Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family

Abstract

We have used expression screening of a genomic Mycobacterium tuberculosis library with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termed mtb39a, that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a family of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A in M. tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculosis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine for M.tuberculosis.

FIG. 1

Purification of rMtb39A and rΔMtb39A proteins. Expression and purification of rMtb39A (A) and rΔMtb39A (B) are shown with uninduced (lane 2) and induced (lane 3) E. coli lysates and 5 μg of purified proteins (lane 4). Molecular mass markers are shown in kDa (lane 1).

FIG. 2

Comparison of amino acid sequences encoded by mtb39 genes. Identical residues are indicated by dots (.) and deletions are indicated by dashes (-). Amino acid residues present in rTbH9 are underlined. The carboxy-terminal portion of the Mtb39C ORF was recovered by database searching and extends from residues 360 to 393.

FIG. 3

Southern blot analysis of mtb39 genes. Genomic DNA (2.5 μg) from mycobacterial strains was digested with PstI, separated by agarose gel electrophoresis, and blotted onto Nytran. The mtb39a gene was labeled with [³²P]dCTP by random oligonucleotide primers and used as a probe. Molecular sizes in kilobases are shown.

FIG. 4

Characterization of native Mtb39A. M. tuberculosis H37Rv lysate (2.5 μg; lane 1), 2.5 μg of culture filtrate protein (lane 2), 50 ng of purified rMtb11 (recombinant 11-kDa M. tuberculosis antigen [unpublished results]) (lane 3), 50 ng of purified rΔMtb39A (lane 4), and 50 ng of purified recombinant antigen 85B (lane 5) were subjected to SDS-PAGE, transferred to nitrocellulose, and reacted with rabbit antisera generated against rΔMtb39A (A) or recombinant antigen 85B (B). Molecular markers in kDa are indicated.

FIG. 5

Proliferation and IFN-γ production by PBMC from PPD-positive and -negative donors to CFP, tetanus toxoid, rΔMtb39A, rMtb39A, and r85B. Proliferation was measured in PBMC from PPD-positive donors (n = 12) and PPD-negative donors (n = 12). PBMC (2 × 10⁵ cells/well) were cultured in the presence of antigen (10 μg/ml) for 5 days. After 5 days, 50 μl of culture supernatant was carefully aspirated for determination of IFN-γ levels by ELISA, and the plates were pulsed with tritiated thymidine. After culture for a further 18 h, cells were harvested, and tritium uptake was determined by using a gas scintillation counter. Proliferation results are reported as an SI (counts per minute of cultures with antigen/counts per minute of medium control cultures). The number of responses that have an SI of greater than 100 is indicated in parentheses.

FIG. 6

Dose response of PBMC from PPD-positive donors to rΔMtb39A and rMtb39A. Proliferation and IFN-γ production to rΔMtb39A and rMtb39A were measured in PBMC from four PPD-positive donors. After 5 days, 50 μl of culture supernatant was carefully aspirated for determination of IFN-γ levels by ELISA, and the plates were pulsed with tritiated thymidine. After culture for a further 18 h, cells were harvested, and tritium uptake was determined by using a gas scintillation counter.

FIG. 7

PBMC responses from PPD-positive donors to rΔMtb39A, rMtb39A, and peptides derived from the amino acid sequence. Proliferation to rΔMtb39A, rMtb39A and peptides based on Mtb39A amino acid sequence was measured in PBMC from four PPD-positive donors. PBMC (2 × 10⁵ cells/well) were cultured in the presence of antigen (10 μg/ml) for 5 days. After 5 days, 50 μl of culture supernatant was carefully aspirated for determination of IFN-γ levels by ELISA, and the plates were pulsed with 1 μCi of tritiated thymidine/well. After culture for a further 18 h, cells were harvested, and tritium uptake was determined by using a gas scintillation counter. Proliferation results to the antigens are reported as an SI (counts per minute of cultures with antigen/counts per minute of medium control cultures).

FIG. 8

Mtb39A is present in Mtb-infected DC. A total of 5 × 10⁴ Mtb39A-specific CD4⁺ T-cell clone D160TbH9-9 cells were incubated with 2 × 10⁴ autologous DC that had either been incubated with PPD, CFP, or rMtb39A at a concentration of 10 μg/ml (A) or incubated for 18 h with M. tuberculosis H37Rv (MOI, 50) (B). Supernatants were collected after 18 h and assessed for the presence of IFN-γ by ELISA.

FIG. 9

mtb39a DNA immunization in the murine protection model. Groups of five C57BL/6 mice were immunized i.m. three times 1 month apart with 100 μg of mtb39a DNA or with control DNA (Vector). In addition, groups of five mice were also immunized with BCG (once) or simply injected with saline. Thirty days after the last immunization, the mice were challenged by the aerosol route with approximately 100 CFU of M. tuberculosis Erdman. Protection was measured by enumerating the bacteriological burden (CFU) in the mouse lungs, shown as log₁₀ CFU.