Changes in Trypanosoma cruzi-specific immune responses after treatment: surrogate markers of treatment efficacy

Abstract

Background: As many as 20 million people are living with Trypanosoma cruzi infection in Latin American, yet few receive any treatment. The major limitation in developing and evaluating potential new drugs for their efficacy is the lack of reliable tests to assess parasite burden and elimination.

Methods: Adults volunteers with chronic T. cruzi infection were evaluated clinically and stratified according to the Kuschnir classification. Individuals with group 0 and group 1 clinical status were treated with benznidazole (5 mg/kg per day for 30 days). The changes in T. cruzi-specific T cell and antibody responses, as well as in clinical status, were measured periodically over the 3-5-year follow-up period and were compared with pretreatment conditions and with values in an untreated control group.

Results: The frequency of peripheral interferon (IFN)-gamma-producing T cells specific for T. cruzi declined as early as 12 months after benznidazole treatment and subsequently became undetectable in a substantial proportion of treated subjects. In addition, decreases in antibody responses to a pool of recombinant T. cruzi proteins also decreased in many of these same subjects. The shift to negative IFN-gamma T cell responses was highly associated with an early increase in IFN-gamma-producing T cells with phenotypic features of effector/effector memory cells in a subset of subjects. Benznidazole treatment also resulted in an increase in naive and early differentiated memory-like CD8(+) T cells in a majority of subjects.

Conclusions: Benznidazole treatment during chronic Chagas disease has a substantial impact on parasite-specific immune response that is likely indicative of treatment efficacy and cure.

Figure 1

Effect of treatment with BZ on T. cruzi-specific T cell responses in chronic infection. IFN- g-producing T cells specific for a parasite lysate/1×10⁶ PBMC were determined by ELISPOT at baseline and 12 months after with BZ. Each line represents an individual subject. (*) P =0.041 Mann-Whitney U test on PT/pre-treatment differences between treated and untreated groups as described in the Materials and Methods.

Figure 2

Monitoring of IFN- g ELISPOT responses in chronic Chagas disease subjects with positive ELISPOT responses at baseline. IFN- g-producing T cells were measured at different time points following BZ treatment or enrolment (for untreated subjects). Plots represent the data for single subjects from a selected group. IFN- g-secreting T cells significantly increased at 2-6 months post treatment and decreased thereafter as determined by Friedman range test (P=0.012).

Figure 3

Monitoring of T. cruzi-specific IFN- g ELISPOT responses in subjects with negative ELISPOT responses at baseline. IFN- g-producing T cells were measured at different time points following BZ treatment or enrollment (for untreated subjects). Plots show representative data for single subjects from a selected group. IFN- g-secreting T cells significantly increased at 2-6 months post treatment and decreased thereafter as determined by Friedman range test (P=0.012). The horizonal line indicates the threshold for positive/negative response.

Figure 4

Monitoring of IFN- g and IL-2 ELISPOT responses in chronic Chagas disease subjects following treatment with BZ. IFN- g (solid line) and IL-2 (broken line)-producing T cells were measured at different time points of follow-up in BZ-treated and untreated subjects. Time “0” indicates assay point just prior to BZ treatment. Plots show representative data for single subjects whose IFN- g ELISPOT responses became negative during follow-up (PP06), decreased > 3 fold, showed a rebound (PP01, PP120, PP179) or remained stable (PP96, PP100 and RD31). The criteria for determining “postive responses are defined in Materials and Methods.

Figure 5

Phenotype of T. cruzi-specific CD4⁺ T cells early after treatment with BZ. A. Distribution of CD122, CD127, CCR7 and KLRG1 expression on CD4⁺ IFN- g⁺ T cells after stimulation with a T. cruzi lysate. A gate was set on total CD4⁺ T cells with side-scatter and forward-scatter light for lymphocytes. The percentage of CD4⁺ IFN- g⁺ T cells with low (upper left quadrant) or high (upper right quadrant) expression for each marker is indicated. B. Mean ±SE of cumulative data on the proportion of T. cruzi-specific CD4⁺ IFN- g⁺ T cells within the different phenotypic cell populations 6 months following treatment with BZ in 7 T. cruzi-infected subjects. The percentage of CD4⁺ IFN- g⁺ T cells expressing each marker is shown. (*) P < 0.0001 vs CD122, CCR7 and KLRG-1; (#) P < 0.0001 vs CCR7 and CD122.

Figure 6

Multiplex analysis of anti-T. cruzi antibodies before and after treatment. Sera obtained at the indicated time points were screened using a bead array-based multiplex serologic assay against 14 recombinant T. cruzi proteins (Antigens 1-14) as well as a negative control protein (green fluorescent protein, GFP) and an T. cruzi amastigote lysate, as previously described in detail (28). Subject not treated (A) or treated (B) with BZ (at time t=0) did not exhibit a consistent change in antibody levels over 36 and 31 months, respectively. Different patterns of alterations in antibody responses was observed in treated subjects, including changes in antibodies specific for all (C), or just some (D), of the T. cruzi proteins. Subject PP118 had a stable pattern of antibodies for 12 months prior to treatment (E) but showed a steady decline in responses to most proteins apparent within 12 months following treatment (F). * no data – insufficient numbers of beads recovered for accurate measurement.