Bioinformatically guided discovery of novel Trypanosoma cruzi epitopes recognised by CD4+ and CD8+ t cells from chronic Chagas disease patients

Abstract

T cell-mediated response has been proven to play a major role in controlling T. cruzi infection and parasite burden. It also seems to be involved in the progression from asymptomatic Chagas disease (ACD) to chronic Chagas cardiopathy (CCC) in chronically infected patients. However, the complexity of the parasite-host interactions hampers the identification and characterization of T cell activating epitopes. We approached this issue by combining in silico and in vitro methods to interrogate patients’ T cells specificity. The sequences of the 53 T. cruzi proteins annotated on the Immune Epitope Database (IEDB) were split in all possible 15-aminoacid long peptides, and 50 candidate peptides were selected using a bioinformatic pipeline (MHCpan, MHCIIpan, PopCover), based on conservation between annotated parasite strains, non-identity with H. sapiens sequences, class I and II MHC molecules binding affinity and HLA polymorphic variants coverage. Candidate peptides were randomised in 5 pools of 10 peptides each, which were used to challenge peripheral blood mononuclear cells (PBMC) from chronic Chagas disease patients, in IFN-γ ELISPOT assays. Positive pool-patient pairs were re-assayed in a single-peptide fashion to identify individual active peptides. A total of 7 peptides induced IFN-γ secretion in at least one patient’s PBMC, 4 of which do not contain any previously described epitope. In combination, response to these peptides covered 33% of the patients cohort in this study (n=51), 40% of the ACD group (n=25) and 27% of the CCC group (n=26). IFN-γ ELISPOT with CD8- or CD4-depleted PBMC showed that 6 of the peptides contain MHC class II epitopes, while 1 contains an MHC class I epitope. The fact that most of these subjects responded to 1 or 0 peptides is a strong sign of HLA restricted epitopes. In summary, we predicted, validated and characterized 7 T cell-activating peptides from T. cruzi antigens, 4 of which contain epitopes first described in this work.