Relationship of the sensitizing and protective properties of pertussis antigens

When comparing delayed hypersensitivity (DN) to B. pertussis corpuscular antigens with the agglutinogenic composition of B. pertussis, as well as to its histamine-sensitizing, leukocyte-sensitizing, adjuvant and hemagglutinating activity, no correlation was detected between the specific sensitizing properties of these antigens and the serotype and studied nonspecific properties of B. pertussis. The DH level correlated with the protective activity of B. pertussis corpuscular antigens and the ultrasonic fractions of B. pertussis. The close correlation of these two phenomena suggest that DH-inducing and protective B. pertussis antigens are identical, though their action has different manifestations, depending on the method of administration of the antigen preparation. On these grounds a new method for evaluating the immunogenicity of B. pertussis antigens is proposed. This method consists in comparing the sensitizing activity of the antigen under test and that of the national standard.     

Separation and characterization of two distinct hemagglutinins contained in purified leukocytosis-promoting factor from Bordetella pertussis.

1. The leukocytosis-promoting factor of Bordetella pertussis was found to contain two hemagglutinins with different susceptibilities to papain and separable from each other by agarose gel filtration with Tris - HCl buffer containing 1 M NaCl. 2. One hemagglutinin, referred to as hemagglutinin HA, had a high hemagglutinating activity, but neither leukocytosis-promoting nor histamine-sensitizing activity. The other hemagglutinin, referred to as hemagglutinin LPF appeared to be identical with the leukocytosis-promoting factor and possessed a low hemagglutinating and high leukocytosis-promoting and histamine-sensitizing activities. 3. The hemagglutinating activity of hemagglutinin HA was highly sensitive to papain. The hemagglutinating, leukocytosis-promoting, and histamine-sensitizing activities of hemagglutinin LPF were fairly resistant to the enzyme. 4. The two hemagglutinins were distinct from each other in immunological and chemical properties. 5. Morphologically, hemagglutinin HA showed itself to be filamentous molecules of approx. 2 X 40 nm, while hemagglutinin LPF comprised of spherical molecules of approx. 6 nm diameter. 6. The molecular weight values of hemagglutinin HA estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis and sucrose density gradient centrifugation were approx. 126 000 and 133 000, respectively. Those of hemagglutinin LPF estimated by polyacrylamide gel electrophoreis at pH 4.5, sucrose density gradient centrifugation and gel filtration on a 10% agarose column were 107 000, 103 000 and 30 000, respectively. A possible reason for obtaining such a low molecular weight value by gel filtration is discussed.     

Isolation and purification of a preparation possessing leukocytosis-stimulating properties from pertussis bacteria]

A homogeneous protein LSF-2 preparation was extracted from the cultural fluid of Bordetella pertussis strains of the 1.0.3 serological type by means of precipitation with ammonium sulphate and electrofocussing; this preparation proved to produce a marked leukocytosis-stimulating and a weak toxic action of delayed type in experiments on animals. Intraperitoneal administration of 5 mug of the LSF-2 preparation caused a rise of leukocytosis in mice to 100,000 cells per 1 mm3, a delay in the gain in weight beginning from the 3rd day of the administration and a late death of the animals in 5% of cases. The LSF-2 preparation protected the mice in infection with a virulent pertussis strain No. 18323 in the amount of from 12 to 91%, depending on the immunizing dose; its ImD50 was equal to 2.0 -2.4 mug of protein. The results of investigations carried out permitted to assess the role of this substance in the formation of specific immunity in pertussis infection.     

Vaccine from the cell fragments of Bordetella pertussis. I. Protective, sensitizing properties and morphological characteristics of the vaccine]

The authors present the results of studying the protective and sensitizing properties of a new preparation made of a ultrasonic disintegrate of pertussis microbes treated by ethyl ether. As shown by electron microscopy, the preparation consisted of the cell wall elements (the membrane), remnants of the cytoplasm and protectosome, i.e. it represented a vaccine consisting of cell fragments. In crude and sorbed condition it possessed marked protective properties (a test on mice). The content of protective units in the adsorbed preparation increased 1.5-3 times. The vaccine produced no sensitizing action, and its histamine-sensitizing activity was 3-5 times lower by protein and 5-10 times--by IOU than that of the whole-cell vaccine prepared form the same microbial suspension.     

