Mapping of Murine Th1 Helper T-Cell Epitopes of Mycolyl Transferases Ag85A, Ag85B, and Ag85C fromMycobacterium tuberculosis

Abstract

BALB/c (H-2d ) and C57BL/6 (H-2 b ) mice were infected intravenously with Mycobacterium tuberculosis H37Rv or vaccinated intramuscularly with plasmid DNA encoding each of the three mycolyl transferases Ag85A, Ag85B, and Ag85C from M. tuberculosis. Th1-type spleen cell cytokine secretion of interleukin-2 (IL-2) and gamma interferon (IFN-␥) was analyzed in response to purified Ag85 components and synthetic overlapping peptides covering the three mature sequences. Tuberculosis-infected C57BL/6 mice reacted strongly to some peptides from Ag85A and Ag85B but not from Ag85C, whereas tuberculosis-infected BALB/c mice reacted only to peptides from Ag85A. In contrast, spleen cells from both mouse strains produced elevated levels of IL-2 and IFN-␥ following vaccination with Ag85A, Ag85B, and Ag85C DNA in response to peptides of the three Ag85 proteins, and the epitope repertoire was broader than in infected mice. Despite pronounced sequence homology, a number of immunodominant regions contained component specific epitopes. Thus, BALB/c mice vaccinated with all three Ag85 genes reacted against the same amino acid region, 101 to 120, that was also immunodominant for Ag85A in M. bovis BCG-vaccinated and tuberculosis-infected H-2 d haplotype mice, but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241 to 260 and 261 to 280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA. Our results underline the need for comparative analysis of all three Ag85 components in future vaccination studies.

Difference in TB10.4 T‐cell epitope recognition following immunization with recombinant TB10.4, BCG or infection with Mycobacterium tuberculosis

“…Although epitopes have been identified in M.tb proteins other than TB10.4 [12,14,23], a detailed comparison between post immunization and post infection epitopes has not been described.…”

A role for IL‐18 in protective immunity against Mycobacterium tuberculosis

“…Cells (1-3 Â 10 6 ) were cultured in a volume of 1 mL complete RPMI medium and stimulated for 5 h with a combination of the peptides Ag85A 241-260 (QDAYNAGGGH NGVFDFPDSG) and Ag85B 240-260 (FQDAYNAAGG HNAVFNFPPN G) both at 10 mM or with 1 mM of the peptide Mtb32/RV0125 309-318 (GAPINSATAM) [45,46]. During the final 4 h of culture, 10 mg/mL Brefeldin A was added.…”

“…40: 396-405 Immunity to infection In vitro re-stimulation of cells and flow cytometric determination of cytokine expression T-cell responses in lung and spleen were determined by intracellular IFN-g staining [44]. Cells (1-3 Â 10 6 ) were cultured in a volume of 1 mL complete RPMI medium and stimulated for 5 h with a combination of the peptides Ag85A 241-260 (QDAYNAGGGH NGVFDFPDSG) and Ag85B 240-260 (FQDAYNAAGG HNAVFNFPPN G) both at 10 mM or with 1 mM of the peptide Mtb32/RV0125 309-318 (GAPINSATAM) [45,46]. During the final 4 h of culture, 10 mg/mL Brefeldin A was added.…”

Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis

“…Lymphocytes were isolated from different tissues and incubated for 5 h with a combination of the peptides Ag85A 241-260 and Ag85B 240 -260 or with Mtb32 309 -318 to stimulate mycobacteria-specific CD4 ϩ and CD8 ϩ T cells, respectively (13,20). T cell responses were determined by intracellular IFN-␥ staining.…”