Detoxification pertussis

Since 1940, vaccination with whole cell vaccines, composed of Bordetella pertussis extracts, has dramatically reduced the incidence of whooping cough. However, the occurrence of side effects has stimulated research that has resulted in the design of new, acellular vaccines. These vaccines are composed of adhesins, such as filamentous haemagglutinin and pertactin, and detoxified pertussis toxin. Detoxification can be achieved by genetic engineering resulting in alterations of specific amino acid residues that are involved in the enzymatic activity or in the target-cell receptor binding activity of the toxin...

Progress in molecular biology and biotechnology is making possible the development of new vaccines or the improvement of already existing ones. Recombinant DNA technology, genetic attenuation of bacterial and viral pathogens and their use as vectors for heterologous proteins, expression of microbial antigens in transgenic edible plants, and naked nucleic acid technology represent the most popular approaches hitherto adopted. A successful biotechnological approach to the development of new and improved vaccines has been based on genetic detoxification of bacterial toxins, such as the toxin of Bordetella pertussis, the heat-labile enterotoxin of Escherichia coli, and the toxin of Vibrio cholerae...

Physico-chemical methods are being developed for use in the control and standardization of acellular pertussis vaccines and their individual components. We have compared native and detoxified preparations of the B. pertussis antigens, pertussis toxin (PT), filamentous haemagglutinin (FHA), and the 69-kDa outer membrane protein (P69) using circular dichroism (CD), fluorescence spectroscopy, SDS-PAGE and FPLC gel filtration chromatography. Upon aldehyde detoxification, PT underwent a large change in its intrinsic fluorescence maximum (8-10 nm red-shift) and a large increase in its apparent size, detected by chromatography...

The quality control of acellular pertussis vaccines presents particular problems related to the differences in composition and method of detoxification used in the various type of preparation. These vaccines are not amenable to potency assay by the active mouse protection test used for whole-cell pertussis vaccines and assurance of protective activity is problematic. In contrast, monitoring of these vaccines for safety is relatively straightforward and is centred on assays for the lipooligosaccharide endotoxin, active pertussis toxin and absence of reversion to toxicity of detoxified product...

NAVA's acellular pertussis vaccine is based on highly purified pertussis toxin (PT) inactivated with H(2)O(2). PT was analysed using advanced biochemical methodology including mass spectroscopy (LC/MS), yielding mass and peptide mapping information on the subunits. Pertactin, adenylate cyclase, and Fim 1, 2 were below detection levels and only trace amounts of filamentous haemagglutinin (FHA) have been identified as a minor impurity. The vaccine does not induce anti-FHA antibodies during the course of a 3-dose primary immunization series in infants...

In 1974, the authors reported the isolation and characterization of protective antigens of Bordetella pertussis in mice. With this information, an acellular pertussis vaccine was developed, composed mainly of pertussis toxin (PT) and filamentous haemagglutinin (FHA). Substances causing side effects, especially lipopoly sacahoride (LPS) or endotoxin that cause fever, were removed, and detoxification of the PT by formaldehyde with retention of potency was achieved. In 1981, an acellular pertussis vaccine called the "Adsorbed Purified Pertussis Vaccine" was approved in Japan, in place of the whole-cell pertussis vaccine...

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...

Until very recently, development of vaccines has been based on an empirical approach. For example, bacterial toxins have been detoxified using empirical chemical treatment. Progress in biotechnology and molecular biology has allowed the fine knowledge of the structure-function relationship of several bacterial toxins. Thanks to this, the genetic attenuation of bacterial toxins has been made possible. Following this approach, a genetically detoxified pertussis toxin has been produced. This molecule is now the component of an acellular pertussis vaccine, which has been shown to be highly immunogenic and efficacious in infants...

The development of acellular pertussis vaccines has raised a number of issues relevant to the control of these products. Of particular importance is the need for robust and accurate in vitro assays for the antigen content of the vaccines which might contain up to five different antigen components, each of which needs to be independently assayed. This paper describes a simple method for the quantification of three component antigens. Because relatively high doses of purified antigens are used in those preparations, the elimination of residual toxicity is a major concern...

Vaccines represent the most cost-effective means to prevent infectious diseases. Most of the vaccines which are currently available were developed long before the era of molecular biology and biotechnology. They were obtained following empirical approaches leading to the inactivation or to the attenuation of microorganisms, without any knowledge neither of the mechanisms of pathogenesis of the disease they were expected to protect from, nor of the immune responses elicited by the infectious agents or by the vaccine itself...

