Rick L. Tarleton

Chagas Disease Etiology: Autoimmunity or Parasite Persistence?

The question of the cause and the mechanisms of disease in chronic Trypanosoma cruzi infection continues to attract debate. Chagas disease, characterized by cardiomyopathy and/or megasyndrome involving the esophagus or colon, occurs in approximately 30% of individuals with chronic T. cruzi infections. Although the pathogenesis of Chagas disease is often attributed to autoimmune mechanisms, definitive proof of anti-self responses as the primary cause of disease in T. cruzi-infected hosts is lacking. Rick Tarleton and Lei Zhang here consider an alternative view that the primary cause of chronic Chagas disease is the failure of the host to clear the infection, resulting in infection-induced, immune-mediated tissue damage.

The challenges of Chagas Disease-- grim outlook or glimmer of hope:

The authors discuss the key challenges that undermine the control of Chagas disease and that must be urgently addressed to ensure long-term, sustainable control.

CD8+ T-Cell Responses to Trypanosoma cruzi Are Highly Focused on Strain-Variant trans-Sialidase Epitopes

CD8+ T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8+ T cells in T. cruzi–infected mice and humans. This analysis demonstrates that in both hosts the CD8+ T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi–infected mice, with more than 30% of the CD8+ T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level.

Drug-induced cure drives conversion to a stable and protective CD8 + T central memory response in chronic Chagas disease

In this study, we document the development of stable, antigen-independent CD8+ T cell memory after drug-induced cure of a chronic infection. By establishing a system for drug cure of chronic Trypanosoma cruzi infection, we present the first extensively documented case of total parasite clearance after drug treatment of this infection. Cure resulted in the emergence of a stable, parasite-specific CD8+ T cell population with the characteristics of central memory cells, based upon expression of CD62L, CCR7, CD127, CD122, Bcl-2 and a reduced immediate in vivo CTL function. CD8+ T cells from treated and cured mice also expanded more rapidly and provided greater protection following challenge than those from chronically infected mice. These results show that complete pathogen clearance results in stable, antigen-independent and protective T cell memory, despite the potentially exhausting effects of prior long-term exposure to antigen in this chronic infection.

A Heuristic Method for Assigning a False-discovery Rate for Protein Identifications from Mascot Database Search Results

MS/MS and database searching has emerged as a valuable technology for rapidly analyzing protein expression, localization, and post-translational modifications. The probability-based search engine Mascot has found widespread use as a tool to correlate tandem mass spectra with peptides in a sequence database. Although the Mascot scoring algorithm provides a probability-based model for peptide identification, the independent peptide scores do not correlate with the significance of the proteins to which they match. Herein, we describe a heuristic method for organizing proteins identified at a specified false-discovery rate using Mascot-matched peptides. We call this method PROVALT, and it uses peptide matches from a random database to calculate false-discovery rates for protein identifications and reduces a complex list of peptide matches to a nonredundant list of homologous protein groups. This method was evaluated using Mascot-identified peptides from a Trypanosoma cruzi epimastigote whole-cell lysate, which was separated by multidimensional LC and analyzed by MS/MS. PROVALT was then compared with the two traditional methods of protein identification when using Mascot, the single peptide score and cumulative protein score methods, and was shown to be superior to both in regards to the number of proteins identified and the inclusion of lower scoring nonrandom peptide matches.

Frequency of Interferon-γ-Producing T Cells Specific for Trypanosoma cruzi Inversely Correlates with Disease Severity in Chronic Human Chagas Disease

This study sought to quantify CD8(+) T cell responses to Trypanosoma cruzi and to identify potential links between these responses and the severity of disease in humans. In the majority of patients with Chagas disease, staining with class I major histocompatibility complex tetramers and analysis of interferon (IFN)- gamma ELISPOT responses to a panel of known cytotoxic T lymphocyte target epitopes from T. cruzi failed to identify parasite-specific CD8(+) T cells. However, the frequency of individuals with positive ELISPOT responses was higher in areas of active transmission. Analysis of IFN- gamma ELISPOT responses to a parasite lysate revealed a very high frequency of responders among patients with mild clinical disease and a very low frequency of responders among those with the most severe form of the disease. These data suggest that the frequency of IFN- gamma -producing T cells in patients with chronic Chagas disease is associated with the history of recent exposure and with the clinical status of the patient.

Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR

A real-time PCR technique that allows for accurate and sensitive quantitation of tissue parasite burden in animals infected with the protozoan parasite Trypanosoma cruzi was developed. The utility of this method was demonstrated by confirmation of higher parasite load in mice with acute infections in comparison to chronically infected mice, detection of tissue-restricted parasite persistence in different parasite:host strain combinations, and the observation of increased tissue parasite burden with higher infective doses. This method should be a useful tool for monitoring parasite burden in hosts under various treatment regimens.

The relative contribution of antibody production and CD8+ T cell function to immune control of Trypanosoma cruzi.

The life cycle of the protozoan parasite Trypanosoma cruzi in mammalian hosts includes both non‐dividing trypomastigote forms which circulate in the blood and replicating intracellular amastigotes which reside within the cytoplasm of a variety of host cells. In this study we have used mice with induced mutations in genes responsible for either antibody production or cytolytic T lymphocyte (CTL) function to examine the relative contributions of these effector mechanisms to control of T. cruzi. Mice deficient in the production of antibodies exhibited a delay in the rise in acute phase parasitaemia and an extended time to death relative to mice lacking CD8+ T cells. Nevertheless, B cell deficient mice eventually succumbed to the infection. Prior infection with an avirulent strain of T. cruzi failed to protect either CD8+ T cell‐deficient mice or B cell deficient mice from challenge infection with virulent parasites. In contrast, mice with disruptions in the genes controlling perforin‐ or granzyme B‐mediated cytolytic pathways had parasitaemia and mortality rates similar to wild‐type mice and were protected from secondary infection by prior exposure to avirulent parasites. These results 1) confirm that antibody production, although secondary in importance to cellular responses, is nevertheless absolutely required and 2) perforin‐ or granzyme B‐mediated lytic pathways are not required for control of T. cruzi infection.

CD8+ T cells in Trypanosoma cruzi infection

CD8(+) T cells have emerged as crucial players in the control of a number of protozoan pathogens, including Trypanosoma cruzi, the agent of human Chagas disease. The recent identification of the dominant targets of T. cruzi-specific T cells has allowed investigators to follow the generation of and document the functionality of T cell responses in both mice and humans. Although slow to develop in the early stages of the infection, T. cruzi-specific CD8(+) T cells reach prodigious levels and remain highly functional throughout chronic infections in mice. Following drug-induced cure during either the acute or chronic stage, these immunodominant T cells persist as stable, antigen-independent memory populations. T. cruzi-specific CD8(+) T cells in humans are less-well-studied but appear to lose functionality and decline in numbers in these decades-long infections. Changes in the frequency of parasite-specific T cell upon therapeutic treatment in humans may provide a new metric for determining treatment efficacy.

Genetic Immunization Elicits Antigen-Specific Protective Immune Responses and Decreases Disease Severity in Trypanosoma cruzi Infection

ABSTRACT Immunity to Trypanosoma cruzi requires elicitation of humoral and cell-mediated immune responses to extracellular trypomastigotes and intracellular amastigotes. In this study, the effectiveness of the T. cruzi trans-sialidase family (ts) genes ASP-1, ASP-2, and TSA-1 as genetic vaccines was assessed. Immunization of mice with plasmids encoding ASP-1, ASP-2, or TSA-1 elicited poor antigen-specific cytotoxic-T-lymphocyte (CTL) activity and T. cruzi-specific antibody responses. Codelivery of interleukin-12 and granulocyte-macrophage colony-stimulating factor plasmids with antigen-encoding plasmids resulted in a substantial increase in CTL activity and antibody production and in increased resistance to T. cruzi infection. In pooled results from two to four experiments, 30 to 60% of mice immunized with antigen-encoding plasmids and 60 to 80% of mice immunized with antigen-encoding plasmids plus cytokine adjuvants survived a lethal challenge with T. cruzi. In comparison, 90% of control mice injected with empty plasmid DNA died during the acute phase of infection. However, the pool of three ts genes provided no greater protection than the most effective single gene (ASP-2) either with or without coadministration of cytokine plasmids. Importantly, the extent of tissue parasitism, inflammation, and associated tissue damage in skeletal muscles during the chronic phase of T. cruzi infection in mice immunized with antigen-encoding plasmids plus cytokine adjuvants was remarkably reduced compared to mice immunized with only cytokine adjuvants or empty plasmid DNA. These results identify new vaccine candidates and establish some of the methodologies that might be needed to develop effective vaccine-mediated control of T. cruzi infection. In addition, this work provides the first evidence that prophylactic genetic immunization can prevent the development of Chagas’ disease.