Immunization with cDNA Expressed by Amastigotes ofTrypanosoma cruziElicits Protective Immune Response against Experimental Infection

Abstract

Immunization of mice with plasmids containing Trypanosoma cruzi genes induced specific antibodies, CD4 ؉ Th1 and CD8؉ Tc1 cells, and protective immunity against infection. In most cases, plasmids used for DNA vaccination contained genes encoding antigens expressed by trypomastigotes, the nonreplicative forms of the parasite. In this study, we explored the possibility of using genes expressed by amastigotes, the form of the parasite which replicates inside host cells, for experimental DNA vaccination. For that purpose, we selected a gene related to the amastigote surface protein 2 (ASP-2), an antigen recognized by antibodies and T cells from infected mice and humans, for our study. Using primers specific for the asp-2 gene, four distinct groups of genes were amplified from cDNA from amastigotes of the Y strain of T. cruzi. At the nucleotide level, they shared 82.3 to 89.9% identity with the previously described asp-2 gene. A gene named clone 9 presented the highest degree of identity with the asp-2 gene and was selected for immunological studies. Polyclonal antisera raised against the C terminus of the recombinant protein expressed by the clone 9 gene reacted with an antigen of approximately 83 kDa expressed in amastigotes of T. cruzi. Immunization of BALB/c mice with eukaryotic expression plasmids containing the clone 9 gene elicited specific antibodies and CD4 ؉ T-cell-dependent gamma interferon secretion. Upon challenge with trypomastigotes, mice immunized with plasmids harboring the clone 9 gene displayed reduced parasitemia and survived lethal infection. We concluded that amastigote cDNA is an interesting source of antigens that can be used for immunological studies, as well as for vaccine development.

Infection with Trypanosoma cruzi Restricts the Repertoire of Parasite-Specific CD8+ T Cells Leading to Immunodominance

“…This fact may accelerate and improve immune response by providing an advantage for T cells specific for H-2K b -VNHRFTLV complexes on the surface of APC. In contrast, the epitopes IYNVGQVSI or TEWETGQI are expressed only by trypomastigotes (infective forms) or amastigotes (intracellular stages), respectively (38,39). This last hypothesis would explain the differences observed following infection or immunization of heterozygote mice with adenovirus-expressing parasite Ag.…”

“…Earlier studies have shown that prolonged Ag exposure influences the generation of immune responses improving priming of T cells specific for immunodominant epitopes (34 -36). Indeed, VNHRFTLV epitope is present in proteins expressed by trypomastigotes (infective forms) and amastigotes (intracellular stages) of the Y strain of T. cruzi (37,38). This fact may accelerate and improve immune response by providing an advantage for T cells specific for H-2K b -VNHRFTLV complexes on the surface of APC.…”

Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells

“…However, the latter EVs contain a number of additional proteins that were preferentially enriched in immunoprecipitates of the TESA EVs. Examples include the amastigote surface proteins ASP2 and ASP3 (51,52), paraflagellar rod proteins, several mitochondrial membrane proteins (ATP synthase, ADP/ATP translocase, and cytochrome c 1 ), the glycosome membrane protein Gim5A, and the Golgi apparatus protein UDP-galactose glycosyltransferase (Tables S2 and S3). Although our studies focused on EVs released into the culture supernatant following trypomastigotemediated rupture of Vero cells, proteomic analysis revealed that EVs contained both Vero and T. cruzi proteins.…”

Genetic Immunization Converts theTrypanosoma cruziB-Cell Mitogen Proline Racemase to an Effective Immunogen

“…As is the case with many infectious disease immunization strategies, the approach to T. cruzi immunization has been focused primarily on the introduction of immunodominant targets that would initiate a strong secondary response upon primary infection with parasite (6,10,19,20,41,52,67,68,70). The novel strategy presented here was to induce an immune response to a parasite-derived immune evasion factor, in this case a B-cell mitogen.…”