CD4+ T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes.

Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4(+) and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

An effective CTL peptide vaccine for Ebola Zaire Based on Survivors' CD8+ targeting of a particular nucleocapsid protein epitope with potential implications for COVID-19 vaccine design.

The 2013-2016 West Africa EBOV epidemic was the biggest EBOV outbreak to date. An analysis of virus-specific CD8+ T-cell immunity in 30 survivors showed that 26 of those individuals had a CD8+ response to at least one EBOV protein. The dominant response (25/26 subjects) was specific to the EBOV nucleocapsid protein (NP). It has been suggested that epitopes on the EBOV NP could form an important part of an effective T-cell vaccine for Ebola Zaire. We show that a 9-amino-acid peptide NP44-52 (YQVNNLEEI) located in a conserved region of EBOV NP provides protection against morbidity and mortality after mouse adapted EBOV challenge. A single vaccination in a C57BL/6 mouse using an adjuvanted microsphere peptide vaccine formulation containing NP44-52 is enough to confer immunity in mice. Our work suggests that a peptide vaccine based on CD8+ T-cell immunity in EBOV survivors is conceptually sound and feasible. Nucleocapsid proteins within SARS-CoV-2 contain multiple Class I epitopes with predicted HLA restrictions consistent with broad population coverage. A similar approach to a CTL vaccine design may be possible for that virus.

Autoimmunity in Chagas' disease. Identification of cardiac myosin-B13 Trypanosoma cruzi protein crossreactive T cell clones in heart lesions of a chronic Chagas' cardiomyopathy patient.

Heart tissue destruction in chronic Chagas' disease cardiomyopathy (CCC) may be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after Trypanosoma cruzi infection. Indirect evidence suggests there is molecular mimicry between T. cruzi and heart tissue. In murine models of CCC, antibodies and CD4+ T cells recognize myosin, the major heart protein. We recently identified a heart-specific epitope of cardiac myosin heavy chain (residues 1442-1447, AAALDK) that is crossreactive with a homologous sequence (AAAGDK) of the immunodominant T. cruzi antigen B13. Furthermore, cardiac myosin-B13 crossreactive antibodies are present in 100% CCC patients vs 14% asymptomatic T. cruzi-seropositive individuals (P = 2.3 x 10(-6)), suggesting a role for molecular mimicry between cardiac myosin and B13 in CCC pathogenesis. In this paper, we obtained heart-infiltrating T cell clones from CCC patients to assess whether molecular mimicry between cardiac myosin and B13 is directly involved in the genesis of heart lesions. We identified T cell clones derived from CCC heart lesions simultaneously responsive to cardiac myosin heavy chain (but not skeletal myosin heavy chain) and B13 T. cruzi protein, but could not find T cell clones primarily reactive to any T. cruzi antigen. Together with the association of myosin-B13 crossreactive antibodies with CCC, the present data strongly suggest the relevance of molecular mimicry between cardiac myosin and the T. cruzi protein B13 in the pathogenesis of heart lesions in chronic Chagas' disease cardiomyopathy.

Proteomic inventory of myocardial proteins from patients with chronic Chagas' cardiomyopathy.

Chronic Chagas' disease cardiomyopathy (CCC) is an often fatal outcome of Trypanosoma cruzi infection, with a poorer prognosis than other cardiomyopathies. CCC is refractory to heart failure treatments, and is the major indication of heart transplantation in Latin America. A diffuse myocarditis, plus intense myocardial hypertrophy, damage and fibrosis, in the presence of very few T. cruzi forms, are the histopathological hallmarks of CCC. To gain a better understanding of the pathophysiology of CCC, we analyzed the protein profile in the affected CCC myocardium. Homogenates from left ventricular myocardial samples of end-stage CCC hearts explanted during heart transplantation were subjected to two-dimensional electrophoresis with Coomassie blue staining; protein identification was performed by MALDI-ToF mass spectrometry and peptide mass fingerprinting. The identification of selected proteins was confirmed by immunoblotting. We demonstrated that 246 proteins matched in gels from two CCC patients. They corresponded to 112 distinct proteins. Along with structural/contractile and metabolism proteins, we also identified proteins involved in apoptosis (caspase 8, caspase 2), immune system (T cell receptor ss chain, granzyme A, HLA class I) and stress processes (heat shock proteins, superoxide dismutases, and other oxidative stress proteins). Proteins involved in cell signaling and transcriptional factors were also identified. The identification of caspases and oxidative stress proteins suggests the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. These results generated an inventory of myocardial proteins in CCC that should contribute to the generation of hypothesis-driven experiments designed on the basis of the classes of proteins identified here.

