Utility of the Trypanosoma cruzi Sequence Database for Identification of Potential Vaccine Candidates by In Silico and In Vitro Screening

Abstract

Glycosylphosphatidylinositol (GPI)-anchored proteins are abundantly expressed in the infective and intracellular stages of Trypanosoma cruzi and are recognized as antigenic targets by both the humoral and cellular arms of the immune system. Previously, we demonstrated the efficacy of genes encoding GPI-anchored proteins in eliciting partially protective immunity to T. cruzi infection and disease, suggesting their utility as vaccine candidates. For the identification of additional vaccine targets, in this study we screened the T. cruzi expressed sequence tag (EST) and genomic sequence survey (GSS) databases. By applying a variety of web-based genomemining tools to the analysis of ϳ2,500 sequences, we identified 348 (37.6%) EST and 260 (17.4%) GSS sequences encoding novel parasite-specific proteins. Of these, 19 sequences exhibited the characteristics of secreted and/or membrane-associated GPI proteins. Eight of the selected sequences were amplified to obtain genes TcG1, TcG2, TcG3, TcG4, TcG5, TcG6, TcG7, and TcG8 (TcG1-TcG8) which are expressed in different developmental stages of the parasite and conserved in the genome of a variety of T. cruzi strains. Flow cytometry confirmed the expression of the antigens encoded by the cloned genes as surface proteins in trypomastigote and/or amastigote stages of T. cruzi. When delivered as a DNA vaccine, genes TcG1-TcG6 elicited a parasitespecific antibody response in mice. Except for TcG5, antisera to genes TcG1-TcG6 exhibited trypanolytic activity against the trypomastigote forms of T. cruzi, a property known to correlate with the immune control of T. cruzi. Taken together, our results validate the applicability of bioinformatics in genome mining, resulting in the identification of T. cruzi membrane-associated proteins that are potential vaccine candidates.

Schistosomes—proteomics studies for potential novel vaccines and drug targets

“…This high throughput approach has been successfully used to propose promising vaccine targets for prokaryote pathogens such as Neisseria meningitides [35] and Streptococcus agalactiae [36]. Reverse vaccinology has also been used for identifying potential vaccine candidates in the protozoa Trypanosoma cruzi and Leishmania [37][38][39]. Use of this approach in more complex eukaryotic pathogens such as schistosomes, however, suffers from several drawbacks: (1) deduction of ORFs from genomes of most eukaryotes is not straightforward owing to mRNA splicing.…”

Serum-Mediated Activation of Macrophages Reflects TcVac2 Vaccine Efficacy against Chagas Disease

“…We have recently shown that prophylactic vaccination before challenge infection (11)(12)(13)(14) or treatment of chronically infected experimental animals with the antiparasite drug benznidazole (15), which resulted in the host's ability to control acute parasitemia and tissue parasite burden (11)(12)(13)(14), consequently prevented myocardial oxidative and inflammatory pathology (15)(16)(17) and led to preservation of the hemodynamic function of the heart (15). Because oxidative/inflammatory adducts result in cellular damage, we hypothesize that the damage-associated molecular patterns (DAMPs) are released in the peripheral blood and can be captured by the phagocyte activation pattern.…”

Immunological Dominance of Trypanosoma cruzi Tandem Repeat Proteins

“…Traditionally, such targets of B-cell responses have been identified from parasites through serological screening of an expression library or by immunoblotting of crude lysate separated by two-dimensional gel electrophoresis. In contrast, only a few attempts have been made to computationally predict serological antigens of pathogens from the proteome based on their sequences, such as predicting secreted or surface proteins (4,11) and identifying proteins with ␣-helical coiled-coil domains (61). Although the prediction of secreted or surface proteins has shown some promise in identifying antigens from T. cruzi (11), it may not be powerful enough to reduce the number of candidates to a practical level when dealing with the whole genome.…”

“…In contrast, only a few attempts have been made to computationally predict serological antigens of pathogens from the proteome based on their sequences, such as predicting secreted or surface proteins (4,11) and identifying proteins with ␣-helical coiled-coil domains (61). Although the prediction of secreted or surface proteins has shown some promise in identifying antigens from T. cruzi (11), it may not be powerful enough to reduce the number of candidates to a practical level when dealing with the whole genome. There are 3,141 T. cruzi genes containing sequences encoding predicted signal peptides, 5,169 with transmembrane domains, and 1,776 containing both.…”