Recombinant Mycobacterium bovis BCG expressing the Sm14 antigen of Schistosoma mansoni protects mice from cercarial challenge

Abstract

The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall. The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability. The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14. Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S. mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli. The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries.

FIG. 1.

Schematic representation of promoter and antigen regions of shuttle vectors pPL71-sm14, pPL73-sm14, and pPL12-sm14. All vectors contain E. coli and mycobacterial origins of replication, a kanamycin resistance gene (aph), pBlaF*, and sm14. pPL71-sm14 has the β-lactamase signal sequence (ssBlam) and pPL73-sm14 has the complete β-lactamase sequence (Blam) fused to the sm14 sequence, while pPL12-sm14 has a conserved mycobacterial Shine-Dalgarno sequence (Mega SD).

FIG. 2.

Expression of Sm14 in rBCG. (a) Total cell extracts of BCG (50 μg) were analyzed by Western blotting using anti-rSm14 antiserum. Lanes: 1, BCG; 2, rBCG(pPL71-sm14); 3, rBCG(pPL73-sm14); 4, rBCG(pPL12-sm14); 5, control rSm14 (1 μg). (b) Localization of heterologous protein in rBCG(pPL73-sm14). Fractions of the extract of rBCG were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using anti-rSm14 antiserum. Lanes: 1, total extract; 2, membrane; 3, cytosol; 4, cell wall. Molecular masses are indicated to the left.

FIG. 3.

Evaluation of infectivity, persistence, and plasmid maintenance of BCG and rBCG(pPL73-sm14) within murine (RAW 264.7) (a) and bovine (b) monocyte cultures. CFU are shown in log₁₀ units. Time zero samples represent the total numbers of viable cells that were added to six wells (of a 24-well plate), i.e., the starting inocula. At each sample point, six wells were lysed and the number of CFU recovered on medium with and without kanamycin was recorded. Values recorded at 3 h represent the numbers of bacteria which had been internalized after 3 h of contact with the monocytes. As such, they provide a value for the infectivity of BCG and rBCG for each culture. The values recorded at 24 h and at 4 and 7 days give an indication of the persistence of each strain for the experimental period.

FIG. 4.

Cellular response induced by immunization with rBCG-Sm14. The data show IFN-γ production by rSm14-stimulated splenocyte cultures from BALB/c mice immunized with BCG or rBCG(pPL73-sm14) (10⁶ CFU). The cytokine content was estimated as described in Materials and Methods. The error bars indicate standard deviations.

FIG. 5.

Scattergram of worm burden in mice immunized with rBCG(pPL73-sm14) according to the immunization schedule shown in Table 1.