Inflammatory and cardiac biomarkers are differentially expressed in clinical stages of Chagas disease.

BACKGROUND: Chagas disease has a long clinically silent period following Trypanosoma cruzi infection and before development of overt clinical pathology; detectable biomarkers of infection and pathogenesis are urgently needed. We tested 22 biomarkers known to be associated with cardiomyopathy to evaluate if a biomarker signature could successfully classify T. cruzi seropositive subjects into clinical Chagas disease stage groups. METHODS: This cross-sectional retrospective case-control study enrolled T. cruzi seropositive blood donors (BD) who were further characterized as having chronic Chagas cardiomyopathy (CC-BD) or not (nonCC-BD) and seronegative (SN) control donors; we also included clinically diagnosed Chagas cardiomyopathy patients (CC-P). All subjects underwent a health history questionnaire, medical examination, electro- and echocardiograms (ECG and Echo) and phlebotomy. Biomarkers were measured on blinded samples by luminex bead array and Ortho VITROS. RESULTS: A clear biomarker pattern was observed only in more severe cardiac disease; this pattern included significantly elevated levels of inflammatory cytokines IFN-γ, IL-6, IL-10 and TNF-α and soluble cardiovascular disease biomarkers CK-MB, troponin, myoglobin, VCAM and NTproBNP while there were lower levels of MPO, PAI-1, and MCP-1. The markers determined to be the most predictive of disease by ROC curve analysis were NTproBNP and T. cruzi PCR status. CONCLUSIONS: Although many biomarkers demonstrated increased or decreased concentrations among the clinical forms of Chagas disease, NTproBNP and T. cruzi PCR were the only tests that would independently be of clinical value for disease staging, in concert with ECG, Echo and clinical assessments.

HLA and beta-myosin heavy chain do not influence susceptibility to Chagas disease cardiomyopathy.

An inflammatory dilated cardiomyopathy occurs in 30% of Chagas' disease patients, chronically infected by Trypanosoma cruzi, while the remaining infected individuals are asymptomatic. Studies have indicated a role for genetic factors in the susceptibility to Chagas' disease cardiomyopathy. In an attempt to identify the genetic factors influencing the development and outcome of Chagas' cardiomyopathy, we compared the frequencies of alleles from two candidate gene loci, class II HLA and a microsatellite marker for the human cardiac beta-myosin heavy chain gene in different clinical groups. Patients were grouped as asymptomatic or with severe or mild cardiomyopathy. The results indicate that the HLA and myosin microsatellite allele profiles in all cardiomyopathy and in asymptomatic groups are similar. In conclusion, these results establish that polymorphism of HLA-DR and -DQ molecules, as well as beta-cardiac myosin, do not influence the susceptibility to different clinical forms of Chagas' disease or the progression to severe Chagas' cardiomyopathy. On the other hand, male sex was identified as a risk factor for progression to the more severe forms of cardiomyopathy (relative risk = 8.75).

Gamma delta T cells play no major role in human heart allograft rejection.

To investigate the role of gamma delta T cells in human heart transplantation, we searched for this T cell population in endomyocardial biopsies as well as in T cell lines and clones derived from graft-infiltrating lymphocytes. The number of gamma delta T cells in endomyocardial biopsies from transplanted patients (n = 55) was mostly low and did not differ significantly from nontransplanted patients (n = 21). Moreover, there was no association of gamma delta T cell distribution with rejection status or with time posttransplantation. Graft-derived T cell lines were established in the presence of autologous feeder cells and recombinant interleukin-2 to favor the growth of in vivo-activated T cells. Twenty T cell lines analyzed by flow cytometry showed low percentages of gamma delta T cells, and we were unable to obtain gamma delta T cell clones for functional studies. These results show that gamma delta T cells are poorly expressed on human heart allograft infiltrates and indicate that, when present, they are not activated in the graft. Our data suggest that gamma delta T cells do not have a major role in human heart rejection.