Bacterial toxins are commonly detoxified by chemical treatment in order to use them in human vaccines. We have used site-directed mutagenesis of toxin genes to obtain bacteria that produce naturally nontoxic mutants of bacterial toxins, such as pertussis toxin (PT), cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT). Genetically detoxified PT showed a superior safety and immunogenicity in animal models, phase I and phase II clinical trials, and a superior protective efficacy in the early and late stage of a phase III efficacy trial, proving in a definitive and extensive way that genetic detoxification of bacterial toxins can, and should, replace chemical treatment...

Adverse reactions to routine vaccines are obstacles to the mass vaccination campaigns. Though the absolute safety of any injectable vaccine cannot be guaranteed, the adverse side effects to vaccines can be minimized by practicing existing scientific knowledge. Adverse side effects to tetanus and diphtheria toxoids have been known for many years and there have been ways to minimize these reactions. These procedures did not get wide acceptance, because the current partially purified tetanus and diphtheria vaccines meet the regulatory requirements and the manufacturers are reluctant to change the established procedures of production due to the amount of work involved in the regulatory issues under the current Good Manufacturing Practices (GMP)...

The effect of detoxification of pertussis toxin (PT) for vaccine usage by either genetic manipulation, hydrogen peroxide or formaldehyde treatment on epitope recognition by a large collection of murine monoclonal pertussis toxin antibodies (PT MAbs) was assessed in a solid-phase and a soluble phase enzyme-linked immunosorbent assay (ELISA). The MAb binding patterns were found to be different in the two assays as the immobilization step appeared to cause conformational alterations in the native as well as the toxoided forms of PT...

Endotoxin [lipopolysaccharide (LPS)], the major antigen of the outer membrane of Gram-negative bacteria, consists of a variable-size carbohydrate chain that is covalently linked to N,O-acylated beta-1,6-D-glucosamine disaccharide 1,4'-bisphosphate (lipid A). The toxic activity of LPS resides in the lipid A structure. The structural features of synthetic peptides that bind to lipid A with high affinity, detoxify LPS in vitro, and prevent LPS-induced cytokine release and lethality in vivo were defined. The binding thermodynamics were comparable to that of an antigen-antibody reaction...

No abstract text is available yet for this article.

Cultures of Bordetella pertussis cultivated in shake flasks were invariably highly toxic for mice, but cultures of the same strain grown in vortex-aerated vessels were nontoxic at the time of harvest. Results reported here indicate that toxin is present during the early log phase in vortex-aerated cultures, but is lost as the cultivation proceeds. The loss of toxicity is apparently due to denaturation of the toxin by the combined influence of vigorous aeration and elevated pH.

No abstract text is available yet for this article.

A new acellular pertussis vaccine, whose official name is "Precipitated Purified Pertussis Vaccine" (PPV), was prescribed in the Japanese Minimum Requirements for Biological Products in 1981. The PPV is composed of partially detoxified fractions of pertussis cells. The histamine-sensitizing (HS) activities of PPV vaccines are less than those of killed whole pertussis cell vaccines (WPV) produced in Japan until 1980, when the histamine challenge was carried out in mice on day four after injection. It was found that when the histamine challenge day was delayed that the HS activities of some lots of PPV gradually increased from those estimated on day four after injection of PPV and reached the maximum in about 12 days, while the activity of the reference whole cell pertussis vaccine reached the maximum several days after injection and then gradually decreased...

The physicochemical and biological properties of antigenic complexes isolated from the supernatant fluid of the culture medium of B. pertussis, strains 305 and 475, were studied. The preparations obtained from both strains contained proteins, carbohydrates, nucleic acids and lipids. Electrophoresis in polyacrylamide gel revealed the presence of filamentous hemagglutinin, 4 subunits of B. pertussis toxin and agglutinogens in the antigenic complexes of both strains. The preparations of both strains possessed similar toxic properties and, after their detoxification, produced a pronounced protective effect...

Rabbits that were injected intradermally with pertussis toxin (PT), produced from Bordetella pertussis, showed slight edema and erythema at the injection sites, but not hemorrhage nor necrosis. The edema lesions were stained blue by the intravenous injection of Pontamine Sky Blue 6B dye, suggesting that PT caused increased vascular permeability, similarly to the permeability factor (PF) of cholera toxin. The reaction of the PF of PT could be determined by measuring the diameter of the blue area. The diameter of the blue area bore a good linear relationship to the logarithm of the dose of PT...