Open and closed conformations of a sub-80 kDa Chagas vaccine candidate defined by a cryo-EM led integrative approach

Abstract

AbstractChagas disease, caused by the protozoan parasiteTrypanosoma cruzi, remains a significant global public health concern. It affects an estimated eight million individuals worldwide, with the majority remaining undiagnosed. 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profound health impact in both endemic and non-endemic areas, no vaccine is available, and the existing therapies are outdated, producing severe side effects.The 80kDa prolyl oligopeptidase ofTrypanosoma cruzi(TcPOP) has been recently identified as a leading candidate for Chagas vaccine development. However, its three-dimensional structure has remained elusive for almost two decades since its discovery. We report the first three-dimensional structure ofTcPOP 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 ofTcPOP, we immunized mice and evaluated both polyclonal and monoclonal responses against theTcPOP 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 ofTcPOP for future Chagas vaccine development and diagnostic applications.Additionally, our integrative approach indicated that stage-tilted acquisition can yield biologically relevant information for challenging sub-80kDa proteins and could adequately resolve the cryoEM structures. Consequently, this comprehensive strategy can significantly enhance the success rate in determining the structures of proteins that present challenges in characterization.