Heart-infiltrating and peripheral T cells in the pathogenesis of human Chagas' disease cardiomyopathy.

Heart tissue destruction in chronic Chagas' disease cardiomyopathy (CCC), occurring in 30% of individuals chronically infected by the protozoan parasite Trypanosoma cruzi, may be caused by autoimmune recognition of patients' heart tissue by a T cell rich inflammatory infiltrate. Recently, our group demonstrated that T cells infiltrating the heart of CCC patients crossreactively recognize cardiac myosin heavy chain and tandemly repetitive T. cruzi antigen B13, and possess an inflammatory T1-type cytokine profile. Susceptibility factors leading 30% of infected patients to develop CCC, while the rest of the patients remain largely asymptomatic (ASY), are still obscure. We compared immunological phenotypes of CCC and ASY patients, who have distinct clinical outcomes despite bearing a similar chronic T. cruzi infection. Preliminary observations indicate that PBMC from CCC patients recognize a set of B13 and cardiac myosin epitopes distinct from that recognized by ASY patients. Moreover, the IFN-gamma response of CCC patients is more intense than that of ASY, both at qualitative and quantitative levels. Taken together, results suggest that heart damage in Chagas' disease cardiomyopathy may be secondary to inflammatory cytokines and a delayed-type hypersensitivity process started and/or maintained by heart-crossreactive T cells. Furthermore, the distinct recognition repertoire and the high frequency of IFN-gamma producing among CCC patients could be important factors leading to the differential development of CCC among T. cruzi infected individuals.

Retro-inverso peptide analogues of Trypanosoma cruzi B13 protein epitopes fail to be recognized by human sera and peripheral blood mononuclear cells.

Retro inverso (RI) analogues of antigenic synthetic peptides, which are made of D-amino acids with a reversed sequence, may mimic the side chain conformation of natural all-L peptides. RI analogues were cross-reactively recognized by antibodies and CD4+ T cells reactive against natural all-L synthetic peptides or native proteins in animal models. Since peptides containing D-amino acids are highly resistant to proteolytic digestion, cross-reactive RI analogues may be ideal for in vivo administration to humans as synthetic peptide vaccines or immunomodulators. B13 is an immunodominant tandemly repetitive protein from Trypanosoma cruzi, a protozoan parasite that is the causative antigen of Chagas' disease. In order to test whether RI peptides can be recognized by human antibody and T cells, we synthesized two all-L peptides containing the immunodominant B (S12) and T (S15.7) cell epitopes of B13 protein from T. cruzi and their retro (R, made of all-L amino acids with reversed sequence), inverso (I, made of all-D amino acids) and RI analogues. Recognition of peptides S12, S12-R, S12-I and S12-RI by anti-B13 antibodies in sera from T. cruzi-infected patients was tested in competitive ELISA assay with recombinant B13 protein as the solid phase antigen. Peptides S15.7 and its topological analogues were tested at the 10-50 microM range in proliferation assays on peripheral blood mononuclear cells (PBMC) from S15.7-responder individuals. The median percentage inhibition of B13 ELISA for peptide S12 was 94%, while those of the RI analogue or the other topological analogues were below 12%. While peptide S15.7 was recognized by PBMC from all subjects tested, none recognized the RI analogue of the S15.7 T cell epitope. Our results indicate that cross-reactivity with natural epitopes is not an universal property of RI analogues. This may limit the general applicability of the use of cross-reactive RI analogues as human vaccines and immunotherapeutic agents.

MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans.

The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

Prostaglandin E2 inhibits proliferation but not interleukin-2 production by phorbol ester plus calcium ionophore-activated murine T cell clones.

1. We have investigated the effect of prostaglandin E2 (PGE2) on the T lymphocyte activation pathway. 2. At physiologically attainable concentrations (approximately 0.1 microM), PGE2 effectively inhibited the proliferation of murine antigen-specific "helper" T cell clones stimulated either with specific antigen in the presence of macrophages or with phorbol ester plus calcium ionophore A23187. The inhibition was not reversed by the addition of exogenous Interleukin 2 (IL-2) in either case. 3. PGE2 treatment at the same concentrations did not inhibit IL-2 production by phorbol ester plus calcium ionophore-stimulated T cell clones as assayed by CTLL proliferation. 4. These results suggest that the major target (or targets) of PGE2 inhibition directly on T cells lies in the IL-2 signal transduction pathway rather than in the early activation events leading to T cell activation.

