Characteristics and potency of an acellular pertussis vaccine composed of pertussis toxin, filamentous hemagglutinin, and pertactin.

In an attempt to develop a safer pertussis vaccine, we successfully purified 3 pertussis protective antigens-pertussis toxin, filamentous hemagglutinin, and a 69-kDa outer membrane protein (also named pertactin), from Bordetella pertussis strain ATCC 9340. The toxicity of pertussis toxin could be effectively reduced by the treatment with formaldehyde 0.07% while preserving of a high degree of immunogenicity. By mixing purified pertussis antigens with diphtheria and tetanus toxoids (DT), we have formulated a DT acellular pertussis (DTaP) vaccine. Toxicity studies on body-weight gain in mouse, histamine sensitization, lymphocyte promoting, and Chinese hamster ovary cell clustering tests suggested that this DTaP vaccine is safer than a whole cell vaccine produced in France (DTP[F]). The formulated vaccine elicited high levels of anti-pertussis toxin antibodies in both mice and monkeys. In mice, a 2-fold neutralization of anti-pertussis toxin antibodies was produced by DTaP compared with DTP(F) vaccine and an acellular vaccine manufactured in Japan (DTaP[J]). More importantly, in intracerebral challenge assay in mouse, this vaccine also provided a better protection than DTaP(J).

Preparation and characterization of Pertussis toxin subunits.

Pertussis toxin (PT), a typical A-B oligomer exotoxin of Bordetella pertussis, has been demonstrated to be an essential protective antigen for acellular pertussis vaccine against whooping cough. In order to investigate the associated functionality ascribed to its components, we have purified A and B oligomers for the activity study. The A oligomer (S1 subunit) of PT was expressed in E. coli B834 (DE3) harboring expression vector (pET-20b) with the insert of S1 coding region and purified by metal-chelating column. The B oligomer was isolated by a single-step purification procedure. Individually, recombinant S1 and B oligomer exhibited quite distinct biological activities in vivo. S1 subunit induced leukocytosis-promoting (LP) activity, but did not affect mouse body weight-gain. On the contrary, B oligomer reduced mouse body weight-gain but did not reveal LP activity. In vitro, the combination of S1 subunit and B oligomer could enhance the toxic activities as exhibited by native PT and showed an additive toxicity in CHO cell clustering test and hemagglutination assay.

Production and purification of Bordetella pertussis toxin.

Pertussis toxin (PT) is the major protective antigen of acellular pertussis vaccine (aP). We have established an optimal culture condition for the growth of B. pertussis and the production of PT in a laboratory scale fermentor. It was found that when the dissolved oxygen in medium was supplied with pure oxygen instead of air, the yield of PT was dramatically increased (i.e. from 2-3 mg/l using air to 8-10 mg/l using pure oxygen). PT was purified by affinity chromatography using hydroxyapatite and fetuin-sepharose columns. SDS-PAGE analysis and CHO cell clustering test showed that the purified PT was comparable to the reference PT in purity and biological activity. The purified PT could be detoxified by formaldehyde (d-PT). The results of CHO cell clustering neutralization assay and ELISA showed that the antibody induced by d-PT in mice was comparable to that induced by PT contained in a commercial DTaP. These results indicated that the immunogenicity of our d-PT was retained after the purification and detoxification procedures.

Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin.

We have examined the protease susceptibility of aortic myosin, the thermal unfolding profiles of myosin rod and light meromyosin (LMM) and the solubility properties of the LMM fragments. Two major protease-susceptible sites were found, located at the head-rod junction and the heavy meromyosin (HMM)-LMM junction. Both tryptic and chymotryptic digestion of aortic myosin rod produced the LMM (80-85 kDa) and short subfragment 2 (S-2) (40-45 kDa) segments, which were similar to those of gizzard myosin rod and differed from the short LMM (70 kDa) and long S-2 (58 kDa) segments produced from skeletal-muscle rod. The thermal unfolding profile of aortic myosin rods exhibited three helix-unfolding transitions, at 47.5, 51 and 54 degrees C, similar to those of gizzard rods yet different from those of skeletal-muscle rods. There was a dramatic difference in the solubility of aortic LMM fragments of various molecular mass, as for gizzard smooth-muscle LMM and rabbit skeletal-muscle LMM. LMM fragments of molecular mass 77 kDa or more were completely insoluble in low-ionic-strength buffer, whereas LMM fragments of molecular mass 73 kDa or less were completely soluble in low-ionic-strength buffer. Proteolytic digestion patterns of LMM showed two additional protease-susceptible sites located 13 and 30 kDa from the ends of the LMM molecule. This suggests the existence of flexible regions within the LMM molecule, which may be responsible for the folded form of aortic myosin.