Immunization with recombinant paraflagellar rod protein induces protective immunity against Trypanosoma cruzi infection.

In the present study, we have produced recombinant paraflagellar rod proteins (PFR) and report their use for successful vaccination of mice against Trypanosoma cruzi. This protection is associated with a highly polarized type 1 cytokine production profile. Additionally, we have analyzed the gene sequence encoding PFR-2 to determine the degree of conservation among seven highly diverse strains of T. cruzi, and found it to be highly conserved. The results presented here indicate that the PFR antigens are highly conserved, and immunization with rPFR-1, PFR-2, or an equimolar mix of the PFR-1, -2, and -3 proteins provides protective immunity against Trypanosoma cruzi.

Humoral and cellular immune responses to Trypanosoma cruzi-derived paraflagellar rod proteins in patients with Chagas' disease.

Sera and peripheral blood mononuclear cells (PBMC) from patients displaying different clinical symptoms as well as from normal uninfected individuals (NI) were used to evaluate the humoral and cellular responses of Chagas' disease patients to Trypanosoma cruzi-derived paraflagellar rod proteins (PFR). Our results show that sera from both asymptomatic Chagas' disease patients (ACP) and cardiac Chagas' disease patients (CCP) have higher levels of antibodies to PFR than sera from NI. Immunoglobulin G1 (IgG1) and IgG3 were the main Ig isotypes that recognized PFR. We also tested three recombinant forms of PFR, named rPAR-1, rPAR-2, and rPAR-3, by Western blot analysis. Sera from seven out of eight patients with Chagas' disease recognized one of the three rPAR forms. Sera from 75, 50, and 37.5% of Chagas' disease patients tested recognized rPAR-3, rPAR-2, and rPAR-1, respectively. PFR induced proliferation of 100 and 70% of PBMC from ACP and CCP, respectively. Further, stimulation of cells from Chagas' disease patients with PFR enhanced the frequencies of both small and large CD4(+) CD25(+) and CD4(+) CD69(+) lymphocytes, as well as that of small CD8(+) CD25(+) lymphocytes. Finally, we evaluated the ability of PFR to elicit the production of gamma interferon (IFN-gamma) by PBMC from patients with Chagas' disease. Fifty percent of the PBMC from ACP as well as CCP produced IFN-gamma upon stimulation with PFR. PFR enhanced the percentages of IFN-gamma-producing cells in both CD3(+) and CD3(-) populations. Within the T-cell population, large CD4(+) T lymphocytes were the main source of IFN-gamma.

Paraflagellar rod protein-specific CD8+ cytotoxic T lymphocytes target Trypanosoma cruzi-infected host cells.

Our previous studies show that in mice immunized with the paraflagellar rod (PFR) proteins of Trypanosoma cruzi protective immunity against this protozoan parasite requires MHC class I-restricted T cell function. To determine whether PFR-specific CD8+ T cell subsets are generated during T. cruzi infection, potential CTL targets in the PFR proteins were identified by scanning the amino acid sequences of the four PFR proteins for regions of 8-10 amino acids that conform to predicted MHC class I H-2b binding motifs. A subset of the peptide sequences identified were synthesized and tested as target antigen in 51Cr-release assays with effector cells from chronically infected T. cruzi mice. Short-term cytotoxic T lymphocyte (CTL) lines specific for two of the peptides, PFR-1(164-171) and PFR-3(123-130), showed high levels of lytic activity against peptide-pulsed target cells, secreted interferon (IFN)-gamma in response to parasite-infected target cells, and were found to be CD8+, CD4-, CD3+, TCRalphabeta+ cells of the Tc1 subset. Challenge of PFR immunized CD8-/- and perforin-deficient (PKO) mice confirmed that while CD8+ cells are required for survival of T. cruzi challenge infection, perforin activity is not required. Furthermore, while lytic activity of PFR-specific CD8+ T cell lines derived from PKO mice was severely impaired, the IFN-gamma levels secreted by CTLs from PKO mice were equivalent to that of normal mice, suggesting that the critical role played by CD8+ T cells in immunity to the parasite may be secretion of type 1 cytokines rather than lysis of parasite infected host cells.