RECOMBINANT ANTI-TRYPANOSOMA CRUZI ANTIBODIES FROM A HUMAN ANTIBODY LIBRARY

NIBORSKI, LL; GRIPPO, V; RAATS, J; LEVIN, MJ

Rosario, Santa Fe, Argentina

Congreso; VII Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2008

Sociedad Argentina de protozoología

Repertoire analysis of VH genes from 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 cDNA was synthesized. Variable chains genes (VH, Vkappa y Vlambda) 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 immobilized T.cruzi epimastigote lysate. To determine the number of different human recombinant antibodies (hu-rAbs), ELISA-positive clones were characterized by DNA fingerprinting and DNA sequencing. A hu-rAb set that belong to the VH 3-30*01 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 the anti T.cruzi recognition pattern of the different bone marrow donors as assessed by Western blot allowed us to identify the origin of hu-rAb. Immunofluorescence assays confirmed the binding of these hu-rAb to the parasite.