Elucidation of anti-T. cruzi recombinant human antibodies specificity using positional scanning combinatorial peptide libraries.

L NIBORSKI; C PINILLA; V JUDKOWSKI ; M GIULIANOTTI ; A MORALES; MJ LEVIN; KA GOMEZ

Buenos Aires

Congreso; IX Congreso Argentino de Protozología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoologia

Protective immune mechanisms against T. cruzi are constituted in part by strong antibody (Ab) responses to the parasite. However, it has also been proposed that some parasite Abs can cross-react to human antigens (Ag), suggesting that autoimmunity may have a role in the pathogenesis of Chagas disease. The aim of this study was to elucidate the Ag specificity of relevant human T. cruzi antibodies. Using phage display technology, two single chain variable fragments (scFvs) 6B and A2 were isolated from Chagas heart Disease patients. Western-blot analysis revealed that scFv 6B and scFv A2 recognized T. cruzi proteins of 175 and 47 kDa respectively. Interestingly, scFv A2 also identified a 47 kDa protein in mouse and rat brain lysate. The antigenic determinant of scFv B6 and scFv A2 antibodies was studied using a Positional Scanning Combinatorial Libraries (PS-CL) composed for 120 different hexapeptide mixtures, each of which consisted of more than 2.5 million peptides totaling more than 50 million individual sequences in approximately equimolar representation. The hexapeptides have one position that is defined by a particular amino acid (aa) while other positions have mixtures of aa. The 120 hexapeptide mixtures of acetylated and non acetylated PS-CL were screened by competitive ELISA for their ability to inhibit scFv binding to T. cruzi lysate. The screening data determined the most active aa at each position of the hexapeptide and a scoring matrix was constructed for each clone used to score and rank all of the sequences contained in the T. cruzi and T. brucei database (TriTrypDB). This biometric analysis compared information from millions of peptides in the library to the millions of peptides in the databases and generated a list of ranked sequences that allowed us the selection of 200 candidate peptides. The 200 hexapeptides were solid phase synthesized and currently tested by competitive ELISA to identify the most active ones with basic residues as their predominant recognition motif. These results showed that PS-CL is an effective method for the elucidation of binding motifs, cross-reactivity, poly-specificity and molecular mimicry found in Ag-Ab interactions. The common neuronal and trypanosomal Ag recognized by scFv A2 may allow us to progress in the understanding of events underlying Chagas disease.