Antibody Recognition and Functional Activity Against a Conserved Trypanosoma cruzi Peptide and Its Natural Variants in Kinetoplastids

Purpose this study aimed to evaluate the potential cross-reactivity of antibodies raised against the conserved TcTLE peptide (TLEEFSAKL) of Trypanosoma cruzi with two natural variants found in Leishmania spp., and to assess the humoral specificity toward this epitope. Methods chemically synthesized TcTLE, TcY (TYEEFSAKL), and TcYA (TYEEFAAKL) peptides were tested by ELISA using both a monoclonal antibody (mAb) and polyclonal rabbit sera raised against TcTLE. Additional analyses included indirect immunofluorescence assay (IFAT) and flow cytometry with T. cruzi epimastigotes (TcTLE), Leishmania amazonensis (TcY), and Leishmania panamensis (TcYA) promastigotes. Results by ELISA, both mAb and polyclonal sera recognized TcY but showed minimal reactivity to TcYA. In IFAT, the mAb failed to bind fixed parasites, whereas in flow cytometry it bound live T. cruzi epimastigotes but not Leishmania promastigotes. The anti-TcTLE mAb significantly reduced T. cruzi invasion in vitro . Alanine scanning revealed that residue 2 is not critical for antibody recognition. Conclusion overall, although the TcY variant was recognized in vitro , antibodies to TcTLE did not detect parasites expressing the KMP-11 variants in vivo , indicating that true cross-reactivity is unlikely.