Construção e avaliação de vacinas contra a leptospirose a partir de abordagem de vacinologia reversa e estrutural

Abstract

Leptospirosis is among the leading zoonotic causes of morbidity worldwide, with a global incidence over one million cases every year, resulting in ~60.000 deaths. There are 15 Leptospira spp. that cause leptospirosis, serologically classified in more than 300 serovars. The current leptospiral vaccines are bacterins. Bacterins confer a short-term immune response that only protects against the serovars included in the vaccine and cause adverse reactions. A new, effective universal vaccine against leptospirosis will likely include surface-exposed epitopes that are conserved among the pathogenic leptospires and able of generating neutralizing immune responses. This study aims to identify exposed and conserved epitopes using a reverse and structural vaccinology approach, constructing synthetic molecules composed by those segments, and to evaluate the vaccine potential of these proteins. Seventeen beta-barrel transmembrane proteins were selected to compose this study. The three-dimensional (3D) structures of the 17 βb-OMPs were predicted by I-TASSER and quality assessed. The amino-acid sequences from these proteins were analyzed using NetMHCII 2.2 to predict immunogenic T cell epitopes. The presence of linear B cell epitopes was identified by BepiPred 1.0. The epitopes were manually mapped onto the 3D models of each βb-OMP in order to found which were exposed in bacterial surface. These regions were analyzed by multiple sequence alignment to confirm their presence in the orthologues in the remaining pathogenic Leptospira spp. Five chimeric proteins were constructed using those epitopes. Each chimera was designed to contain surface-related epitopes from 3-4 βb-OMPs, comprising six to eleven different surface-related immunogenic regions. The DNA coding sequences for each of the chimeras were codon optimized for Escherichia coli expression, synthetized and cloned into the expression vector pAE. Human convalescent sera recognized four of the five recombinant proteins. The protective capacity of these antigens was evaluated by lethal challenge in hamster model using aluminium hydroxide adjuvant. None of the proteins were able to confer immune protection. This study is the first report using structural approaches towards the development of a leptospirosis vaccine.