Strategies for the indentification of targets of cellular immune responses in chronic Chagas disease

LAUCELLA S, POSTAN M, ALBAREDA MC, ARMENTI A, BARBIERI G, TARLETON R

Rosario, Argentina

Congreso; XX Reunión Anual de la Sociedad Argentina de Protozoología; 2004

The primary clinical consequence of T. cruzi infection is a chronic chagasic cardiomyopathy which manifests in approximately in 30% of infected individuals many years after the initial infection. Although the etiology of Chagas disease is controversial, a significant body of evidence supports the hypothesis that T. cruzi persists at sites of disease and is thus the stimulus for the chronic inflammatory response that results in tissue damage. Then, the stimulation of an effective set of immune responses that efficiently limit the parasite load in tissues should result in less-severe disease. However, support for this latter hypothesis , with regard to human patients is very limited. The aim of the present study was to quantify CD8+ T cell responses specific for T. cruzi-derived peptides in the peripheral blood of patients with chronic Chagas disease, who differed with respect to degree of cardiac dysfunction. To accomplish this goal, we used 2 tools that have become standards for assessing CD8+ T cell function in human infections: staining with class I major histocompatibility complex tetramers and analysis of peptide-induced ELISPOT responses of cytokine-secreting cells. Trans-sialidase, calcium binding (CaBP), and LYT-1-derived peptides were selected for this study because they were reported to be target of citotoxic responses (CTL) in mice and humans. Trans-sialidase and LYT-1 proteins were also shown to protect against a lethal infection with T. cruzi The analysis was restricted to HLA-A2.1-binding peptides due to the high frequency of expression of this allele in individuals of various ethnic backgrounds, including those living in areas endemic for T. cruzi infection. Staining with class I MHC-tetrameric complexes containing T. cruzi trans-sialidase-derived peptides failed to identify parasite-specific CD8+ T cells in chronic chagasic patients living in non-endemic area. Twenty one percent [6 out of 29] of chagasic patients with asymptomatic [Group 0] or mild disease [Group 1] showed significant numbers of IFN-gamma secreting CD8+ T lymphocytes in response to trans-sialidase or CaBP-derived peptides. Patients with severe forms of the disease [Groups 2 and 3] did not response (0 out of 8) to any of the target proteins assayed. However the frequency of individuals with positive ELISPOT responses was higher in individuals living in areas of active transmission versus those in areas where the possibility of re-exposure to infection was minimal. Analysis of IFN-gamma ELISPOT responses to a wider range of parasite antigens using a lysate from T. cruzi amastigotes as stimulus, revealed substantial CD8+ and CD4+ T-cell responses in the individuals living in non-endemic area, who had been largely unresponsive to the T. cruzi peptides, with 71% (22 out of 31) of individuals in the G0 and G1 groups responding but only 14% (2 out of 14) individuals in the G3 group responding. Since antigen abundance might influence natural immune responses, it is possible that the low responses to T. cruzi peptides recorded in chronic chagasic patients be due to a poor representation of it in the T. cruzi genome. A whole genome screened for homologues of 2 trans-sialidase-derived peptides that are CTL targets in humans was performed and it was also determined the frequency of occurrence of these epitopes in the T. cruzi genome sequences. Seventy one peptides with a high frequency of occurrence and good HLA-A2.1-binding were synthesized and their antigenicity assayed by IFN-gamma ELISPOT in A2.1 transgenic mice infected with T. cruzi. Peptides with the higher frequency of occurrence and the higher A2-1-binding affinity turned to be the more antigenic in mice. IFN-gamma ELISPOT responses to these peptides were evaluated in the circulation of chronic chagasic patients. Ten out of 17 (59%) G0 patients displayed positive responses to 1-23 peptides from a total of 28 peptides assayed. These data demonstrate that the frequency of IFN-gamma producing T cells in chronic chagasic patients is determined in part by the history of recent re-exposure, by the clinical status of the patient and also by the frequency of occurrence of the epitopes in the T. cruzi genome. The identification of CD8+ T cell targets in the natural infection with T. cruzi has important implications for the development of a vaccine against T. cruzi.