Six different secretory proteins of molecular weights (15, 26, 30, 41,
55 and 70 kDa) were isolated from 8-day-old culture filtrate of Mycob
acterium tuberculosis H37Ra using different column chromatography tech
niques. These proteins were further examined for their ability to indu
ce cell mediated (T-cell proliferation assay) and humoral immune respo
nse (ELISA) in mice immunized with total culture filtrate proteins. Ou
t of six proteins, three proteins showed good reactivity. However, the
activity was at a maximum with 30 kDa antigen. The immune response in
duced by 30 kDa antigen emulsified in Freund's incomplete adjuvant (FI
A) was investigated and was found to be dose dependent. The T-cell res
ponse induced by this protein was skewed rewards T-helper (Th1) cells
as determined by the pronounced secretion of interleukin-2 (IL-2) and
gamma-interferon (IFN-gamma). The protective activity of the 30 kDa pr
otein was also evaluated and compared with reference to Bacillus Calme
tte Guerin (BCG) vaccine in the mice challenged with virulent M. tuber
culosis H(37)Rv. The degree of protection afforded by the 30 kDa antig
en on the basis of mortality and the significant decrease in c.f.u.'s
recovered from different organs (lung, liver, spleen) after 30 days of
challenge with LD50 of M. tuberculosis H(37)Rv was significantly high
er in comparison to BCG vaccinated animals. However, the degree of imm
unity induced by this antigen decreased with time (when challenged 8 a
nd 12 weeks post-immunization) but it was still comparable with BCG, T
hese findings suggest that 30 kDa secretory protein of M. tuberculosis
is the key immunoprotective antigen and may be a suitable candidate f
or the development of an alternative subunit vaccine against tuberculo
sis. (C) 1997 Elsevier Science Ltd.