Antigens of Bordetella pertussis. IV. Effect of heat, merthiolate, and formaldehyde on histamine-sensitizing factor and protective activity of soluble extracts from Bordetella pertussi.s

Munoz, J. (Rocky Mountain Laboratory, Hamilton, Mont.), and B. M. Hestekin. Antigens of Bordetella pertussis. IV. Effect of heat, Merthiolate, and formaldehyde on histamine-sensitizing factor and protective activity of soluble extracts from Bordetella pertussis. J. Bacteriol. 91:2175-2179. 1966.-Both histamine-sensitizing and protective activities of soluble preparations from Bordetella pertussis cells are destroyed by heating at 80 C for 0.5 hr. The histamine-sensitizing activity appeared to be more susceptible to inactivation by heat than the protective activity. Formaldehyde in a final concentration of 0.5% rapidly diminished the histamine-sensitizing ability of saline extract (SE) held at 37 C. The protective activity was clearly more resistant to inactivation by formaldehyde at similar temperature. The inactivating action of formaldehyde was slower when the concentration of SE was increased or when the mixture was kept at 2 to 5 C. Merthiolate in a final concentration of 1:10,000 had no demonstrable deleterious effects on either protective or histamine-sensitizing activity of SE.     

Endotoxin-induced hypersensitivity to histamine in mice. I. Contrasting effects of bacterial lipopolysaccharides and the classical histamine-sensitizing factor of Bordetella pertussis

Pieroni, Robert E. (Massachusetts Department of Public Health, Boston), Edward J. Broderick, and Leo Levine. Endotoxin-induced hypersensitivity to histamine in mice. I. Contrasting effects of bacterial lipopolysaccharides and the classical histamine-sensitizing factor of Bordetella pertussis. J. Bacteriol. 91:2169-2174. 1966.-The capacity of typhoid and possibly of pertussis endotoxins to induce histamine-shock susceptibility in some of the mice that survive graded doses of these endotoxins was confirmed. It was demonstrated, however, that pertussis endotoxin cannot be the main source of the typical histamine sensitization of pertussis vaccine. The following points are made. (i) With typhoid and pertussis endotoxins as inducers of histamine shock, no systematic relation between deaths and induction dose could be found, and 100% mortality could not be achieved. In contrast, with pertussis protective fraction as inducer, there was clear dose-response regression, with 100% mortality possible. (ii) The major part of the histamine-sensitizing activity of pertussis vaccine or its extracts was destroyed by trypsinization or by heating for 30 min at 100 C. These processes do not affect the histamine-sensitizing activity of the endotoxins. The implication for purified pertussis vaccine with high histamine-sensitization capacity is that endotoxin need not necessarily be present. The significance and possible mechanisms of action of endotoxin-induced histamine sensitivity are briefly discussed.     

Highly sensitive histamine-sensitization test for residual activity of pertussis toxin in acellular pertussis vaccine

Histamine-sensitization test method based on histamine-sensitizing death is widely used for controlling residual activity of pertussis toxin in acellular pertussis vaccines. The test method evaluates the residual activity according to the death of mice injected with a test vaccine after histamine challenge and the test result, therefore, depends on the sensitivity of mice. A highly sensitive test method based on change in rectal temperature of mice has been used in Japan for many years but has limited feasibility in other countries. We examined the possibility of a test method using dermal temperature measured by infrared thermometer to reduce animal suffering instead of rectal temperature. The dermal temperature method was shown to be as sensitive as the rectal temperature method. Furthermore, the dermal as well as rectal temperature methods can evaluate the activity of a test vaccine in relative to a reference preparation so as to allow direct comparison of the test results among different laboratories. The activity by means of the dermal temperature method was also found to be well consistent with that by the rectal temperature method.     

Enhancement of reaginic and hemagglutinating antibody production by an extract of Bordetella pertussis containing histamine sensitizing factor.

The effect of an extract containing the histamine-sensitizing factor (HSF) of Bordetella pertussis on the immune response of mice to ovalbumin was investigated with respect to dose of antigen and adjuvant. Of particular interest was the enhancement of reaginic antibody production. In comparison to the Al(OH)3 induced production of reaginic antibody where low doses of antigen and adjuvant yield high titers of reagin, the HSF extract demonstrated optimal adjuvant activity at high doses of both adjuvant and antigen. The reaginic antibody response was maximal usually by 2 to 3 weeks post-immunization and persisted for long periods of time. The hemagglutinating antibody response was maximal at 8 to 10 weeks post-immunization. The initial treatment of mice with HSF extract plus antigen resulted in the production of memory cells since a subsequent immunization with ovalbumin alone evoked a secondary reaginic response. These observations may have implications in clinical allergy since substances similar to the pertussis factor might be produced by other microbial organisms and these substances could modulate the immunologic response of individuals to common allergens.     

