RESUMO
In an effort to devise a safer and effective pertussis acelullar vaccine, outer membrane vesicles (OMVs) were engineered to decrease their endotoxicity. The pagL gene from Bordetella bronchiseptica, which encodes a lipid A 3-deacylase, was expressed in Bordetella pertussis strain Tohama I. The resulting OMVs, designated OMVs(BpPagL), contain tetra- instead of penta-acylated LOS, in addition to pertussis surface immunogens such as pertactin and pertussis toxin, as the wild type OMVs. The characterized pertussis OMVs(BpPagL) were used in murine B. pertussis intranasal (i.n.) challenge model to examine their protective capacity when delivered by i.n. routes. Immunized BALB/c mice were challenged with sublethal doses of B. pertussis. Significant differences between immunized animals and the PBS treated group were observed (p<0.001). Adequate elimination rates (p<0.005) were observed in mice immunized either with OMVs(BpPagL) and wild type OMVs. All OMV preparations tested were non toxic according to WHO criteria; however, OMVs(BpPagL) displayed almost no weight loss at 3 days post administration, indicating less toxicity when compared with wild type OMVs. Induction of IL6- and IL1-expression in lung after i.n. delivery as well as neutrophil recruitment to airways showed coincident results, with a lower induction of the proinflammatory cytokines and lower recruitment in the case of OMVs(BpPagL) compared to wild type OMVs. Given their lower endotoxic activity and retained protective capacity in the mouse model, OMVs(BpPagL) obtained from B. pertussis seem as interesting candidates to be considered for the development of novel multi-antigen vaccine.
Assuntos
Antígenos de Bactérias/imunologia , Bordetella pertussis/imunologia , Hidrolases de Éster Carboxílico/imunologia , Vesículas Citoplasmáticas/imunologia , Vacina contra Coqueluche/imunologia , Animais , Bordetella pertussis/enzimologia , Vesículas Citoplasmáticas/enzimologia , Feminino , Imunidade Inata , Lipopolissacarídeos/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Acelulares/imunologia , Aumento de Peso , Coqueluche/imunologia , Coqueluche/prevenção & controleRESUMO
Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. The proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. The proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. The proteases were stable and presented enzymatic activity at 37 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.
Assuntos
Bordetella pertussis/enzimologia , Corynebacterium diphtheriae/enzimologia , Meios de Cultura/química , Peptídeo Hidrolases/análise , Peptídeos/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/análise , Bordetella pertussis/crescimento & desenvolvimento , Soluções Tampão , Cromatografia em Gel , Corynebacterium diphtheriae/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Filtração , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/isolamento & purificação , Peptídeos/química , Toxina Pertussis/análise , Cloreto de Sódio/química , Especificidade por Substrato , Trometamina/químicaRESUMO
Bordetella pertussis produces extracytoplasmic adenylate cyclase toxin (AC) which has received considerable attention as a potential vaccine candidate. Great interest from laboratories involved in production, purification and quality control of acellular pertussis vaccine is focused on finding an appropriate technique for rapid and accurate quantitation of AC antigen. In this paper a competitive ELISA is proposed. A polystyrene microplate coated with purified AC was incubated with the sample to be tested plus anti-AC serum. The bound anti-AC antibodies were measured by sequential reaction with alkaline phosphatase-labelled anti-mouse IgG and p-nitrophenylphosphate. This method showed high specificity, with the 50% inhibition corresponding to 4 micrograms/ml of AC. It also proved to be useful to assess the presence of AC in culture supernatants, with high reproducibility.