Glutaraldehyde inactivated pertussis vaccine: a less histamine sensitizing vaccine

The effects of different inactivating agents on the biological activity of the histamine sensitization factor of Bordetella pertussis toxin were examined. The agents were used for inactivation in the preparation of whole cell pertussis suspension. The histamine sensitizing activity was reduced to 36.9-13.3% by treatment with glutaraldehyde, to about 50% by treatment with formaldehyde and by the acetone-II treatment, relative to the reduction by heat treatment. Treatment with thimerosal and the acetone-I treatment did not reduce the histamine sensitizing activity as the 50% histamine sensitizing doses of the heat inactivated pertussis preparation, the thimerosal inactivated pertussis preparation and the acetone-I treated pertussis preparation were very similar. Glutaraldehyde has thus been found to be a better inactivating agent for the preparation of a safe pertussis suspension as it considerably reduced the histamine sensitizing activity of pertussis toxin.     

Removal of lipopolysaccharide from acellular Bordetella pertussis vaccine by detergent treatment

Protective antigen was extracted from Bordetella pertussis cells with 1.0 M NaCl and precipitated with ammonium sulfate, 20-40% saturation (designated fraction 15A-1B). The protective antigen was purified further by detergent (Emulphogene BC720) treatment and adsorption to aluminum hydroxide gel (designated fraction 15A-108A). Compared with B. pertussis vaccine and fraction 15A-1B, fraction 15A-108A retained protective activity as assessed by the mouse protection test, but had reduced protein and markedly reduced endotoxin content. Fraction 15A-108A also had reduced leukocytosis-promoting, histamine sensitizing splenomegaly-inducing, and adjuvant activities. Emulphogene treatment provided a relatively simple method for removing endotoxin from a potential acellular B. pertussis vaccine.     

The use of chemiluminescence for assessing the physiological status of macrophages activated by pertussis preparations

The work deals with the comparative analysis of oxygen-dependent metabolic processes in the peritoneal macrophages of intact and immune, mice after their interaction with whole-cell pertussis vaccine (WCPV), dialyzed pertussis antigen (DPA) and fraction 2 of this antigen. The study revealed that WCPV, DPA and its fraction 2 modulated the production of active forms of oxygen by peritoneal macrophages, evaluated by means of luminol-dependent chemiluminescence (CL). The influence of WCPV of oxygen-dependent metabolic processes in macrophages after the first contacts with them had a dose-dependent character: low concentrations activated and higher concentrations suppressed the "respiratory" explosion. The immunization of animals abolished the effect of extinguishing CL on the contact of macrophages with high doses of WCPV and essentially increased the level of their CL response to all pertussis preparations in comparison with those for intact cells. DPA and its fraction 2 were not inferior to WCPV in their capacity for inducing the "respiratory" explosion, and in high doses they essentially surpassed WCPV doses in this capacity, especially on the first contact with macrophages.     

Consistency of Bordetella pertussis vaccine seed strains and potency of whole-cell pertussis vaccine still in use in Poland

In Poland, where the wP vaccine has been used since 1960, pertussis rates increased in the mid-1990s. In 2012, the rate of pertussis recognised by surveillance was unexpectedly found to be two-fold higher than in the previous decade. Quality measures on potency and vaccine working seeds were introduced, to confirm the possible impact of manufacturing inconsistency or potency lowering on the observed increase in pertussis. Shewhart charts on potency values for lots released between 2001 and 2013 did not reveal any significant fluctuations. Working seeds of three vaccine strains used within last decade for wP manufacturing belong to the PFGE group III and were highly related. According to PFGE and SDS-PAGE data, all vaccine strains were found consistent according profiling on the genomic and protein levels. According to the sequencing data, they harboured ptxA2, ptxC1, prn1, fim2-1, fim3-1, tcfA2, ptxP1 and were assigned as MLST-2 type. Other factors apart from vaccine manufacturing inconsistency might be responsible for the increase in pertussis noted in 2012 in Poland.     