Immunogenicity and toxicity of soluble trichloroacetic acid precipitate from pertussis microbes and its fractions]

The authors studied the biological properties of the preparations of pertussis protective antigens obtained by the disintegration of the microbial mass of Bordetella pertussis, with the subsequent purification with trichloracetic acid (TCA-preparations). TCA-preparations proved to possess a stable protective activity and by the ratio of the protective dose to toxic and histamine-sensitizing doses considerably exceeded the corpuscular vaccine. A TCA-preparation fraction with a greater immunogenic activity than the initial preparation was obtained by chromatography on sepharose 4B.     

Comparative characteristics of the toxic properties of the whooping cough microbes]

The author presents materials concerning the study of the toxic properties of 12 pertussis cultures used in the USSR for the production of vaccines. As a result of studying the toxic properties of the microbes in the test of the change of the weight of mice, and in experiments of testing the anaphylactogenic and histamine-sensitizing activities it was revealed that the strains of pertussis microbes differed by the quantitative content in them of the thermolabile toxin, endotoxin, lymphocytosis-stimulating and histamine-sensitizing factors; therefore, the period of keeping the pertussis suspensions for 1.5 months from the moment of detoxication proved to be inadequate for some of the strains. Since the toxic properties of the strains failed to correlate with their protective activity, in recommendation of pertussis strains for the production of pertussis vaccine preference should be given to the strains with lesser toxic properties.     

Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-sepharose affinity chromatography

A general procedure for purifying biologically active pertussis toxin from Bordetella pertussis fermentation broth using affinity chromatography on heat-treated fetuin-Sepharose CL-4B is described. Diethanolamine is used as eluent in this single-step purification to prepare endotoxin-free pertussis toxin in good yield (70%) and high purity (greater than 95%). This one-step affinity chromatography procedure can be easily applied for large-scale preparation of pertussis toxin S1 subunit and its B-component. The affinity-purified S1 subunit is devoid of any of the histamine-sensitizing activity normally associated with pertussis toxin. The chromatographically purified pertussis toxin and its subunits retained their immunogenicity and could induce high levels of anti-toxin neutralizing antibodies.     

The pyrogenicity of pertussis vaccine in mice and the factors in the vaccine responsible for this effect

The injection of whole cell pertussis vaccine into mice produced a biphasic fever reaction with two peaks appearing after about one and four hours, respectively. A method for the quantitative determination of each peak fever activity was developed and the factor responsible for each activity was investigated. The first and the second peak fever activities did not parallel each other in individual vaccines. The earlier fever activity appeared to correlate with endotoxin activity in individual vaccines while the later appeared to correlate with histamine-sensitizing factor (HSF) activity. The later peak fever activity was greatly reduced by heating the vaccine at 100 degrees C for 30 min while the first was little affected by such treatment. It was concluded that the fever activity of pertussis vaccine in mice may be ascribed to the combined actions of endotoxin and a heat-labile substance, possibly HSF.     

Correlation between growth rate and loss of histamine-sensitizing factor during antigenic modulation in Bordetella pertussis

The pattern of loss of histamine-sensitizing factor (HSF) during antigenic modulation of Bordetella pertussis in Hornibrook medium was examined. The aim was to determine the possible underlying mechanism involved in modulation. Normal (X-mode) B. pertussis cells were grown in Hornibrook medium in which 0.5% (w/v) NaCl had been replaced with 0.5% (w/v) MgSO4. 7H2O (C-medium). At various time intervals during growth, the viable cell numbers and optical densities of both cultures in the X- and C-media were estimated. Whole cells were harvested from the cultures at the same time intervals and aliquots from the cultures were assayed for the levels of their histamine-sensitizing properties. Correlation of the increase in viable cell numbers with rate of loss of histamine-sensitizing activity in both the cells and whole cultures indicated that components responsible for the histamine-sensitizing activity were not synthesized during modulation. Moreover, the loss of HSF from B. pertussis cells was faster than can be explained by dilution of the original factor in the inoculum among progeny cells. Modulation may involve cessation of synthesis and selective degradation or denaturation of some envelope polypeptides immediately upon inoculation of normal X-mode B. pertussis cells into C-medium.     

