Análise de anticorpos anti-glicolipídeos em soros de pacientes com doença de chagas e paracoccidioidomicose. Estudos e propriedades do anticorpo monoclonal bst-1 dirigido a resíduos de β-d-galactofuranose de trypanosoma cruzi

Abstract

In this study it was analyzed the reactivity of sera obtained from patients with paracoccidioidomycosis (PCM) and Chagas disease with glycolipids presenting or not galactofuranose residues (Galf). Studies with glycolipids isolated from Paracoccidioides brasiliensis by ELISA, showed that all samples of PCM sera tested reacted with Pb-1 antigen (Galfβ1-6(Manpα1-3)Manpα1- 2Ins-P-Cer). As expected, it was not observed any reactivity of PCM sera with Pb-2 antigen (Manpα1-3Manpα1-2Ins-P-Cer) or glucosylceramide. To evaluate the prognosis of patients with PCM, sera of patients without previous treatment and under therapy with antifungal drugs were tested with Pb-1 antigen. High titers of anti-Pb-1 antibodies were found in sera of both patients without treatment, as well as in those under therapy from 1 up to 4 months. Immunoglobulins IgG (IgG1), IgM and IgA directed to Pb-1 were detected in sera of patients with PCM. Sera from patients with Chagas disease are highly reactive with glycolipids from T. cruzi epimastigotes (strain CL). It was also observed that sera of patients with Chagas disease present a low reactivity with glycolipids from epimastigotes of Tulahuen strain, probably due to the absence of galactofuranose residues in those glycolipids.Immunoglobulins IgG, IgM and IgA directed to epimastigote glycolipids (strain CL) were detected in sera of patients with Chagas disease. IgG1 was detected in 100% of sera of Chagas disease, and IgG3 in about 57% of patients. It should be noted that Galf residue is the immunodominant glycoepitope, however Galf residues present in Pb-1 antigen is not recognized by sera of patients with Chagas disease, indicating that linkage β1-3 between the terminal residue of galactofuranose and subterminal residue of mannose is a key feature for the antibody recogntion. A monoclonal antibody (mAb), termed BST-1 (IgG3), directed to glycolipids from epimastigote of strain CL was produced. By ELISA, it was verified that mAb BST-1 recognizes the major glycolipids in epimastigotes of strain CL and Y, as well as minor glycolipid components on strain G, but does not recognize any glycolipid antigen on Tulahuen strain. It was demonstrated that the epitope recognized by BST-1 is contained within the structure Manα1-2(Galfβ1-3)Manα1-2Manα. By “Western blotting” it was demonstrated that mAb BST- 1 recognizes only glycolipids expressed by epimastigote forms of strains CL and Y. On the other hand, mAb BST-1 recognizes glycoprotein antigens of 115 and 85 kDa in epimastigotes of strain G, and glycoproteins of 50 and 85 kDa in epimastigote forms of Tulahuen strain. By indirect immunofluorescence it was detected a strong fluorescence with mAb BST-1 on trypomastigotes andamastigotes of the four strains analyzed in this thesis. By Western blot, high molecular weight glycoproteins (180-210 kDa) were identified as main antigens recognized by mAb BST-1 in these parasites. mAb BST-1 was able to inhibit about 70% of trypomastigotes adhesion/infection in Vero cells, indicating that the epitope recognized by mAb BST-1 may be involved in trypomastigote infectivity. In order to determine if T. cruzi glycolipids are essential for parasite survival/division the effect of an inhibitor of inositol phosphoceramide synthase, Aureobasidin A (AbA), was analyzed on epimastigotes and intracellular amastigotes growth, as well as in trypomastigotes infectivity. It was verified that AbA (10 μg/mL) inhibits about 97% epimastigotes growth and in about 90% the growth of intracellular amastigotes. However, when the inhibitor is removed from the culture medium, the parasites are able to resume their growth. The study of biosynthesis of sphingolipids in T. cruzi opens new perspectives for the development of more specific potent therapies for Chagas disease.

Nesta tese analisamos anticorpos presentes em soros de pacientes com paracoccidioidomicose (PCM) e doença de Chagas reativos com glicolipídeos contendo ou não resíduos de galactofuranose (Galf). Por ELISA, verificamos que 100% dos soros de pacientes com PCM foram reativos com o glicolipídeo Pb-1, isolado de Paracoccidioides brasiliensis, cuja estrutura é: Galfβ1- 6(Manpα1-3)Manpα1-2Ins-P-Cer, mas não reconheceram os glicolipídeos Pb-2 e glucosilceramida. Soros de pacientes com PCM antes e durante o tratamento com antifúngicos foram testados quanto a reatividade com o antígeno Pb-1. Foram verificados altos títulos de anticorpos anti-Pb-1 em pacientes antes do tratamento, e tratados por um período de 1 a 4 meses. IgG (IgG1), IgM e IgA foram as principais imunoglobulinas de imunoglobulinas dirigidas ao antígeno Pb-1. Soros de pacientes com doença de Chagas foram altamente reativos com glicolipídeos de epimastigotas de T. cruzi da cepa CL, apresentando menor afinidade por glicolipídeos purificados de epimastigotas da cepa Tulahuen. IgG1, IgM e IgA foram as principais imunoglobulinas presentes em soros de pacientes com doença de Chagas dirigidas aos glicolipídeos de epimastigotas. Com o propósito de se obter uma ferramenta importante para avaliar o papel dos glicolipídeos contendo resíduos de Galf na patogenia da doença de Chagas, foi produzido um anticorpo monoclonal (mAb), denominado de BST-1, dirigido a glicolipídeos de epimastigotas da cepa CL. Por ELISA, foi verificado que o mAb BST-1 é reativo com glicolipídeos majoritários expressos em epimastigotas das cepas CL e Y, reconhecendo um componente minoritário da cepa G e não reconhecendo glicolipídeos da cepa Tulahuen.Por “Western blotting” demonstrou-se que o mAb BST-1 reconhece somente glicolipídeos em epimastigotas das cepas CL e Y, enquanto que em epimastigotas das cepas G e Tulahuen, o mAb BST-1 reconhece componentes glicoproteicos. Já, tripomastigotas e amastigotas de cultura foram reconhecidos pelo o mAb BST-1, e forte imunofluorescência foi detectada em todo o parasita. Componentes de alto peso molecular (180-210 kDa) foram identificados como os principais antígenos reconhecidos pelo mAb BST-1 nestes parasitas. O mAb BST-1 inibe em cerca de 74% a adesão/infecção de tripomastigotas em células Vero. Nesta tese demonstrou-se que a Aureobasidina A (AbA), inibidor da enzima inositol fosfoceramida sintase, inibe significativamente o crescimento de epimastigotas e amastigotas intracelulares. O estudo de esfíngolipídeos de parasitas e fungos abre novas perpectivas para o desenvolvimento de quimioterápicos mais específicos e potentes para doença de Chagas e micoses sistêmicas.