Resolving multiple conformations of a sub-80 kDa Chagas vaccine candidate by cryo-EM led integrative approach

Abstract Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a significant global public health concern. Despite its profound health impact in both endemic and non-endemic areas, no vaccine is available, and the existing therapies are outdated, producing severe side effects. The 80kDa prolyl oligopeptidase of Trypanosoma cruzi (TcPOP) has been recently identified as a leading candidate for Chagas vaccine development. We report the first three-dimensional structure of TcPOP in open and closed conformation, at a resolution of 3.0 and 2.5 Angstroms respectively, determined using single-particle cryo-electron microscopy. Multiple conformations were observed and were further characterized, using plasmonic optical tweezers. To assess the immunogenic potential of TcPOP, we immunized mice and evaluated both polyclonal and monoclonal responses against the TcPOP antigen and its homologues. The results revealed unexpected cross-reactivity across prolyl POPs from other closely related parasites, but intriguingly, not towards the human homologue. Altogether, our findings provide critical structural insights necessary to understand the immunogenicity of TcPOP for future Chagas vaccine development and diagnostic applications.