Acellular and whole cell pertussis vaccines protect against the lethal effects of intracerebral challenge by two different T-cell dependent humoral routes

Athymic (nu/nu) and euthymic (+/nu) BALB/c mice were immunized with a whole cell pertussis vaccine or with an acellular vaccine which contained detoxified pertussis toxin (PT) and filamentous hemagglutinin (FHA). Only the euthymic mice were protected against intracerebral challenge with virulent Bordetella pertussis which implies involvement of T-cells. As a cell transfer from mice immunized with whole cell or acellular vaccine prior to the challenge did not protect naive euthymic recipients, cellular immunity seems to be non-protective as an effector mechanism. Mice could be protected passively against a challenge by administration of immune sera. Therefore, T-cell dependent humoral immune responses to B. pertussis appear to be crucial for protection. The humoral response was further studied with athymic and euthymic mice. In euthymic mice the whole cell vaccine induced antibodies to FHA, pililipopolysaccharides (LPS) and an outer membrane protein (OMP) preparation, whereas the acellular vaccine induced antibodies to PT, FHA and OMP. Both IgM and IgG could be detected. From the nude mice only those immunized with the whole cell vaccine showed an antibody response which consisted of low titres of IgM directed to LPS. Sera from both +/nu and nu/nu mice immunized with the whole cell vaccine were bactericidal in vitro. These data demonstrate that in the mouse model protection to intracerebral challenge with B. pertussis is T-cell dependent as is the humoral response to PT, FHA, OMP and pili. The T-independent B-cell activation by the whole cell preparation is due to the presence of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine-sensitizing Factor, Mouse-protective Antigens, and Other Antigens of Some Members of the Genus Bordetella

The three species of the genus Bordetella-B. pertussis, B. parapertussis, and B. bronchiseptica-have many antigens in common. Studies on representative strains of these species have shown that there are only a few specific antigens in each species. Whole-cell vaccines and extracts from B. pertussis contained specific mouse-protective antigen and a histamine-sensitizing factor. In addition, whole-cell vaccines and some saline extracts protected mice against intracranial challenge with B. bronchiseptica. Cells and a saline extract of B. parapertussis also protected against B. bronchiseptica but not against B. pertussis. Whole cells of B. bronchiseptica protected against B. bronchiseptica, but only one of three saline extracts protected against this challenge. Neither whole cells nor saline extracts from B. bronchiseptica protected against B. pertussis. The antigen in B. pertussis responsible for cross-protection against B. bronchiseptica was less resistant to heat than the protective antigen in B. bronchiseptica. Since histamine-sensitizing factor was not detected in B. bronchiseptica or B. parapertussis cells or extracts, this factor is not required to protect mice against B. bronchiseptica challenge. Whether B. pertussis vaccines protected against B. bronchiseptica by a nonspecific mechanism was not established, but it is clear that the specific antigen responsible for protection against B. pertussis was found only in B. pertussis and not in B. bronchiseptica or B. parapertussis.     

Polymorphonuclear leukocyte-inhibitory factor of Bordetella pertussis. I. Extraction and partial purification of phagocytosis- and chemotaxis-inhibitory activities.

A new factor that inhibited phagocytosis to opsonized targets and chemotaxis of PMN was extracted from B. pertussis cells, and named PMN-inhibitory factor (PIF). Cells in phase I produced 10 times more PIF than those in phase III, and like other phase I-associated components--the hemagglutinin, the histamine-sensitizing factor and agglutinogens--PIF showed degenerative, phenotypic variation during in vitro culture of phase I bacteria. PIF was partially purified by four steps, including adsorption chromatography on Dansyl-aminononamethylene Sepharose. The resulting fraction was heterogeneous but showed little histamine-sensitizing and cytotoxic activities and was free from LPS, the hemagglutinin and a leukocyte agglutinin. The inherent resistance of B. pertussis cells, in either phase I or III, as demonstrated also in the present study, and PIF-mediated defiance against immunological defense mechanism may constitute a complex host-parasite relation in experimental infections with B. pertussis.     

Effect of immunomodulators of various origin on the histamine-sensitizing activity of whole-cell pertussis vaccine]

Changes in the histamine-sensitizing activity of whole-cell pertussis vaccine (PV) under the action of immunomodulators (IM) of bacterial (peptide and peptidoglycans), synthetic (peptidoglycan) and vegetable origin have been studied. The study has revealed that these IM, introduced orally and parenterally, exhibit histamine-sensitizing activity, depending on the nature of IM and the optimum selection of the doses of IM and PV.     

Immunochemical study of the antigenic structure of bacteria of genus Bordetella. IV. Fractionation of B. pertussis extracts and study of the immunochemical and biological properties of the isolated fractions]

Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.     

An international collaborative study of the effect of active pertussis toxin on the modified Kendrick test for acellular pertussis vaccines

Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose–response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.