Crystal structure of the complex mAb 17.2 and the C-terminal region of Trypanosoma cruzi P2ß protein: implications in cross-reactivity.

Patients with Chronic Chagas' Heart Disease possess high levels of antibodies against the carboxyl-terminal end of the ribosomal P2ß protein of Trypanosoma cruzi (TcP2ß). These antibodies, as well as the murine monoclonal antibody (mAb) 17.2, recognize the last 13 amino acids of TcP2ß (called the R13 epitope: EEEDDDMGFGLFD) and are able to cross-react with, and stimulate, the ß1 adrenergic receptor (ß1-AR). Indeed, the mAb 17.2 was able to specifically detect human ß1-AR, stably transfected into HEK cells, by flow cytometry and to induce repolarisation abnormalities and first degree atrioventricular conduction block after passive transfer to naïve mice. To study the structural basis of this cross-reactivity, we determined the crystal structure of the Fab region of the mAb 17.2 alone at 2.31 Å resolution and in complex with the R13 peptide at 1.89 Å resolution. We identified as key contact residues on R13 peptide Glu3, Asp6 and Phe9 as was previously shown by alanine scanning. Additionally, we generated a model of human ß1-AR to elucidate the interaction with anti-R13 antibodies. These data provide an understanding of the molecular basis of cross-reactive antibodies induced by chronic infection with Trypanosoma cruzi.

Polymerase chain reaction detection and serologic follow-up after treatment with benznidazole in Bolivian children infected with a natural mixture of Trypanosoma cruzi I and II.

Thirty-five Bolivian children (5-10 years of age) seropositive for infection with T. cruzi underwent specific chemotherapy with benznidazole. Before treatment, 57.1% had a positive parasitologic diagnosis. Some patients presented an early conversion by polymerase chain reaction of blood samples, while others were still positive four and seven months after the end of the treatment, which indicated an absence of parasite clearance. Strain typing showed that most patients were infected by a mixture of clones I and II of T. cruzi. Serologic conversion in conventional tests and antibodies to shed acute-phase antigen were observed in two and four patients, respectively. For the other patients, the average rate of antibody decay was half the initial rate. The parasitologic and serologic data indicated that chemotherapy acts throughout the course of infection in a long-lasting process in which the decrease of specific antibody production is related to the reduction of the live parasite load.

Selection of ovine PrP high-producer subclones from a transfected epithelial cell line.

The hallmarks of prion diseases are the conversion of the normal prion into an abnormal protease resistant isoform and its brain accumulation. Purification of the native abnormal prion isoform for biochemical and biophysical studies has been hampered by poor recovery from brain tissue. An epithelial cell transfected with the ovine VRQ allele prion, called Rov9, has been used to select prion high-producer cells by flow cytometry. The representative clone 4 described here produced 6.2 microg of cellular prion protein per mg of total protein extract, representing 8- to 10-fold the amount produced by the Rov9 parental cells. After exposure to the scrapie agent (PG128/98), clone 4 produced 2.6 microg of abnormal isoform per mg of total protein. When infected clone 4 cell cultures were treated with tunicamycin, 80% of the abnormal isoform was deglycosylated. The infectivity of the prions produced in clone 4 cultures was confirmed in a mouse bioassay. Such high-producer clones represent new tools for producing large amounts of glycosylated and/or non-glycosylated PrP(Sc) and for a powerful screening of clinical samples' infectivity.

Seroprevalence of Trypanosoma cruzi infection in French Guiana

A survey was carried out on 1487 individuals to assess the seroprevalence of Trypanosoma cruzi infection in French Guiana. The overall prevalence of T. cruzi specific IgG was 0.5 percent. In multivariate analysis, residence in areas where housing is favorable for the presence of triatomine bugs was the only factor associated with the presence of T. cruzi antibodies. These results have implications for public health since blood donors are not routinely screened for T. cruzi infection in French Guiana.

Seroprevalence of Trypanosoma cruzi infection in French Guiana.

A survey was carried out on 1487 individuals to assess the seroprevalence of Trypanosoma cruzi infection in French Guiana. The overall prevalence of T. cruzi specific IgG was 0.5%. In multivariate analysis, residence in areas where housing is favorable for the presence of triatomine bugs was the only factor associated with the presence of T. cruzi antibodies. These results have implications for public health since blood donors are not routinely screened for T. cruzi infection in French Guiana.

