Peptídeos e anticorpos combinatoriais imunorreativos às leishmanioses visceral e tegumentar e implicações diagnósticas e terapêuticas

Abstract

CHAPTER II: Leishmania amazonensis is a causative agent of cutaneous leishmaniasis in the New World. Despite the fact that the intracellular parasites represent a major cause of illness and despite years of efforts, no effective vaccine was developed. Surface antigens, secreted and excreted were tested for their prophylactic potential for being the first factors to interact with the parasite\'s immune system and are usually involved in the establishment of infection. Once the humural response is induced by epitopes on the surface of an antigen, instead of a whole antigen, it is important to locate these epitopes with the goal of designing an effective vaccine. To this end, Phage Display technology was used for the selection of combinatorial antibody binding in the parasite s surface. An antibody named La7h has been isolated and their reactivity against the parasite was tested by different immunological assays demonstrated high affinity. Likely antibody binding epitopes were characterized by selecting recombinant peptide expressed in phages, which allowed the characterization of a possible antigenic region common to different surface proteins characterized as the main proteins in Leishmania sp. By immunoprecipitation with this recombinant antibody, was possible to isolate a protein with approximately 42KDa. This finding confirms the linear alignments of recombinant peptides to surface proteins, the study of three-dimensional structure of this protein has contributed to the characterization of antigenic sites mapped by the antibody of fundamental interest in finding vaccine targets more directed and effective. CHAPTER III: Leishmaniasis are an important tropical disease with increasing incidence worldwide, because there are no vaccines available and its control is based on the diagnosis and treatment which often has severe side effects. The diagnostic tests are not effective, they fail to detect the sensitivity and specificity in the vairos forms of the disease, and don t identify releasy from infection. Therefore, highly accurate diagnostic tests, inexpensive, simple and fast are crucial for the control and treatment of leishmaniasis, but new antigens would be required for the improvement of such tests. In this sense we propose to develop mimetic peptides of Leishmania chagasi and Leishmania amazonensis by Phage Display technology as a strategy for the proteomic discovery of new antigens more effective and sensitive. Phage Display random peptide selections were carried out agaist purified IgG from visceral and cutaneos leishmaiasis in order to mapping antigens that recognize the specific immune response in both clinical forms. The clones were purified, amplified, sequenced and analyzed by bioinformatics. Several recombinant peptides were characterized and pre-validated by ELISA, competitive dot-blot and functional tests with macrophage culture. Our results showed that specific recombinant peptides were selected for both disease forms and they are related to important antigens used for diagnostic tools and vaccine targets.