Identificação de alvos antigênicos na ATP difosfohidrolase solúvel de Schistosoma mansoni e possível aplicação funcional de peptídeos sintéticos

Abstract

The antigenicity of both soluble S. mansoni ATP diphosphohydrolase and membraneassociated isoforms has been recently described. Cross-immunoreactivity between potato apyrase and IgG antibody from S. mansoni-infected mice or schistosomiasis patients strongly suggested that the epitopes shared between the vegetable and parasite proteins are antigenic. By bioinformatics analysis, a strategic comparative study of the conserved domains shared between the soluble ATP diphosphohydrolase isoform and potato apyrase predicted a homologue antigenic-domain available for antibody and HLA-DR binding. Synthetic peptides belonging to the domain of the parasite (Sm) and vegetable (pot) proteins were then obtained. By ELISA, the serum samples from the schistosomiasis patients (n= 146) showed high seropositivity of IgG1 (58%) and IgG4 (58%) antibody sub-classes for potato apyrase. Seropositive patients (n= 35) were then selected for analyses of antibody reactivity against the synthetic peptides. Significant seropositivity of IgG (potB1LJ, 9%; potB1MP, 54%; potB2LJ, 54%; SmB1LJ, 23%; SmB1MP, 86%; SmB2LJ, 34%), and IgG1 (potB1LJ, 57%; potB1MP, 67%; potB2LJ, 46%; SmB1LJ, 57%; SmB1MP, 10%; SmB2LJ, 31%) or IgG4 (potB1LJ, 71%; potB1MP, 40%; potB2LJ, 71%; SmB1LJ, 54%; SmB1MP, 63%; SmB2LJ, 49%) was observed for the homologue peptides in accordance with the existence of shared antigenic epitopes between the parasite and vegetable proteins, and demonstrating that in schistosomiasis the domain r155-194 from soluble ATP diphosphohydrolase isoform is rich in B-cell epitopes. The reactivity of the IgG1 and IgG4 antibody from the serum samples of patients (n= 26) cured after chemotherapy with praziquantel was analyzed against potB1LJ e potB2LJ, before and six months after treatment. Individual analysis revealed variable IgG1 and IgG4 reactivity against each peptide, suggesting that different antigenic epitopes could be involved in susceptibility or resistance to S. mansoni reinfection. By ELISA, polyclonal antibody to potato apyrase of BALB/c or CBA/J mice reacts with potB1LJ, potB1MP and/or potB2LJ. Similar reactivity was also observed between these immune sera and SmB1LJ, SmB1MP and/or SmB2LJ, confirming again the cross-immunoreactivity between parasite and vegetable proteins. In addition, inoculation of each peptide in healthy Swiss mice had significant immunostimulatory activity, increasing the amount of total IgG and/or IgG1 and IgG2a antibody, as observed by ELISA and/or Dot blots, suggesting that each peptide has two distinct epitopes capable of inducing a Th1 or Th2-type immune response. By Western blots, serum immune anti-SmB2LJ recognized two polypeptides of approximately 63 and 55 kDa in SEA or SWAP, indicating the presence of the soluble isoform of ATP diphosphohydrolase in these antigenic preparations, as well as the sensitivity and specificity of this immune serum. The potato apyrase and potB1LJ were evaluated in delayed-type hypersensitivity reaction (DTH), using ovalbumin (OVA) as standard antigen, by footpad thickness measurement, titration of antibody sub-class by ELISA, and histological analysis. Seemingly, the inflammatory reaction was similar between potato apyrase and OVA, while potB1J inhibited the DTH reaction. Further studies of these new biomolecules could contribute to a better understanding of host-parasite interactions, and may to be explored as new tools in schistosomiasis research.