[The immunological responses induced by Mycobacterium tuberculosis heat shock protein 16.3 and its synthetic peptide in mice].

OBJECTIVE

To evaluate the immune responses and resistance against Mycobacterium tuberculosis (MTB) infection in the mice induced by HSP16.3 of MTB and its synthetic peptide.

METHODS

BALB/c mice were immunized subcutaneously 3 times at 2 week interval at the base of tail. The doses of HSP16.3 protein and synthetic peptide were both 50 microg each time. A single dose of BCG (5 x 10(6) CFU/mouse) was used to immunize the mice. The concentrations of specific antibodies in serum obtained at 0, 2, 4, 6, 8 weeks after the first immunization and the titer of serum obtained at 8th week, were analyzed by enzyme linked immunosorbent assay (ELISA). Four weeks after the final immunization, 8 mice from each group were sacrificed and single-cell suspensions of splenocytes were prepared, some of which were used for lymphocyte proliferation by MTT colorimetry with HSP16.3 stimulation, and the remaining cells were used for IFN-gamma level assay by sandwich ELISA. The remaining mice in each group were challenged intravenously with 10(5) colony forming units (CFU) of MTB H(37)Rv and were sacrificed 4 weeks after infection, and the number of bacteria in the spleens and lungs were determined by plating serial dilutions of homogenized tissue on Middlebrook 7H10 agar. The statistical significance of differences among means was assessed by an LSD-t test.

RESULTS

The level of specific antibody to HSP16.3 protein and the peptide increased rapidly in the former 4 weeks and moderately in the later weeks. The average antibody-specific titers of 3 experiment groups (HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA) were higher than the BCG group. The indexes of spleen lymphocyte proliferation (SI) of the 3 experiment groups (3.13 +/- 0.18, 3.21 +/- 0.21 and 2.40 +/- 0.15) were significantly higher than the BCG group (1.67 +/- 0.12) and the saline group (1.04 +/- 0.09) respectively. The SI of HSP16.3 protein + DDA + MPL group (3.13 +/- 0.18) and synthetic peptide + DDA + MPL group (3.21 +/- 0.21) were higher than the synthetic peptide + IFA group (2.40 +/- 0.15). The IFN-gamma levels induced among the 3 experiment groups [(182 +/- 6), (194 +/- 9) and (179 +/- 8) mg/L] were lower than the BCG group [(275 +/- 10) mg/L], but higher than the saline group [(71 +/- 3) mg/L]. The IFN-gamma level induced among the 3 experiment groups did not show any marked difference. Although the protection induced by HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA all showed resistance against MTB H(37)Rv infection in the spleens or lungs (the bacterial logarithmic loads of spleen: 6.74 +/- 0.14, 6.60 +/- 0.13 and 6.81 +/- 0.28; the bacterial logarithmic loads of lung: 5.81 +/- 0.21, 5.74 +/- 0.27 and 6.65 +/- 0.32), none of them was better than the conventional BCG (the bacterial logarithmic loads of spleen and lung: 5.95 +/- 0.17 and 5.62 +/- 0.23).

CONCLUSIONS

Both HSP16.3 and its synthetic peptide can be considered as TB vaccine candidates or effective components in TB vaccines.