, of which little is known.
Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette-Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP-10 in M. leprae (ML0050) is identified and characterized. Interferon-g production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP-10 of M. leprae is a potent antigen that crossreacts with CFP-10 of M. tuberculosis at the T-cell level. This crossreactivity has implications for the use of CFP-10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.
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