Résumé
We have previously demonstrated that sera from dogs vaccinated with excreted/secreted antigens (ESA) of Leishmania infantum promastigotes (LiESAp) mainly recognized an immunodominant antigen of 54 kDa. An anti-LiESAp-specific IgG2 humoral response was observed and associated to Th1-type response in vaccinated dogs. This response was highly correlated with a long-lasting and strong LiESAp-vaccine protection toward L. infantum experimental infection. In addition, it was also shown that dogs from the vaccinated group developed a selective IgG2 response against an immunodominant antigen of 45 kDa of Leishmania amazonensis ESA promastigotes (LaESAp). In order to identify and characterize these immunodominant antigens, a mouse monoclonal antibody (mAb F5) was produced by immunization against LaESAp. It was found to recognize the major antigenic targets of both LaESAp and LiESAp. Analysis with mAb F5 of L. amazonensis amastigote and promastigote cDNA expression libraries enabled the identification of clones encoding proteins with significant structural homology to the promastigote surface antigens named PSA-2/gp-46. Among them, one clone presented a full-length cDNA and encoded a novel L. amazonensis protein of 38.6 kDa calculated molecular mass (LaPSA-38S) sharing an amino acid sequence consistent with that of the PSA polymorphic family and a N-terminal signal peptide, characteristic of a secreted protein. We then screened a L. infantum promastigote DNA cosmid library using a cDNA probe derived from the LaPSA-38S gene and identified a full-length clone of a novel excreted/secreted protein of L. infantum with a calculated molecular mass of 49.2 kDa and named LiPSA-50S. The fact that a significant immunological reactivity was observed against PSA, suggests that these newly identified proteins could have an important immunoregulatory influence on the immune response. This hypothesis is supported by the fact that (i) these proteins were naturally excreted/secreted by viable Leishmania promastigotes and amastigotes, and (ii) they are selectively recognized by vaccinated and protected dogs.