cruzi infection, we decided to immunize mice with naked DNA or recombinant proteins. For DNA immunization and recombinant protein production, plasmids were generated containing DNA coding for TcSP, TcSPA TcSPR or TcSPC (Table 1). The his-tagged recombinant proteins rTcSP,

rTcSPA, rTcSPR and rTcSPC were purified, and their identity was confirmed by Western blotting with anti-histidine antibodies (Figure 1). Recombinant proteins were also assayed with sera from the mice infected with T. cruzi, and the results revealed that the antibodies generated against the native TcSP protein Hydroxychloroquine were directed primarily against the central amino acid repeated sequence (rTcSPR) (Figure 2). The apparent molecular weight of rTcSPR was higher than expected based on the primary amino acid sequence, but this behaviour has also been observed in studies of other proteins [29, 30]. However, the origin of such behaviour remains unknown. The mice immunized with rTcSP or rTcSPR showed similar serum levels for the analysed IgG isotypes. selleck products These serum levels were higher than those observed in the mice immunized with rTcSPA or rTcSPC (P < 0·001 in all cases, except

for IgG2b in rTcSPR vs. rTcSPC). In the latter two groups, the IgG1 and IgG2a serum levels were comparable, while the serum levels of IgG2b and IgG3 were higher in the mice immunized with rTcSPC than rTcSPA (P < 0·001) (Figure 3a). Serum antibody levels were lower in the mice immunized with naked DNA when compared with the serum antibody levels in the mice immunized with the corresponding proteins (Figure 3b). However, significant differences were detected in the humoral response when the mice were immunized with the plasmid pBKTcSP. Specifically, the IgG1 and IgG2b levels differed from the antibody levels in the mice immunized with plasmids containing DNA coding for the A, R or C domains of TcSP (P < 0·001 in all cases except for IgG1 P < 0·01 in pBKTcSP vs. pBKTcSPA) (Figure 3b).

In contrast, the levels of IgG2a and IgG3 remained low in the mice immunized with the various plasmids. Interestingly, in the animals immunized with the plasmids pBKTcSP, pBKTcSPR or pBKTcSPC, the proportion of immunoglobulins was IgG2b>IgG1 with a ratio >1, thus suggesting MTMR9 a predominantly Th1 immune response. Analysis of serum cytokines revealed a similar profile when the mice were immunized with almost all the recombinant proteins. However, immunization by rTcSP produced a different response, in that IL-2 and INF-γ were absent and IL-5, IL-10 and TNF-α were detected at lower levels (P < 0·001) (Figure 4a). These results suggest that recombinant proteins induce a mixed Th1/Th2 response. In contrast, the study of cytokines induced by immunization with plasmid DNA showed that IL-2 was induced only by pBKTcSPA, IL-5 by pBKTcSP and pBKTcSPA, and none of the cytokines were detected after immunization by pBKTcSPC.