Molecular mimicry between cardiac myosin and Trypanosoma cruzi antigen B13: identification of a B13-driven human T cell clone that recognizes cardiac myosin.

Previous reports from our group have demonstrated the association of molecular mimicry between cardiac myosin and the immunodominant Trypanosoma cruzi protein B13 with chronic Chagas' disease cardiomyopathy at both the antibody and heart-infiltrating T cell level. At the peripheral blood level, we observed no difference in primary proliferative responses to T. cruzi B13 protein between chronic Chagas' cardiopathy patients, asymptomatic chagasics and normal individuals. In the present study, we investigated whether T cells sensitized by T. cruzi B13 protein respond to cardiac myosin. T cell clones generated from a B13-stimulated T cell line obtained from peripheral blood of a B13-responsive normal donor were tested for proliferation against B13 protein and human cardiac myosin. The results showed that one clone responded to B13 protein alone and the clone FA46, displaying the highest stimulation index to B13 protein (SI = 25.7), also recognized cardiac myosin. These data show that B13 and cardiac myosin share epitopes at the T cell level and that sensitization of a T cell with B13 protein results in response to cardiac myosin. It can be hypothesized that this also occurs in vivo during T. cruzi infection which results in heart tissue damage in chronic Chagas' disease cardiomyopathy.

Cytokine production profile of heart-infiltrating T cells in Chagas' disease cardiomyopathy.

The hallmark of chronic Chagas' disease cardiomyopathy (CCC) is the finding of a T cell-rich inflammatory mononuclear cell infiltrate in the presence of extremely few parasites in the heart lesions. The scarcity of parasites in affected heart tissue casts doubt on the direct participation of Trypanosoma cruzi in CCC heart tissue lesions, and suggests the possible involvement of autoimmunity. The cells in the infiltrate are presumably the ultimate effectors of tissue damage, and there is evidence that such cells recognize cardiac myosin in molecular mimicry with T. cruzi proteins rather than primary reactivity to T. cruzi antigens (Cunha-Neto et al. (1996) Journal of Clinical Investigation, 98: 1709-1712). Recently, we have studied heart-infiltrating T cells at the functional level. In this short review we summarize the studies about the role of cytokines in human and experimental T. cruzi infection, along with our data on heart-infiltrating T cells in human Chagas' cardiomyopathy. The bulk of evidence points to a significant production of IFN-gamma and TNF-alpha which may be linked to T. cruzi-induced IL-12 production.

Malignant, non-Hodgkin's lymphomas in Trypanosoma cruzi-infected rabbits treated with nitroarenes.

Use of 2-nitroimidazole, 5-nitrofuran and 5-nitroimidazole compounds in T. cruzi-infected rabbits resulted in a reduction in duration of parasitaemia in comparison with untreated, infected rabbits. The chronic myocarditis associated with Chagas' disease was not, however, prevented in nitroarene-treated rabbits; lymphocytic infiltrates associated with cardiac cell lysis, in the absence of parasites in situ, were present in both treated and untreated rabbits. The carcinogenic effect of each trypanocidal nitroarene used in this study was also assessed. Administration of nitroarenes to rabbits resulted in the appearance of solid tumours in 37.8 per cent of animals that received drug therapy. Untreated, control rabbits in this series did not show tumour growth. Furthermore, malignant, mixed-cell type, non-Hodgkin's lymphomas were seen in 32.4 per cent of the treated rabbits. It seems that a direct relationship could be present between the presence of the nitro group, the trypanocidal cytotoxicity and the prevalence of tumours. Benznidazole cleared up parasitaemias in the shortest time and was associated with 41.6 per cent of lymphoma growths, whereas MK-436 required twice as much time to clear blood parasites, and showed lymphomas in 25 per cent of experimental rabbits. The demonstration of a high prevalence of malignant tumours in addition to the chronic myocarditis of Chagas' disease in nitroarene-treated rabbits is important since indiscriminate use of such compounds currently used to treat T. cruzi infections in man could increase the risk of lymphoma.

Autoimmunity in Chagas' heart disease.

