Sm14 of Schistosoma mansoni in fusion with tetanus toxin fragment C induces immunoprotection against tetanus and schistosomiasis in mice

Abstract

We have constructed vectors that permit the expression in Escherichia coli of Schistosoma mansoni fatty acid-binding protein 14 (Sm14) in fusion with the nontoxic, but highly immunogenic, tetanus toxin fragment C (TTFC). The recombinant six-His-tagged proteins were purified by nickel affinity chromatography and used in immunization and challenge assays. Animals inoculated with TTFC in fusion with or coadministered with Sm14 showed high levels of tetanus toxin antibodies, while animals inoculated with Sm14 in fusion with or coadministered with TTFC showed high levels of Sm14 antibodies. In both cases, there were no changes in the type of immune response (Th2) obtained with the fusion proteins compared to those obtained with the nonfused proteins. Mice immunized with the recombinant proteins (TTFC in fusion with or coadministered with Sm14) survived the challenge with tetanus toxin and did not show any symptoms of the disease. Control animals inoculated with either phosphate-buffered saline (PBS) or Sm14 died with severe symptoms of tetanus after 24 h. Mice immunized with the recombinant proteins (Sm14 in fusion with or coadministered with TTFC) showed a 50% reduction in worm burden when they were challenged with S. mansoni cercariae, while control animals inoculated with either PBS or TTFC were not protected. The results show that the expression of other antigens in fusion at the carboxy terminus of TTFC is feasible for the development of a multivalent recombinant vaccine.

FIG. 1.

Analysis of the recombinant expressed protein from NaCl-induced E. coli BL21-SI by SDS-12% PAGE. Protein bands were visualized by Coomassie blue staining. Lane M, molecular weight protein markers; lane 1, noninduced culture; lane 2, induced culture; lanes 3 and 4, inclusion body pellet and supernatant after bacterial cell lysis and centrifugation, respectively; lane 5, purified protein eluted from Ni²⁺-charged chelating Sepharose column with 1 M imidazole.

FIG. 2.

Antibody response induced by recombinant proteins. BALB/c mice were immunized subcutaneously at an interval of 14 days with two doses of purified recombinant proteins (TTFC, TTFC-Sm14 fusion protein, TTFC plus Sm14, or Sm14), tetanus toxoid, or PBS adsorbed to aluminum hydroxide. On the 27th day after the first immunization, mice were bled and pooled sera were analyzed by ELISA. (A) Microdilution plates were coated with tetanus toxin and incubated with a serial dilution of serum from the immunized mice for IgG measurements. The standard error did not exceed 10% of the mean values (data not shown). (B) Sera (1/320 dilution) from immunized mice were subjected to anti-tetanus toxins IgG1 and IgG2a.

FIG. 3.

Antibody responses induced by recombinant proteins. Swiss mice were immunized at intervals of 7 days with three doses of purified recombinant proteins (Sm14, TTFC-Sm14 fusion protein, Sm14 plus TTFC, or TTFC) or PBS adsorbed to aluminum hydroxide. On the 72nd day after the first immunization, mice were bled and pooled sera were analyzed by ELISA. (A) Microdilution plates were coated with Sm14 and incubated with a serial dilution of serum from immunized mice for IgG measurements. The standard error did not exceed 10% of the mean values (data not shown). (B) Sera (1/160 dilution) from immunized mice were subjected to anti-Sm14 IgG1 and IgG2a.

FIG. 4.

Protection of mice against challenge with S. mansoni cercariae. Swiss mice were immunized with three doses of 10 μg of recombinant proteins adsorbed to aluminum hydroxide at intervals of 7 days. Two of the control groups were immunized with PBS plus alum or TTFC plus alum, and one group, the control of the infection, was not immunized. All mice were challenged subcutaneously 60 days after the last immunization with 100 cercariae/mouse and perfused 45 days later. The mean worm burden was calculated using results from three experiments. Percent protection in the three independent experiments was calculated by comparing their results with the results obtained from the control groups. Statistical analysis was done with Student's t test. Results are expressed as means ± standard errors. P was <0.05 compared with results from the control group.