Produção do Oligômero B da toxina pertussis para uso como adjuvante: etapas iniciais e perspectivas

Abstract

Butantan Institute produces a whole cell pertussis vaccine through the fermentation, purification and inactivation processes of Bordetella pertussis cells. B. pertussis is a gram- negative cocobacillus that produces several virulence factors, including adhesins and toxins. Pertussis toxin (PT) is a major virulence factor of B. pertussis and it is considered a key component of the protective immune response against B. pertussis. It has been suggested that the protective efficacy of the whole-cell vaccine may in part be related to residual active PT, and the B oligomer of PT has already been demonstrated to act as adjuvant. The present work aimed to produce the B oligomer and evaluate it as an adjuvant for vaccines produced at Butantan Institute. To obtain oligomer B, the bacterium B. pertussis was grown in a solid medium Bordet & Gengou followed by cultivation in liquid medium to be inoculated in the horizontal Wave bioreactor. The production cultures were performed with 100 mL of the above inoculum in a total of 1 L fermentation, in the Wave horizontal bioreactor for at least 20 hours with controlled temperature and agitation. During the upstream steps, we performed the in-process control, evaluating the following parameters: pH, dissolved oxygen, opacity, optical density at 590 nm (OD590nm), microscopy (Gram staining) and presence of agglutinogens. After cultivation, in the downstream process, centrifugation was performed followed by sterile filtration of the supernatant. To check the production of PT toxin, SDS- PAGE electrophoresis and Western Blotting analysis were performed using a NIBSC reference PT, JNIH-5 (10 mg/mL) and the primary antibody NIBSC Anti-Pertussis PT JNIH- 12. For purification of the pertussis toxin, we performed ion exchange chromatography, using HiTrap CM Fast Flow column. The sample was applied to the column in 50 mM sodium phosphate buffer containing 2 M urea (equilibration buffer), pH 6.0. Then, PT was eluted from the column with Buffer A (pH 7.4) and after elution of PT, FHA was eluted in a gradient of 0-50% buffer A and B (50 mM sodium phosphate pH 7.4 containing Urea 2 M and 1 M NaCl) in a flow of 3 mL/min. After the samples were eluted, the column was washed with 100% buffer B (5 column volumes). Growth in the Wave horizontal bioreactor resulted in cultures with an opacity of 50 UOp/mL, O.D (590nm) of 2.47 and a change in pH between 6.5 and 7.8 during the time of cultivation. The agglutinogen test showed the presence of Agg1, Agg2 and Agg3. Microscopic analysis revealed characteristic gram-negative cells of B. pertussis. The analysis by SDS-PAGE and Western blotting showed bands compatible with those of the positive control, corresponding to the PT subunits. In chromatography, the proteins were eluted separately from the same column and the SDS-PAGE and Western blotting showed separate bands of PT and FHA. As a perspective, after the purification of oligomer B, we will evaluate its potential as a vaccine adjuvant.