The time scale dissociation between high parasitemia and tissue pathology, allied to the absence of parasites in the heart lesions of chronic Chagas' disease cardiopathy, casted doubt on the direct participation of Trypanosoma cruzi in tissue lesions. Moreover, the heart tissue lesions in chronic Chagas' disease cardiopathy are associated to an inflammatory mononuclear cell infiltrate, presumably the ultimate effectors of tissue damage. It has been hypothesized that the inflammatory cell infiltrate could mediate a delayed hypersensitivity process directed to the heart tissue components, an autoimmune response triggered by immunological cross-reactivity in the course of a protective immune response against some T. cruzi antigen homologous to heart proteins. However, little is known about the effector role of the T cells in the infiltrate, or about the nature of the antigen that lead to their accumulation in tissue. In this paper, we will review the published evidence on autoimmunity and immunological cross-reactivity between T. cruzi and the mammalian host, along with data generated in our laboratory. The definition of the precise role played by autoimmunity in the pathogenesis of Chagas' disease cardiopathy may have important consequences both for immunoprophylaxis and for the therapeutic approach of chronic Chagas' disease.

Locally produced survival cytokines IL-15 and IL-7 may be associated to the predominance of CD8+ T cells at heart lesions of human chronic Chagas disease cardiomyopathy.

Human chronic Chagas disease cardiomyopathy (CCC) is an inflammatory-dilated cardiomyopathy occurring years after infection by the protozoan Trypanosoma cruzi. The heart inflammatory infiltrate in CCC shows a 2:1 predominance of CD8(+) in relation to CD4(+) T cells, with a typical Th1-type cytokine profile. However, in vitro expansion of infiltrating T cells from heart biopsy-derived fragments with interleukin-2 (IL-2) and phytohaemagglutinin leads to the outgrowth of CD4(+) over CD8(+) T cells. We hypothesized that survival cytokines, such as IL-2, IL-7 and IL-15 might be differentially involved in the growth and maintenance of heart-infiltrating and peripheral CD8(+) T cells from CCC patients. We found that IL-7 and IL-15 were superior to IL-2 in the expansion and viability of CD8(+) T cells from both PBMC and heart-infiltrating T-cell lines from CCC patients, and the combination of the three cytokines showed synergic effects. Heart-infiltrating CD8(+) T cells showed higher expression of both IL-15R alpha and gamma(c) chain than CD4(+) T cells, which may explain the improvement of CD8(+) T-cell growth in the presence of IL-2 + IL-7 + IL-15. Immunohistochemical identification of IL-15 and the higher mRNA expression of IL-15R alpha, IL-7 and gamma(c) chain in CCC heart tissues compared with control individuals indicate in situ production of survival cytokines and their receptors in CCC hearts. Together, our results suggest that local production of IL-7 and IL-15 may be associated with the maintenance and predominance of CD8(+) T cells, the cells effecting tissue damage in CCC hearts.

Identification of paramyosin T cell epitopes associated with human resistance to Schistosoma mansoni reinfection.

Paramyosin, a Schistosoma mansoni myoprotein associated with human resistance to infection and reinfection, is a candidate antigen to compose a subunit vaccine against schistosomiasis. In this study, 11 paramyosin peptides selected by TEPITOPE algorithm as promiscuous epitopes were produced synthetically and tested in proliferation and in vitro human leucocyte antigen (HLA)-DR binding assays. A differential proliferative response was observed in individuals resistant to reinfection compared to individuals susceptible to reinfection in response to Para (210-226) peptide stimulation. In addition, this peptide was able to bind to all HLA-DR molecules tested in HLA-DR binding assays, confirming its promiscuity. Para (6-22) and Para (355-371) were also shown to be promiscuous peptides, because they were able to bind to the six and eight most prevalent HLA-DR alleles used in HLA-DR binding assays, respectively, and were also recognized by T cells of the individuals studied. These results suggest that these paramyosin peptides are promising antigens to compose an anti-schistosomiasis vaccine.

Autoimmunity in chagas disease cardiomyopathy: Fulfilling the criteria at last?

Here, Jorge Kalil and Edécio Cunha-Neto review the recent evidence for autoimmunity in chronic Chagas cardiomyopathy (CCC) involving molecularly defined antigens and immunopathological mechanisms. They also discuss the criteria for assignment of CCC as an organ-specific autoimmune disease.