Identificação e caracterização de peptídeos miméticos dos antígenos de Mycobacterium leprae por phage display

Abstract

CHAPTER I: Leprosy presents a clinical spectrum that spans from a strong cellular mediated immunity and bacili growth control of Mycobacterium leprae at the tuberculoid pole to a poor T cell immunity with extensive bacterial load at the lepromatous pole. The antigenic profile characterization in both clinical forms is a necessary step for discover and evaluation of recombinant peptides that are mimotopes of M. leprae antigens, which may present immunogenic potential, in order to obtain a beTer understanding of the disease evolution, and allowing the improvement of diagnosis, prognosis and therapeutics. Mimotopes of M. leprae antigens were selected from a random heptapeptide conformational library expressed in fusion with the pIII protein of the M13 phage, using as the ligand target the total purified IgM from leprosy patients of clinical forms, tuberculoid (TT) and lepromatous (LL), and a healthy control population. Recombinant peptides were selected by glycine elution (unspecific) and by mitsudin elution (specific). All clones, after bioinformatic analyses, were validated by immunoenzymatic and colony reduction assays. For the glycine selection, the TT pole presented 24 distinct fagotypes, while the LL pole presented only 14 fagotypes. For the mitsudin selection, only 4 fagotypes were obtained in the LL pole, and 8 fagotypes in the TT pole. The two most frequent mimotopes, in both TT and LL poles and in both elution protocols, were coincidents. The peptides LFPAMHQ and VERHPST were more frequent in the LL pole, and the peptides KNPTTGT and ETHPTTR were more frequent in the TT pole. The three cited first mimotopes had an accentuated plaque reduction with incubation of sera from LL patients; however, the mimotope ETHPTTR presented the highest reduction with sera from TT patients. The immunogenic potential of these mimotopes may be useful in the development of new diagnostic and therapeutic strategies for leprosy control in the future. CHAPTER II: The phenolic glycolipid 1 (PGL-1) is a membrane antigen highly specific of M. leprae, and it is not found in other mycobacteria. Serological tests with PGL-1 have been performed to detect circulating antibodies in multibacillary patients. Due to its importance in the clinical classification of leprosy patients, we have developed mimotopes of this non-proteic antigen through phage display in order to improve serological assays. In this investigation, a random heptapetide conformational library was selected to obtain specific ligands to the monoclonal antibody anti-PGL-1 CS-48. After three rounds of selection, 14 clones were sequenced and translated, generating six distinct peptides. The mimotope HWMLPED was the most frequent one (57%), suggesting an immunodominance. Serological tests with this mimotope were performed in comparison to the synthetic PGL-1 for 39 patients, classified according to their clinical form, together with their 44 household contacts, classified according to their index cases. Results were similar for T and LL patients. However, the intermediate forms were variable, differently from those results with PGL-1, which presented positivity only for multibacillary patients. The clinical form BT presented a seropositivity of 55.5% for the mimotope against 0% for the PGL-1, probably due the greater avidity of the mimotope to circulating antibodies, or due to the greater spepcificity of the peptide in detecting pure neural reaction, or yet a cross reaction with other human antibodies. The serological tests of household contacts for this same phage clone presented an average positivity of 20.5% versus an average of 7.5 for the PGL-1 antigen. It is probable that there was cross reaction of the mimotope with other antibodies; once it presented partial identity with other M. leprae proteins. The mimotopes must be further investigated, once they may be putative targets for the PGL-1 action in the immune humoral response, and also they could be useful in diagnostics and therapeutic strategies.