RECOMBINANT ANTI-TRYPANOSOMA CRUZI ANTIBODIES FROM A HUMAN ANTIBODY LIBRARY

NIBORSKI LL; GRIPPO VANINA; RAATS J; LEVIN MJ

San Pablo

Congreso; XXIV Reunion da Sociedade Brasileira de Protozoologia XXXV Reunion sobre Pesquisa Basica em Doenia de Chagas; 2008

Analysis of the repertoire of VH genes of patients with Chronic Chagas Heart Disease (cChHD) demonstrates that neither autoimmune-prone VH segments nor polyclonal B cell activation evidences were present in these patients. On the contrary, the repertoire showed a high level of hypermutations in CDR regions suggesting a Trypanosoma cruzi driven selection. To further characterize the human antibody response against the parasite, we constructed single chain variable fragment (scFv) libraries derived from cChHD patients. Total RNA was isolated from bone marrow and peripheral blood, and DNA was synthesized. Variable chains genes (VH, Vk y Vl) were amplified by PCR and cloned into a phagemid vector. ScFv were expressed on the surface of M13 viral particles and phage-displayed scFv were affinity-selected by using immobilizated T.cruzi epimastigote lysate. To determine the number of different human recombinant antibodies (hu-rAbs) that recognized the parasite lysate, ELISA-positive clones were characterized by DNA fingerprinting and DNA sequencing. Soluble hu-rAbs bearing (his)6 tag at C-terminus were produced in E.coli and purified from periplasmic extracts. A hu-rAb set that belong to the VH 3-30*18 family recognized a 175 kDa protein and another hu-rAb that belongs to the VH 3-73*01 family recognized two proteins of about 47.5 kDa in T.cruzi Western blot. Comparisons of T.cruzi recognition pattern of the different bone marrow donors allowed us to identify the origin of hu-rAb. Immunofluorescence assays confirmed the binding of these hu-rAb to the parasite. These results showed that phage display technology was an effective method for selection of hu-rAbs against parasite antigens. This approach may allow us to progress in the understanding of pathogenesis of Chagas disease and in the development of an effective immunoprofilaxis.