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 genome-mining tools to the analysis of approximately 2,500 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 parasite-specific 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.

FIG. 1.

Overall strategy for the identification of putative vaccine candidates in T. cruzi. Abbreviations: ER, endoplasmic reticulum; PM, plasma membrane; OS, outside.

FIG. 2.

Phylogenetic conservation of TcG1-TcG8 in various T. cruzi strains. Genes TcG1-TcG8 were amplified in a PCR using gene-specific primers from genomic DNA of CL/Brenner (CL), SylvioX10/4 (S), Brazil (Br), and Y strains of T. cruzi. Plasmid containing the specific gene (P) was used as a template in gene-specific PCR to confirm the accuracy of the reaction. The GPI8 gene was amplified as a positive control. Shown are the amplicons resolved on 1% agarose gel.

FIG. 3.

Expression of TcG1-TcG8 in different developmental stages of T. cruzi. Total RNA isolated from epimastigote (E), trypomastigote (T), and amastigote (A) stages of T. cruzi was reverse transcribed, and the cDNA was amplified by PCR amplification with the gene-specific forward and reverse primers. Amplicons were resolved on 1% agarose gel. The GPI8 gene, constitutively expressed in all three stages of T. cruzi, was amplified as a positive control. No amplification was obtained when template cDNAs were incubated in a PCR with gene-specific forward or reverse primers only.

FIG. 4.

Flow cytometric analysis to demonstrate surface expression of the selected antigens in T. cruzi. The staining pattern of T. cruzi epimastigote, trypomastigote, and amastigote forms by polyclonal serum to TcG1 (D, E, F), TcG2 (G, H, I), TcG3 (J, K, L), TcG4 (M, N, O), TcG5 (P, Q, R), and TcG6 (S, T, U) was determined by flow cytometry. Background staining with NMS (filled gray areas), antibody to parasite-specific intracellular protein GPI8 (dark solid lines), and nonspecific Y3 antibody to MHC class I (broken lines) are shown in panels A, B, and C. Positive staining with C10 antibody to GP50/55 surface protein (panel A, solid line) and CMS (A, B, C, broken line) is shown. The percentage of fluorescent positive parasites (M2) staining above the background level (M1) are shown in each panel. In panels A, B, and C, the percent positive fluorescence is given for parasites stained with CMS.

FIG. 5.

TcG1-TcG6-encoded antigens elicit parasite-specific antibody response. ELISA was performed with polyclonal sera obtained from mice immunized with TcG1-TcG6-encoding expression plasmids. Sera from normal mice (NMS) and mice immunized with pCDNA 3.1 alone were utilized as negative controls. Serum from chronically infected mice (CMS) was used as a positive control.