RESUMO
The localization of T and B cell epitopes on a well characterized 33-kDa protein of the filarial nematode Onchocerca volvulus (Ov33) was studied using peripheral blood mononuclear cells (PBMC) and sera from a total of 52 onchocerciasis patients with the generalized form of infection. A proportion of the PBMC samples proliferated in response to recombinant Ov33-GST fusion protein and to fusion free Ov33-6xHis. Proliferative responses of patient PBMC to seven truncated Ov33-6xHis polypeptides and to three synthetic peptides revealed at least one major and two minor T cell epitopes in the protein. The dominant T cell stimulating domain was localized between amino acids 113 and 143. ELISA studies with the Ov33-GST fusion protein revealed that patient sera contained Ov33-specific IgG1, IgG4, IgE, and IgM antibodies. Analysis of the IgG4 response with 10 truncated Ov33 polypeptides identified four B cell stimulating domains in the N-terminal, central, and C-terminal region of the molecule. The B cell domain recognized by the majority of sera was localized between amino acids 113 and 143. The data indicate that this region of the protein is the major T and B cell stimulating domain of Ov33 and might be relevant for vaccine development and for improved immunodiagnosis of onchocerciasis.
Assuntos
Antígenos de Helmintos/química , Linfócitos B/imunologia , Proteínas de Helminto/imunologia , Onchocerca volvulus/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Técnicas In Vitro , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Onchocerca volvulus/genética , Oncocercose/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
To identify the target antigens implicated in the adherence and killing of microfilariae (mf) by leucocytes, we incubated nodular mf and leucocytes in the presence of anti-cuticular monoclonal antibodies (MOVs) and fresh serum. Leucocyte donors were patients with a mean age of 37 years (with 0-1 mf/snip), who had lived in the endemic village studied for at least 10 years. After 16-20 h of incubation, up to 74% of the mf could be seen with 10 or more cells adhering to them. By 36-40 h up to 54% of the mf had been killed by the leucocytes in the presence of a cocktail of monoclonal antibodies termed MOV GIV. The degree of killing in control experiments with the monoclonal antibody MOV 1 remained lower (P less than 0.05), ranging from 0.0 to 4.5% of mf with initial viability of 90-95%. Western blotting revealed MOV GIV prominent target antigens of 10.5, 18.0, 23.5 and 27 kDa in crude surface extracts of female O. volvulus. The detected antigens may play a role in host protection.