Humoral and Cellular Immune Response in Asymptomatic Dogs with Visceral Leishmaniasis: A Review.

Visceral leishmaniasis is one of the deadliest parasitic diseases in the world and affects both humans and dogs. The host immune response to Leishmania infection plays a critical role in the evolution of canine visceral leishmaniasis (CVL) and consequently in the manifestation of clinical signs. The asymptomatic form of the disease is a major concern in the diagnosis of CVL and in the transmission control of Leishmania infection. Asymptomatic dogs are found in large proportions in endemic areas and are an unquantifiable source of infection. The present review analyzes the possible relationship between the activation of the antigen-specific immune response of the host and resistance or susceptibility to CVL. The review focuses on works that address the characterization of the humoral and cellular immune response profile, at both the functional and phenotypic levels, in infected dogs. Most studies relate the absence of clinical symptomatology to an increased proliferative response and a Th1 cytokine profile. Despite the numerous findings pointing to a differential immune response in asymptomatic dogs, the contradictory results reported in this review highlight the importance of establishing a precise clinical classification of the disease, performing more longitudinal studies, and including a higher number of animals in trials.

Impact of benznidazole treatment on the functional response of Trypanosoma cruzi antigen-specific CD4+CD8+ T cells in chronic Chagas disease patients.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi. The persistence of the parasite is associated with the disease chronicity and the impairment of the cellular immune response. It has been reported that the CD4+CD8+ T cell population expands in chronic Chagas disease patients. Few studies have focused on this subset of cells, and very little is known about the impact of antiparasitic treatment on this population. METHODOLOGY: Thirty-eight chronic Chagas disease patients (20 asymptomatic and 18 symptomatic) and twelve healthy controls were enrolled in this study. Peripheral blood mononuclear cells were stimulated with soluble T. cruzi antigens to analyze the production of cytokines and cytotoxic molecules by CD4+CD8+ T cells before and after benznidazole treatment. Additionally, expression and co-expression of five inhibitory receptors in these patients after treatment were studied using a multiparameter flow cytometry technique. PRINCIPAL FINDINGS: The frequency of CD4+CD8+ T cells was higher in chronic Chagas disease patients compared with healthy donors. Furthermore, a higher ratio of CD4+CD8low/CD4+CD8high subpopulations was observed in chronic Chagas disease patients than in healthy donors. Additionally, CD4+CD8+ T cells from these patients expressed and co-expressed higher levels of inhibitory receptors in direct proportion to the severity of the pathology. Benznidazole treatment reduced the frequency of CD4+CD8+ T cells and decreased the ratio of CD4+CD8low/CD4+CD8high subpopulations. The co-expression level of the inhibitory receptor was reduced after treatment simultaneously with the enhancement of the multifunctional capacity of CD4+CD8+ T cells. After treatment, an increase in the frequency of T. cruzi antigen-specific CD4+CD8+ T cells expressing IL-2 and TNF-α was also observed. CONCLUSIONS: CD4+CD8+ T cells could play an important role in the control of T. cruzi infection since they were able to produce effector molecules for parasite control. Benznidazole treatment partially reversed the exhaustion process caused by T. cruzi infection in these cells with an improvement in the functional response of the T. cruzi antigen-specific CD4+CD8+ T cells.

A 12-mer repetitive antigenic epitope from Trypanosoma cruzi is a potential marker of therapeutic efficacy in chronic Chagas' disease.

OBJECTIVES: The objective was to characterize a Trypanosoma cruzi repetitive amino acid sequence that can be used as a marker of therapeutic drug efficacy in patients with chronic Chagas' disease. METHODS: Reactivities to the 3973 peptide were measured in 85 patients with Chagas' disease (41 in the asymptomatic stage and 44 in the cardiomyopathy stage) before and 9 and 24 months after benznidazole administration. Additionally, the levels of IL-6 and C-reactive protein were measured in serum samples from patients with cardiomyopathy. RESULTS: In 85% of the asymptomatic patients and 73% of the symptomatic chronic patients, modifications of the reactivity to the 3973 peptide were observed at 9 and 24 months post-benznidazole treatment. Significant variations in reactivities to the total antigens of T. cruzi were not observed at these times. Significant decreases in the reactivity to the 3973 peptide were observed after treatment in 20 of 41 (49%) asymptomatic patients and 15 of 44 (34%) cardiac chagasic patients (P < 0.001). In these patients, the decreases in reactivity at 24 months post-treatment were at least 40% lower than those detected before treatment. No correlations were found of the detected modifications in reactivity to the 3973 peptide after treatment with the levels of C-reactive protein or IL-6. CONCLUSIONS: Decreases in reactivity to the 3973 peptide may be relevant in the post-treatment follow-up of chronic chagasic patients.

Promiscuous Recognition of a Trypanosoma cruzi CD8+ T Cell Epitope among HLA-A2, HLA-A24 and HLA-A1 Supertypes in Chagasic Patients.

BACKGROUND: TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide. METHODOLOGY/PRINCIPAL FINDINGS: From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8+ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8+ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8+ T cells with frequencies ranging from 0.07-0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8+ T cells producing at least one cytokine after TcTLE peptide stimulation. CONCLUSION/SIGNIFICANCE: These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy.

Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein.

The TcTLE peptide (TLEEFSAKL) is a CD8(+) T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8(+) T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8(+) T cells in HLA-A2(+) chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8(+) T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope.

Biomarkers of therapeutic responses in chronic Chagas disease: state of the art and future perspectives

The definition of a biomarker provided by the World Health Organization is any substance, structure, or process that can be measured in the body, or its products and influence, or predict the incidence or outcome of disease. Currently, the lack of prognosis and progression markers for chronic Chagas disease has posed limitations for testing new drugs to treat this neglected disease. Several molecules and techniques to detect biomarkers in Trypanosoma cruzi-infected patients have been proposed to assess whether specific treatment with benznidazole or nifurtimox is effective. Isolated proteins or protein groups from different T. cruzi stages and parasite-derived glycoproteins and synthetic neoglycoconjugates have been demonstrated to be useful for this purpose, as have nucleic acid amplification techniques. The amplification of T. cruzi DNA using the real-time polymerase chain reaction method is the leading test for assessing responses to treatment in a short period of time. Biochemical biomarkers have been tested early after specific treatment. Cytokines and surface markers represent promising molecules for the characterisation of host cellular responses, but need to be further assessed.

Trypanosoma cruzi paraflagellar rod proteins 2 and 3 contain immunodominant CD8(+) T-cell epitopes that are recognized by cytotoxic T cells from Chagas disease patients.

The protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. To date, no vaccine is available for protection against T. cruzi infection. The CD8(+) T cells immune response against specific antigens has shown to efficiently control the spread of the parasite in murine experimental infection. However, data concerning CD8(+) response in Chagas patients are still restricted to a few epitopes. We have studied the existence of immunodominant CD8(+) T cell epitopes in the paraflagellar rod proteins 2 and 3 (PFR2 and PFR3) from T. cruzi in a mouse model, and analyzed their recognition by cytotoxic T lymphocytes from Chagas disease patients. Immunization of C57BL/6-A2/K(b) transgenic mice with plasmids coding for the fusion proteins PFR2-HSP70 and PFR3-HSP70 induced a specific CTL response against two PFRs epitopes (PFR2(449-457) and PFR3(481-489)), and showed specific lysis percentages of 24 and 12, respectively. Moreover, the PFR2(19-28), PFR2(156-163), PFR2(449-457), PFR3(428-436), PFR3(475-482) and PFR3(481-489) peptides were observed to have a high binding affinity to the HLA-A*02:01 molecule. Remarkably, these HLA-A*02:01-binding peptides are successfully processed and presented during natural infection by T. cruzi in the context of MHC class I as evidenced by using peptide-pulsed K562-A2 cells as antigen presenting cells. The T cells from Chagas disease chronic patients specific for PFR2/PFR3 selected CD8(+) epitopes showed a pro-inflammatory cytokine secretion profile (IFN-γ, TNF-α and IL-6). A positive Granzime B secretion was observed in three out of 16 patients in response to PFR2(156-163) and PFR2(449-457) peptides, two out of 11 patients in response to PFR2(19-28) peptide and one out of 14 and 11 patients in response to PFR3(428-436) and PFR3(481-489) peptides, respectively. The PFRs-specific cytotoxic activity in purified PBMC was only detected in patients in the indeterminate phase of the disease.

Identification of HLA-A∗02:01-restricted CTL epitopes in Trypanosoma cruzi heat shock protein-70 recognized by Chagas disease patients.

CD8(+) cytotoxic T lymphocyte (CTL) response is critical for controlling the infection of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Since only a few CD8 antigens have been described in Chagas disease patients, the identification of new class I-restricted epitopes is urgently needed for the development of immunotherapies against T. cruzi infection. In this study, bioinformatic methods were used to predict HLA-A∗02:01-binders, and 30 peptides were selected, synthesized and tested for HLA-A∗02:01 binding. Among them, sixteen peptides with medium-to-high affinity were assayed for their recognition by CTL from HSP70-immunized or T. cruzi-infected transgenic B6-A2/K(b) mice. Our results show that four immunodominant epitopes (HSP70(210-8), HSP70(255-63), HSP70(316-24) and HSP70(345-53)) are contained in the T. cruzi HSP70 antigen. Indeed two of them (HSP70(210-8) and HSP70(316-24)) were also recognized by CTL of HLA-A∗02:01(+) Chagas disease patients, indicating that these peptides are processed and displayed as MHC class I epitopes during the natural history of T. cruzi infection. The HLA-A∗02:01 restriction was evidenced using peptide-pulsed K562-A2 cells as antigen-presenting cells. Both cytotoxic and cytokine-secreting activities were detected in response to the former two peptides and, moreover, 10/12 patients (83%) recognized at least one of these two HSP70-derived CD8(+) epitopes.

Monocyte-derived dendritic cells from chagasic patients vs healthy donors secrete differential levels of IL-10 and IL-12 when stimulated with a protein fragment of Trypanosoma cruzi heat-shock protein-70.

