Rn. Coler et al., MOLECULAR-CLONING AND IMMUNOLOGICAL REACTIVITY OF A NOVEL LOW-MOLECULAR-MASS ANTIGEN OF MYCOBACTERIUM-TUBERCULOSIS, The Journal of immunology (1950), 161(5), 1998, pp. 2356-2364
Polypeptide Ags present in the culture filtrate of Mycobacterium tuber
culosis were purified and evaluated for their ability to stimulate PBM
C from purified protein derivative (PPD)-positive healthy donors. One
such Ag, which elicited strong proliferation and IFN-gamma production,
was further characterized, The N-terminal amino acid sequence of this
polypeptide was determined and used to design oligonucleotides for sc
reening a recombinant M. tuberculosis genomic DNA library, The gene (M
tb 8.4) corresponding to the identified polypeptide was cloned, sequen
ced, and expressed in Escherichia coli. The predicted m.w. of the reco
mbinant protein without its signal peptide was 8.4 kDa, By Southern an
alysis, the DNA encoding this mycobacterial protein was found in the M
, tuberculosis substrains H37Rv, H37Ra, Erdman, and ''C'' strain, as w
ell as in certain other mycobacterial species, including Mycobacterium
avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur)
. The Mtb 8.4 gene appears to be absent from the environmental mycobac
terial species examined thus far, including Mycobacterium smegmatis, M
ycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum
, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced signi
ficant proliferation as well as production of IFN-gamma, IL-10, and TN
F-alpha, but not IL-5, from human PBMC isolated from PPD-positive heal
thy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. F
urthermore, immunogenicity studies in mice indicate that Mtb 8.4 elici
ts a Th1 cytokine profile, which is considered important for protectiv
e immunity to tuberculosis. Collectively, these results demonstrate th
at Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.