Characterization of autoantibodies generated in mice by immunization with the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins

The R13 peptide sequence (EEEDDDMGFGLFD) that corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins differs from the eukariotic P consensus sequence EESDDDMGFGLFD (H13) only in a nonconservative amino acid substitution. Since the immunization with R13 peptide coupled to a carrier protein like OVA would break the tolerance to a self-sequence and generate autoantibodies, we characterized the antibodies induced in mice by R13 immunization, analyzing by ELISA their capacity to bind to R13 and the self-sequence H13. Besides, we studied the course of these reactivities a long time after immunization. It was found that all R13-immunized mice had antibodies against H13 and this reactivity was always lower than R13 reactivity. The anti-H13 reactivity evaluated by competitive ELISA demonstrated that the H13 peptide is able to inhibit the binding of immune sera to R13 at high doses. When the levels and the avidity of anti-R13 and anti-H13 were evaluated at 10 and 80 days post third immunization, it was observed that anti-R13 levels were higher than anti-H13 levels in all sera from 10 days after the third immunization. However, avidity of both antibodies was high. In sera from 80 days post third immunization, anti-R13 and anti-H13 levels and avidity either remained elevated or showed a rise, whereas anti-OVA levels declined. Moreover, when ECG traces were obtained from immunized mice, the heart functional alterations observed at 10 days continued at 80 days after the third immunization, showing an association with the levels of the antibodies. In addition, the isotype pattern that recognizes R13 and the self-sequence H13 is different. For anti-R13 response, IgG1 reactivity was higher than IgG2; meanwhile, for anti-H13 response IgG2 reactivity was higher than IgG1. These results indicate that sera from R13-immunized mice bind the H13 sequence and this autoreactivity may be self-perpetuating.