Acellular pertussis vaccine

Protective, immunogenic, toxic, and sensitizing properties of acellular pertussis vaccine (aPV) developed according to original technology were studied, aPV had marked protective activity which lasted more than 2 years. Sera of mice immunized by aPV also possess protective properties, and they were more prominent than in sera of mice immunized by pertussis bacteria suspension (PS). Immune sera to aPV neutralized cytopathogenic effect of pertussis toxin (PT) on ovarian Chinese hamster cells in 1:250 dilution, whereas neutralizing activity of sera to PS was very low. Level of antibodies to PT was higher in rabbits immunized, according to schedules and dosage recommended for children, by aPV than by PS. High immunogenicity of aPV was proved also by levels of IgG to PT in sera of mice immunized three times by aPV in human dosage. During experiments on mice and guinea pigs aPV had mild toxicity, did not induce autoimmune process, did not have anaphylactogenic properties compared with bacterial suspension characterized by high anaphylactogenic activity. Histamine-sensitizing abilityof aPVwas 40 times lower than that of PS. Assessment of pyrogenic properties of aPV and PS performed on rabbits showed that aPV was 1,000 times less pyrogenic than PS. Obtained results demonstrate high protective and immunogenic properties of domestic acellular pertussis vaccine and its low toxic and sensitizing characteristics.     

Bordetella pertussis serotypes in Canada.

A study was done to determine the major antigenic factors of Bordetella pertussis strains isolated throughout Canada and whether these isolates have the same antigenic structure as the bacilli in the currently used vaccines. Testing for the major pertussis antigens, factors 1, 2 and 3, was conducted with 440 freshly isolated strains of B. pertussis received from seven canadian provinces between August 1976 and February 1978 and six batches of pertussis vaccine or immunizing agents containing pertussis vaccine. With the aid of specific antisera prepared in rabbits, the antigenic factors were detected by a slide agglutination technique. Almost all (98.9%) of the pertussis strains examined were serotype 1,3.All six batches of pertussis vaccine or immunizing agents containing pertussis vaccine proved to be rich in each of the three main pertussis agglutinogens.     

Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.     

Antigenic heterogeneity in subunit S1 of pertussis toxin

Culture supernates containing pertussis toxin (PT) from four strains of Bordetella pertussis were examined for both immunological reactivity and biological activity. PT from all four strains sensitized mice to histamine and toxin was detectable in supernates of all strains when examined by Western blotting with polyclonal antiserum to PT. In supernates of three of the four strains, PT was detectable by an enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody to subunit S1 of PT as the third antibody layer. However, supernates from one strain, 18323, failed to react in ELISA. Electroblots probed with the monoclonal antibody labelled subunit S1 of PT from all strains except that of strain 18323. PT of strain 18323, whilst retaining histamine-sensitizing activity, differed antigenically from that of other strains.     

Molecular genetic characteristics of the B. pertussis strains isolated in different periods of the pertussis epidemic process

Despite the fact that the mass immunization of the children population with the DPTs vaccine has been carried out in the Russian Federation since 1959, the pertussis infection persists to be one of the pressing problems for the children population. Although the vaccination coverage of the children population with pertussis vaccines is high in Russia, at present time the pertussis incidence rates are increasing among schoolchildren and remain high among infants younger than 12 months old. Many researchers believe that the variability of the genetic structure of the pertussis causative agent may be one of the causes of increasing pertussis incidence rates. This investigation provides the molecular genetic characteristics of 97 B. pertussis strains isolated in pertussis patients in Moscow in different periods of pertussis epidemic process since the 1950s up to present time. It shows the changes in the structures of genes, which are encoding the main protective antigens of the pertussis microbe that are the pertussis toxin (ptxS1) and the pertactin (pm). The structurre of the ptxS1 and pm gene of the B. pertussis vaccine strains was compared with the structures of these genes in the B. pertussis strains isolated from the pertussis patients at present time and also in past years. All B. pertussis strains isolated in the prevaccination period (1948-1959) and most strains (95%) isolated during the first twenty years of the mass immunization in Russia are characterized by the presence of the so called "vaccine" alleles of the pertussis toxin and pertactin genes that are ptxS1 B or ptxS1 D and pm 1 alleles that corresponds to the genetic structure of the vaccine producing strains. In the early 1970s the B. pertussis strains of another toxin and pertactin genetic structures with so-called "non-vaccinal" alleles ptxS1 A and pm 3 (pm 2 since 1980s) began to appear. The B. pertussis strains with "non-vaccinal" alleles have completely displaced the "old" strains. At present time in Moscow the pertussis disease is caused by the B. pertussis strains bearing ptxS1 A and pm 2 or pm 3 alleles of pertussis toxin and pertactin genes. There was no correlation between the genotype and serotype. Thus, the structure of the B. pertussis toxin and pertactin genes in strains which have been isolated since the 1980s up to now differs from the structure of these genes in strains which are used for producing DPTs vaccine. The data obtained in this investigation suggest that the genetic structure specificity of circulating B. pertussis strains that are producing the disease at present time should be used as one of the criteria for selecting vaccine producing strains.     

