Produção da proteína ribossomal L31 de Streptococcus pneumoniae na forma recombinante e avaliação do seu potencial como vacina protéica

Abstract

Introduction. The Streptococcus pneumoniae, known as pneumococcus, is an encapsulated gram-positive bacterium, possessing more than 90 known serotypes. Pneumococcus causes diseases such as pneumonia, meningitis, sepsis and acute otitis media. While there are vaccines available, all are based on polysaccharide antigens (conjugated or not), making them high cost and low coverage. Objective. This study aims to evaluate the immunogenic potential and protective capacity of ribosomal protein L31 recombinant in murine model, aimed at producing a vaccine based on the conserved protein antigens. Methodology. The choice of ribosomal protein L31, occurred from an in silico study conducted by the Department of Molecular Biology and Bioinformatics at FUNED. It, was made the prediction of possible protein antigens vaccine candidates based on the next features: the exposure on the cell surface, the conservation between the various serotypes and the possible virulence. Thus, the gene encoding ribosomal protein L31 was amplified from the genome of S. pneumoniae, cloned in expression vector pET-21a and confirmed by DNA sequencing. The protein expressed in Escherichia coli was purified by affinity chromatography and its immunogenicity was evaluated by Western blot using sera of patients with meningitis and rabbit serum immunized with the protein L31. Four distinct experiments were performed involving immunization of mice with ribosomal protein L31 recombinant, to evaluat e: (1) the humoral response, comparing the pathway of administration; (2) humoral response comparing adjuvant; (3) cytokine production; and (4) bacteremia and survival after challenge with S. pneumoniae. Results. The gene was amplified and cloned successfully, confirmed by sequencing. Expression and purification of the protein L31 were performed with success, with a good yield after purification. The purified protein L31 proved to be immunogenic and was able to confer high titers of IgG1 in mice, but it was not enough to reduce bacteremia and induce protection in mice challenged with S. pneumoniae. Conclusion. Although the ribosomal protein L31 recombinant proved its immunoreactivity and induced high levels of IgG1, it was not able to induce protection. Thus, others studies will be conducted in search of new protein antigens, to evaluate the immunogenicity and protective capacity.