DNA immunization with the ribosomal P2beta gene of Trypanosoma cruzi fails to induce pathogenic antibodies.

Patients with chronic Chagas' heart disease (cChHD) develop a strong IgG response against the C-terminal region of the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta). These antibodies have been shown to exert an in vitro chronotropic effect on cardiocytes through stimulation of the beta1-adrenergic receptor (beta1-AR). Moreover, the presence of antibodies recognizing the TcP2beta C-terminus was associated with cardiac alterations in mice immunized with the corresponding recombinant protein. Here, we demonstrate that DNA immunization could be used to modulate the specificity of the anti-TcP2beta humoral response in order to avoid the production of pathogenic antibodies. After DNA injection, we detected IgG antibodies that were directed only to internal epitopes of the TcP2beta molecule and that did not exert anti-beta1-AR functional activity, measured as an increase in intracellular cAMP levels of transfected COS-7 cells. Accordingly, DNA-immunized mice did not present electrocardiographic alterations. These data demonstrate that anti-TcP2beta antibodies elicited by DNA immunization are completely different in their specificity and functional activity from those produced during T. cruzi infection.

Identification of HLA-A*0201-restricted cytotoxic T-cell epitopes of Trypanosoma cruzi TcP2beta protein in HLA-transgenic mice and patients.

Trypanosoma cruzi-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of parasite growth and will play an important part in therapeutic and prophylactic T. cruzi vaccines. The identification of parasite-specific epitopes that are efficiently recognized by CTLs is the first step in the development of future vaccines. HLA-A2 transgenic mice (HHD) were shown to provide a powerful model for studying the induction of HLA-A*0201-restricted immune responses in vivo, since these mice are endowed with a CTL repertoire representative of HLA-A2.1 individuals. Here, we describe the immunological characterization of T-cell epitopes of the T. cruzi ribosomal P2 protein (TcP2beta) that are recognized by HLA-A*0201-restricted CTLs in HLA-transgenic mice and humans. Epitopes identified in the present study do not share sequence homology with the homologous human or murine counterparts and so they should not induce any autoreactive response. Moreover, HHD mice vaccinated with these peptide epitopes have reduced parasitemia after challenge with a lethal T. cruzi infection. Hence, these epitopes represent potential subunit components of multi-protein vaccines to prevent Chagas' disease.

Integrate study of a Bolivian population infected by Trypanosoma cruzi, the agent of Chagas disease.

A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3%) ranging from around 12% in < 5 years to 94.7% in > 45 years, and a high sensitivity (83.8%) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5% only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37% of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6% of the patients without clonet 39 (16 cases).

Integrate Study of a Bolivian Population Infected by Trypanosoma cruzi, the Agent of Chagas Disease

A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3 percent) ranging from around 12 percent in < 5 years to 94.7 percent in > 45 years, and a high sensitivity (83.8 percent) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5 percent only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37 percent of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6 percent of the patients without clonet 39 (16 cases)

Evaluacion de la respuesta humoral de pacientes con lesiones diversas asociadas a la enfermedad de chagas frente a diferentes antigenos de T. Cruzi / Humoral reaction in patients with Chagas disease with several antigens of T. cruzi

Con el fin de encontrar mejores antigenos de T. cruzi y posibles marcadores inmunologics, para el diagnostico de las diferentes formas clinicas del mal de chagas, se provaron amastigotes y la fraccion microsomal de epimastigotes de T. cruzi. Se utilizaron tambien clonesrecombinantes (jL5, jL7, jL8) y un peptido sintetico R-13, que compromete 13 residuos C-terminales del recombinante jL5. La habilidad de los anticuerpos, en sueros de individuos con enfermedad de chagas cronica para reaccionar con diferentes antigenos, varia de acuerdo al estado clinico del paciente. Los resultados señalan que los niveles de anticuerpos IgA-anti amastigotes de T. cruzi, son significativos para los pacientes con afeccion digestiva; contrariamnete anticuerpos de pacientes con cardiopatia, fueron predominantemente dirigidos contra la fraccion microsoma, recombinante jL5 y peptido R-13. En conclusion , los resultados del presente trabajo, indican que la forma digestiva del mal de chagas puede ser difinida por la respuesta IgA-antiamastigote, mientras que las formas cardiacas son mejor determinadas por las respuestas IgA antimicrosomal o anti-R13. .