We analyzed the effect of the truncated heat-shock protein 70 from Trypanosoma cruzi on maturation of human dendritic cells (DCs) derived from monocytes of peripheral blood mononuclear cells from healthy donors and chagasic patients. The results show that the T-HSP70 is capable of maturing human DCs inducing an increase in the expression level of the CD83, CD86 and human leukocyte antigen-DR surface markers, as well as in the secretion of interleukin (IL)-12, tumor necrosis factor-alpha (TNF-alpha) and IL-6 cytokines. Results also show the existence of a differential functional activity of matured DCs from chagasic patients vs healthy donors in response to T-HSP70 protein and to HSP-70-derived A72 peptide, as only T-HSP70-matured DCs from chagasic patients have an enhanced secretion of IL-10 and a reduced secretion of IL-12. Moreover, the addition of A72 peptide to immature DCs from chagasic patients induced an increase in the percentage of cells expressing CD83 and CD86 molecules regarding to the expression level observed by cells from healthy donors. These findings suggest that T. cruzi HSP70 protein may induce a specific maturation profile on chagasic patients' DCs, which would favor the persistence of the parasite in the human host.

[Expression of markers on dendritic cells from chronic chagasic patients stimulated with the KMP-11 protein and the K1 peptide from Trypanosoma cruzi]. / Expresión de marcadores en células dendríticas de pacientes chagásicos crónicos estimuladas con la proteína KMP-11 y el péptido K1 de Trypanosoma cruzi.

INTRODUCTION: The kinetoplastid membrane protein 11, KMP-11, from Trypanosoma cruzi elicits humoral and cellular immunity in mice that protects them from infection against further parasite challenge. OBJECTIVE: To characterize the expression of surface markers on dendritic cells from chronic chagasic patients and healthy individuals, in response to the KMP-11 protein from Trypanosoma cruzi and its N-terminal peptide K1. MATERIALS AND METHODS: Monocyte-derived dendritic cells from seven chronic chagasic patients and seven healthy individuals were stimulated with the KMP-11 protein and the K1 peptide. Seven days after culturing, the CD83, CD86, and HLA-DR membrane expression as well as the production of cytokines were evaluated by flow cytometry. RESULTS: Neither KMP-11 protein nor K1 peptide elicited the expression of the maturation marker CD83 on dendritic cells of patients or healthy control individuals. Dendritic cells from chronic chagasic patients exposed to K1 and LPS at the same time presented a significant reduction in CD86 and CD83 membrane expression in contrast to the cells exposed to LPS alone, whereas dendritic cells from healthy individuals did not show this behavior. The secretion of interleukin-12 was decreased in the cultures of dendritic cells from chronic chagasic patients but not from healthy controls. CONCLUSIONS: KMP-1 1 protein does not affect the maturation of dendritic cells, but in the presence of LPS the K1 peptide leads to a decreased expression of CD86 and CD83 as well as interleukin-12 production, This phenomenon may be associated with an impaired T cell stimulation.

Caracterización parcial de los genes codificantes para la proteína de choque térmico HSP70 de Trypanosoma rangeli / Partial characterization of coding genes for Trypanosoma rangeli HSP70

Objetivo: dada la relevancia inmunológica de la región amino terminal de la proteína HSP70 de la Tripanosoma cruzi, así como el hecho de que la inmunización de ratones con Tripanosoma rangeli protege a los animales contra la infección por T. cruzi, el presente trabajo se centró en el aislamiento y caracterización molecular del fragmento homólogo en T. rangeli. Materiales y métodos: la región amino terminal del gen codificante para la HSP70 de T. rangeli fue amplificada mediante PCR utilizando los oligonucleotidos TrHSP70f/R2, diseñados con base en la secuencia homóloga de T. cruzi. El fragmento amplificado fue purificado, clonado en el vector pGEM® –Teasy (Promega) y secuenciado en un 373 Automatic DNA sequencer (Applied Biosystems). La organización genómica de los genes HSP70 se determino mediante ensayos de “Southern blot” y PFGE. Resultados: los genes HSP70 de T. rangeli tienen un tamaño aproximado de 2.400 pb, se encuentran repetidos en tandem y se localizan en un cromosoma de 1.030 y 1.090 Kb en la cepa H14, KP1(+) y de 1.100 KB en la cepa Tre, KP1 (-). La secuencia de nucleótidos correspondiente a la región amino terminal de la proteína tiene una identidad del 99 por ciento entre las cepas H14 y Tre de T. rangeli y del 94 por ciento entre éstas y T. cruzi, preséntando además polimorfismos para diversas enzimas de restricción. La secuencia de amiácidos de dicha región entre ambos parásitos tiene una identidad del 95 por ciento. Conclusiones: el extremo amino terminal de las proteínas HSP70 de T. cruzi y T. rangeli guardan una elevada identidad de secuencia lo que abre un camino a la posible utilización de la HSP70 de T. rangeli en inmunoterapia. Asimismo y dado que en esta región se localizan epítopes B y T inductores de respuesta inmune en pacientes chagásicos, la HSP70 puede estar implicada en la reacción inmunológica cruzada entre estos parásitos