| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1132-1 | ||||
Resumo:Lower respiratory tract infections are important causes of morbidity and mortality worldwide, with Streptococcus pneumoniae (pneumococcus) being a significant etiological agent, responsible for the annual death of about 400,000 children under 5 years of age. To date, more than 100 pneumococcal serotypes were described based on the capsular polysaccharides, with no significant cross-reactivity among them. Still, the available vaccines are composed of polysaccharides from prevalent disease-causing strains, conjugated with carrier proteins. Pneumococcal conjugate vaccines protect against invasive diseases and nasopharyngeal colonization but their effect is limited to the serotypes present in the formulations. Our research group has been studying alternative vaccines composed of protein antigens that may confer serotype-independent protection. One of the candidates are Extracellular Vesicles (EVs) that originate from pneumococcal membranes, have sizes equivalent to nanoparticles and carry protein antigens. In our initial studies, EVs derived from the non-encapsulated R6 pneumococcal strain (R6 EVs) confer protection to mice against infections with pneumococci from different serotypes. Sera from mice immunized with R6 EVs showed reactivity with two proteins of approximately 50kDa and 38kDa, in western-blot assays. Data from the literature suggest that they are two pneumococcal lipoproteins, MalX and PrsA. In the present work, we have expressed these two proteins in E. coli and purified them for the production of antisera and for the characterization of EVs and the immune responses induced by them. Proteins were expressed in E. coli BL-21 Star-pLyS using the pAE expression vector and were purified by nickel affinity chromatography. Polyclonal antisera were produced by the immunization of BALB/c mice using Alum hydroxide as adjuvant. Western-blot analyzes using the anti-PrsA and the anti-MalX sera confirmed that the R6 EVs express both proteins. In addition, both sera recognized PrsA and MalX in extracts from different pneumococcal isolates, confirming their antigenic conservation. On the other hand, ELISA and Western-blot assays have shown that sera from mice immunized with R6 EVs contain significant levels of IgG reacting with PsrA but very low levels of IgG reacting with MalX. Thus, although both antigens are present in the R6 EVs, PrsA seems to be much more immunogenic than MalX. Our results suggest that at least PrsA may be involved in the protection of EVs against pneumococcal infections in mice, through the induction of IgG. In summary, we find that pneumococcal EVs are promising candidates for vaccines. We are currently exploring the contribution of the individual proteins on the EVs efficacy. Palavras-chave: Streptococcus pneumoniae, Vaccine, Recombinant proteins, Extracellular vesicles Agência de fomento:CNPq, FAPESP, Fundação Butantan | |||||