Characterization and expression of recombinant human antibodies against Trypanosoma cruzi

NIBORSKI LL; GRIPPO VANINA; LEVIN MJ

Congreso; Reunion Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

Patients chronically infected with Trypanosoma cruzi (T. cruzi)  develop Chagas heart Disease (cChHD). Their antibody response is suspected to be involved in cardiac pathogenesis. Due to the clinical relevance of the anti-T. cruzi antibodies (Abs), more knowledge about their repertoire, epitope recognition and fine specificity is required. For this purpose, recombinant human anti-T. cruzi single chain variable fragments (scFvs) Abs were isolated from a cChHD patients-derived libraries using phage display technology. The libraries were subsequently panned against T. cruzi total extract and the selected monoclonal anti-T. cruzi antibodies bearing (his)6 tag at C-terminus, were produced in E.coli and purified from periplasmic extracts. We have characterized two human monoclonal anti-T. cruzi scFv, scFv 6B(6) belongs to the VH 3-30*18 family recognizes a 175 kDa protein and scFv A2R1, that belongs to the VH 3-73*01 family recognizes two proteins of about 47.5 kDa in T. cruzi Western blot. Flow cytometry and microscopic analysis revealed that both scFv recognize only permeabilized parasites indicating cytoplasmatic protein targeting. Also the selected scFv clones showed cross-reactivity with other kinetoplastids as T. brucei and C. fasciculata. These results show that phage display technology is an effective method for selection of human recombinant Abs against parasite antigens.Trypanosoma cruzi (T. cruzi)  develop Chagas heart Disease (cChHD). Their antibody response is suspected to be involved in cardiac pathogenesis. Due to the clinical relevance of the anti-T. cruzi antibodies (Abs), more knowledge about their repertoire, epitope recognition and fine specificity is required. For this purpose, recombinant human anti-T. cruzi single chain variable fragments (scFvs) Abs were isolated from a cChHD patients-derived libraries using phage display technology. The libraries were subsequently panned against T. cruzi total extract and the selected monoclonal anti-T. cruzi antibodies bearing (his)6 tag at C-terminus, were produced in E.coli and purified from periplasmic extracts. We have characterized two human monoclonal anti-T. cruzi scFv, scFv 6B(6) belongs to the VH 3-30*18 family recognizes a 175 kDa protein and scFv A2R1, that belongs to the VH 3-73*01 family recognizes two proteins of about 47.5 kDa in T. cruzi Western blot. Flow cytometry and microscopic analysis revealed that both scFv recognize only permeabilized parasites indicating cytoplasmatic protein targeting. Also the selected scFv clones showed cross-reactivity with other kinetoplastids as T. brucei and C. fasciculata. These results show that phage display technology is an effective method for selection of human recombinant Abs against parasite antigens.