Evaluation of whole-cell and acellular pertussis vaccines effectiveness in clearance of experimental B. pertussis infection in mice

The study is based on assumption that B. pertussis strains harbouring different allele variants of genes encoding subunit S1 of pertussis toxin and pertactin might be eliminated with different efficiency from lung tissue of mice which were immunized with whole-cell and acellular pertussis vaccines. It has been assumed that strains containing combinations of genes alleles which were not prevalent since 1990-ties are consisting of mutated strains in respect to pertussis toxin subunit S1 and pertactin, and are capable to decrease efficiency of pertussis vaccines. Experiments performed in vivo dealt with activity of tested vaccines against B. pertussis strains of different combinations of ptxS1/prn. The study indicated for lowered efficiency of whole-cell and acellular pertussis vaccines in elimination of mutated strains of B. pertussis from animal lung tissue in comparison with strains currently used for vaccine production.     

Comparison of some immunologic properties of differentHaemophilus pertussis strains

Conclusion The experiments made show that fairly strong variations occur in the properties of fiveH. pertussis strains investigated. It is not possible to demonstrate a clear correlation between some of these characteristics. However, it seems that the ability of strains to produce phase-I agglutinins in the rabbit is related to their protective potency in experimental murine pertussis. Presumably both these properties have been the accepted criteria for selecting strains for the preparation of a vaccine.In our experiments one strain was found to be superior to the others. This strain was chosen for the vaccine production. The antibody-response of children, injected with this vaccine, was satisfactory. Four weeks after the last injection 28 of 33 vaccinated children showed titres higher than 300.     

Development of pertussis vaccine production and control in the National Institute of Public Health in the Netherlands during the years 1950–1962

The development of the pertussis vaccine production in the National Institute of Public Health in the Netherlands since 1953, and the results with the consecutive lots of vaccine in the mouse protection test and the U.S.A. toxicity test are described. The results in the latter test are compared with the results of a locally developed guinea pig toxicity test. Special attention is given to the difficulties encountered when the U.S.A. toxicity test is used for adsorbed DPT vaccines. The potency data of all lots of DPT vaccines produced since 1958 fall within the limits of the potency test as prescribed in the U.S.A. Minimum Requirements. There are indications that the increased potency of the vaccine may have led to a lower mortality rate of pertussis.     

The effects of different inactivating agents on the potency, toxicity and stability of pertussis vaccine

The effect of heat (56 degrees C for 10 min), formaldehyde (0.1% at 37 degrees for 24h), glutaraldehyde (0.05% at room temperature for 10 min), thimerosal (0.02% at 37 degrees C for 24h), acetone-I (three treatments at room temperature) and acetone-II (three treatments at room temperature and fourth treatment at 37 degrees C), when used as inactivating agents in the preparation of pertussis suspension, was studied with regard to potency, toxicity and stability. Five batches each of Bordetella pertussis strains 134 and 509 were used for the study. The thimerosal inactivated pertussis (TIP) preparation was 1.5-2 times more potent than the heat inactivated pertussis (HIP) preparation. The potency values of the formaldehyde inactivated pertussis (TIP) and glutaraldehyde inactivated pertussis (GIP) preparations were similar to those of the HIP preparation, while the potencies of the acetone-I treated pertussis (A(I)TP) and acetone-II treated pertussis (A(II)TP) preparations were about half those of the HIP preparation. The FIP preparation was the least toxic showing maximum weight gain in the mouse weight-gain test (MGWT), while the TIP preparation did not pass the MWGT. The weight gains shown the GIP, A(I)TP and A(II)TP preparations were greater than those shown by the HIP preparation. The potency of pertussis component in the adsorbed diphtheria-pertussis-tetanus (DPT) vaccine was stable at 4-8 degrees C and 25 degrees C for three months for all types of pertussis vaccine. There was about 54-65% loss in the potency of the samples after three months at 35 degrees C. The inactivating agents used in the manufacture of pertussis preparations had no effect on the stability of the vaccine.     

