DURING CANINE VISCERO-CUTANEOUS LEISHMANIASIS THE ANTI-HSP70 ANTIBODIES ARE SPECIFICALLY ELICITED BY THE PARASITE PROTEIN

A Leishmania infantum cDNA library was screened with sera from dogs wi th viscero-cutaneous leishmaniasis. Sequence analysis of a positive cl one isolated from the library revealed that it coded for the carboxyl- terminal region of a member of the 50-kDa heat-shock protein family. T he full-length sequence of the L. infantum hsp70 gene was determined a fter isolation of genomic clones. This protein shows a high degree of sequence conservation with the homologous protein from other organisms . To test its antigenicity a recombinant Hsp70 protein fused to the ma ltose-binding protein was produced in Escherichia coli using the expre ssion vector pMAL-cRI. By FAST-ELISA assays it was observed that while the complete recombinant protein was recognized by 100% of the sera, the 20 carboxyl-terminal amino acids of the protein were only recogniz ed by 30% of those sera. Thus, although a B-cell epitope must be prese nt within the carboxyl terminal end of the protein other antigenic det erminant(s) must reside out of this region. The analysis of the cross- reactivity with mouse Hsp70 by Western blotting strongly suggests that the anti-Hsp70 antibodies generated by infection with L. infantum are directed at specific determinants of the L. infantum Hsp70. Thus, our results indicate that anti-Hsp70 autoantibodies are not induced durin g Leishmania infection.