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

Abstract

The TcTLE peptide (TLEEFSAKL), located at the amino-terminus of the Trypanosoma cruzi Kinetoplastid Membrane Protein-11 (KMP-11), induces robust humoral and cellular immune responses, suggesting its potential for serological diagnostics or immunoprophylaxis. However, natural variants of this sequence exist in Leishmania spp. This study aims to assess the humoral specificity toward this epitope. KMP-11 sequences were retrieved from the UniProtKB database. Multiple sequence alignment was conducted using the CLUSTALW alignment tool. In silico analysis of the peptides was performed using I-TASSER. Alanine scan derivates as well as 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. Invasion assays were carried out with susceptible cells and T. cruzi trypomastigote CFSE labeling. By ELISA, both mAb and polyclonal sera recognized TcY but showed minimal reactivity to TcYA. Alanine scanning revealed that residue 2 is not critical for antibody recognition. 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 cell invasion in vitro. Overall, although the TcY variant was recognized in vitro, antibodies to TcTLE did not detect parasites expressing the KMP-11 variants, indicating that true cross-reactivity with Leishmania is unlikely.