Potency of pertussis component in the DTP vaccine--an overview of three decade study in Poland.

In Poland, similar to many highly immunized Western countries, a recent increase in cases of pertussis has been observed. This study aims to evaluate the level of potency fluctuations of the pertussis component of Polish-produced DTP vaccine due to the changes having occurred in production and potency testing procedures. We compared the potency of the pertussis component of DTP vaccine lots produced and evaluated in similar periods and with similar production and testing procedures. Records of Kendrick test results performed over a 30-year period were available for analysis. This study confirms the role of different manufacturers, changes in vaccine strain compositions, in-house reference preparations used as reference vaccines in the Kendrick tests, and in mice of single strain sources in the potency values obtained. In addition, the comparisons performed revealed a downtrend in potency levels since 1992. Potency decrease in vaccine lots produced during 1992-1997 has been positively correlated to the lowering of the number of IOU/dose. Strain compositions of the DTP vaccine pertussis component and in-house references have been found to be associated with the fluctuation in potency estimations, and confirmed their crucial role in ensuring vaccine efficacy. Our study reveals that relative efficacy of the DTP vaccine produced in 1992-1997 might be lower than that of vaccines produced in other periods. This might in turn explain the increase in pertussis cases among children aged 5-15 years which is presently being observed in Poland.     

The detoxification of Bordetella pertussis with glutaraldehyde

To improve the whole-cell pertussis vaccine we have studied the inactivation of the biological properties characteristic of Bordetella pertussis phase I bacteria, i.e. histamine-sensitizing, lymphocytosis-promoting and mouse protective activities, by treating a concentrated bacterial suspension with various concentrations of glutaraldehyde. Under the experimental conditions, treatment with 10 mM glutaraldehyde at 37 degrees C for 30 min resulted in a marked reduction of the toxic activities without grossly diminishing the protective potency. Further tests were performed on the stability of the protective potency, on the agglutinin production in mice, and on the freedom from abnormal toxicity in guinea-pigs.     

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.     

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii‐specific serum antibodies might play an important role in protection. Immuno‐proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA‐like protein of B. holmesii. An aBH vaccine prepared from a BipA‐like protein‐deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA‐like protein plays an important role in the protective efficacy of aBH vaccine.     

Development of acellular pertussis vaccines.

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. The acellular pertussis vaccine has been widely accepted as safer and more efficacious in Japan. Since 1981, intense surveillance has shown that there are only rare adverse reactions and that pertussis has virtually been eliminated in Japan. Evaluation of active immunization with highly purified and pharmacologically inert PT and FHA and passive immunization with polyclonal and monoclonal antibodies, provide quantitative data about the vaccine-induced immunity in mice. Finally, it was discovered that the PT toxoid in the vaccine is the major and essential protective antigen. The toxoid of PT should be sufficient for protection against pertussis.     

Immunobiologic activity of Bordetella pertussis strains defective in various virulence factors

The immunobiological properties of mutant strains, selectively deprived of certain antigens (hemagglutinin, B. pertussis toxin, dermonecrotic toxin, hemolysin, adenylate cyclase), have been studied with the aim of finding out the relationship between the presence of certain antigens in microbial strains and their protective properties. The results of these studies suggest that the protective potency of pertussis vaccine may be related to the presence of some antigenic substances, including those not pertaining to the known factors of virulence.