Physicochemical and immunobiological properties of the dialysate antigen of Bordetella pertussis

The study of the physicochemical and immunobiological properties of B. pertussis dialysate antigen indicates that the antigen has a complex composition and possesses hemagglutinating and lymphocytosis-promoting activity, which permits further studies with a view to developing diagnostic and prophylactic preparations on the basis of this antigen.     

Modern strains of Bordetella pertussis: immunobiological properties and vaccine improvement

Strains of B. pertussis isolated from patients in Moscow in 2001-2005 as well as strains included in locally produced diphtheria-tetanus-whole cell pertussis (DTP) vaccine were studied. Nucleotide sequences in genes of pertactin and S1-subunit of pertussis toxin of isolated strains, their immunobiological properties and opportunity to use for producing of the acellular pertussis vaccine were determined. Genes of pertactin and S1-subunit of pertussis toxin in the isolated wild strains differed from the same genes in strains included in the local DTP vaccine. Majority of the isolated strains belonged to serotype 1.0.3 and were markedly virulent.     

Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form.     

Extracted protective antigen of Bordetella pertussis. I. Preparation and properties of the solubilized surface of components

Bordetella pertussis microorganisms were treated with several extracting agents followed by ultracentrifugation to remove particulate matter. Analysis of the resulting supernatants by SDS gel electrophoresis showed one major component after simple salt extraction, and much more complex, although consistent pattern following detergent treatment. The yield of the solubilized protein in detergent extracts exceeded by far the values recorded for salt extracts. In order to prevent irreversible precipitation of the solubilized proteins upon removal of the denaturing agent, a novel procedure was developed. After extraction with urea-salt, the solubilized material was absorbed on a mineral carrier prior to the separation of the denaturing agent. The resulting absorbed vaccine was highly potent in the mouse-protection test, whereas the toxic reactions, elicited upon injection into experimental animals, were reduced in the comparison to the starting material. This diminished reactogenic potential was accompanied by the partial loss of the leukocytosis-promiting factor, whose activity was greatly diminished by urea-salt at alkaline pH-values. The procedure described may be applied to large-scale processing of Bordetella persussis microorganisms. Clinical trials now in progress should confirm or rebut the thesis that increased tolerability of the product, inferred from animal experiments, is reflected by fewer adverse reactions in humans. In the former case, the detergent extract vaccine may constitute a realistic alternative to conventional whole-cell vaccines against whooping-cough.     

Role of the protein fraction of Bordetella pertussis in inducing experimental allergic encephalomyelitis

B. pertussis protein fraction obtained by precipitation with trichloroacetic acid (TCA) stimulated the development of clinically and histologically pronounced experimental allergic encephalomyelitis (EAE) in guinea pigs when introduced together with a heterologous cerebral antigen, the two compounds forming an encephalitogenic mixture. The adjuvant activity of the TCA-precipitated fraction depended on its dose. The sera of the animals with EAE induced by the encephalitogenic mixture containing B. pertussis cells or TCA-precipitated fraction showed a cytopathogenic effect in the monolayer culture of newborn rat cerebellum cells. The cytopathogenic effect was more pronounced in the sera obtained at the period of the development of the clinical symptoms of the disease (days 14-18), while the cytotoxic effect of the sera obtained on day 30 after immunization decreased irrespective of the manifestations of EAE, this decrease being in correlation with the dose of the TCA-precipitated fraction in the encephalitogenic mixture.     

Mouse-protective properties of Bordetella pertussis serotypes in passive tests

In a passive protection procedure in which the ed(50) values of Bordetella pertussis antisera were determined, groups of mice were given graded intraperitoneal doses of serum, followed the next day by intracerebral challenge with 100,000 organisms. Antiserum produced with B. pertussis culture 5373, serotype 1.3, protected mice against challenge with culture 18-323, serotype 1.2.3, as effectively as did an antiserum produced with a serotype 1.2.3 culture. When two groups of mice similarly treated with pertussis immune serum were challenged with culture 353Z (serotype 1) and 18-323, respectively, much lower ed(50) values were obtained with the animals challenged with 353Z. Passive protection tests with adsorbed antiserum gave equivocal results, suggesting that some of the adsorbing antigen remained in the serum and interfered with the tests. There was no evidence that serotype is related to protection.     

Effect of methyl-cyclodextrin on adenylate cyclase activity of Bordetella pertussis

The activity of Bordetella pertussis extracytoplasmic adenylate cyclase (AC) decreased during decelerating growth phase in a Stainer-Scholte medium. Neither proteolytic activity nor virulence variation (phase variation; antigenic modulation) appears to be responsible for the observed activity fall. The addition of methyl--cyclo-dextrin enhances AC activity and prevents the inhibition of AC activity by fatty acids. Cyclodextrin could entrap inhibitors increasing in this way the AC activity. These results show that the inclusion of cyclodextrin in the culture medium increases the AC activity.D.F. Hozbor and O.M. Yantorno are with the Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, (1900) La Plata, Argentina. A. Samo is with the Comisión de Investigaciones Cientificas de la Provincia de Buenos Aires.     

Elaboration of a method of stabilizing the protective fraction of pertussis microbes

A method for stabilizing adsorbed preparations of the protective fraction of B. pertussis has been developed; according to this method, a colloid suspension of protective protein in phosphate buffer is obtained, the protein is then adsorbed on aluminium hydroxide gel and lyophilized with 10% of sucrose. If stored at 4 degrees C, these dried preparations have been found to retain their immunogenicity for 1 year.     

一株减毒百日咳鲍特菌的构建及生物学特性分析

百日咳是传染性强、感染率高的急性呼吸道传染病,主要感染婴幼儿,是婴儿死亡的主要原因之一。百日咳鲍特菌（Bordetella pertussis）是引起百日咳的最主要病原菌。近年来世界各地多次出现百日咳暴发,迫切需研制更加有效的新型百日咳疫苗。本研究构建了一株减毒百日咳活疫苗BPTM1,利用同源重组方法敲除编码百日咳鲍特菌主要毒力因子百日咳毒素（pertussis toxin,PTX）和皮肤坏死毒素（dermonecrotic toxin,DNT）的基因,并用大肠埃希菌的同源基因置换了负责气管细胞毒素（tracheal cytotoxin,TCT）转运的基因ampG。通过聚合酶链反应验证了毒素及相关基因的敲除和置换,蛋白免疫印迹法检测表明PTX的S1亚基未表达。体外生长曲线和体内定植曲线均表明,相比于野生型百日咳鲍特菌BPMM,减毒BPTM1的生长和定植能力未受影响,其所致肺部病理效应减轻,而所诱导的百日咳鲍特菌特异性IgG、IgG1、IgG2a抗体保持高水平。本研究表明,减毒百日咳鲍特菌BPTM1有可能成为百日咳疫苗的候选疫苗。