ANTI-TRYPANOSOMA CRUZI HUMAN RECOMBINANT ANTIBODIES

GRIPPO VANINA; NIBORSKI LL; LEVIN MJ

Mar del Plata

Congreso; XLIII Reunion Anual de la Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular; 2007

To characterize the human antibody response against the parasite Trypanosoma cruzi (T. cruzi) we constructed single-chain variable fragment (scFv) libraries derived from patients with Chronic Chagas Heart Disease (cChHD). Total RNA was isolated from bone marrow, and cDNA was synthesized. Variable regions (VH, Vky Vl) were amplified by PCR and cloned in the phagemid pHEN. The libraries were subsequently panned against T.cruzi total extract by phage display. We obtained different human recombinant antibodies (hu-rAbs) to T. cruzi antigens, as assessed by sequence data. Furthermore, the expressed hu-rAbs were able to recognize the parasite extract in ELISA. Interestingly, a hu-rAb set that belong to the VH 3-30*18 family recognized a 175 kDa protein in T.cruzi Western blots. Comparison of T.cruzi recognition pattern of the different bone marrow donors allowed us to identify the origin of the hu-rAb. The molecular target of the anti-175 hu-rAb was assessed by indirect immunofluorescence (confocal microscopy) and parasite subcellular fraction Western blots. In this study, we report the construction of scFv libraries derived from cChHD patients. We have shown that phage display technology was effective to isolate the first hu-rAbs against parasite antigens. This approach may allow us to progress in the understanding of pathogenesis towards an effective immunoprofilaxis of Chagas disease.