The development of a new generation of pertussis vaccine

The results of the weight gain test on mice have shown that acellular pertussis vaccine is less toxic than the pertussis component of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine due to a lower content of endotoxin in the acellular vaccine; but the leukocytosis-promoting and histamine-sensitizing activities of JNIH-6 and adsorbed DPT vaccines are indicative of incomplete inactivation of Bordetella pertussis toxin. The content of incompletely inactivated B. pertussis toxin is practically the same in both preparations, constituting 1/100-1/200 of the calculated initial activity. For this reason, the use of the new pertussis vaccine also involves a risk of development of serious postvaccinal reactions and/or complications caused by this toxin. Search for the optimum method of inactivation of B. pertussis main toxin should be continued. As shown by the enzyme immunoassay, acellular pertussis vaccine used in the same immunizing dose as adsorbed DPT vaccine induces a more intensive immune response to hemagglutinin and B. pertussis toxin. This is due to higher residual toxicity of the corpuscular component of adsorbed DPT vaccine. Induction of antibodies to B. pertussis toxin has been shown to decrease in response to injection of acellular pertussis vaccine containing a certain residual amount of incompletely inactivated B. pertussis toxin.     

Determination of the epinephrine-refractory hypoglycaemic activity of whole-cell pertussis vaccine in mice

A new assay method has been developed for the quantitative estimation of the inhibitory effect of pertussis vaccine on epinephrine-induced hyperglycaemia in mice. The statistical analysis of the assay was based on logarithm-transformed estimates of the blood glucose levels. The method was sufficiently sensitive to detect the activity of 0.004 millilitre of commercial combined diphtheria-tetanus-whole cell pertussis vaccine. The estimated common variance was as small as 0.0034 and the assay was highly reproducible. Among commercial vaccines there was a significant difference in activity. The activity of a stock pertussis vaccine was inactivated by 5 mM glutaraldehyde at 37 degrees C for 30 min, but resisted treatment with 40 mM formaldehyde at 37 degrees C for 5 days. The extent of inactivation with the chemicals was calculated by a parallel line assay as the activity relative to that of untreated control pertussis vaccine.     

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.     

Acetone-treated pertussis vaccine--a potent and safer new pertussis vaccine

A vaccine was prepared from the growth of Bordetella pertussis by repeated treatment with acetone. The vaccine has been designated as acetone-treated pertussis vaccine (ATPV). A total of ten batches of ATPV were prepared, five each from B. pertussis strains 134 and 509. These strains are routinely employed at this Institute for the production of conventional whole-cell pertussis vaccine (WCPV) for blending in diphtheria-pertussis-tetanus vaccine. The mouse protective and histamine sensitizing activities of ATPV and WCPV were compared. The ATPV showed 1.5- to 2-fold higher potency than the WCPV. The histamine sensitizing activity of ATPV was much reduced compared with that of the WCPV. No appreciable difference was observed in the results of a mouse weight-gain tests between the ATPV and WCPV. The details of the preparation of ATPV have been described. Because of higher potency and reduced histamine sensitizing activity, the ATPV may prove more acceptable in immunization programmes against pertussis, even in countries where WCPV is unpopular due to its suspected reactogenicity.     

Prophylaxis of pertussis: development and use of acellular pertussis vaccine

Modern data substantiating the expediency of the use of acellular pertussis vaccine were analyzed. Serious postvaccinal complications caused by the action of the corpuscular pertussis component of adsorbed DPT vaccine served as the basis for the development of acellular pertussis vaccine (APV). During the period of 1990-1996 as many as 8 international field trials of the effectiveness of APV were carried out. The results of these trials and studies were evaluated in accordance with the unified programs and criteria. The vaccines under test differed by the composition of Bordetella pertussis purified antigens they contained, the methods of their purification and the detoxification of pertussis toxin. All tested APV, with the exception SKB-2, possessed pronounced prophylactic activity.     

Complete genome sequence of Bordetella pertussis CS, a Chinese pertussis vaccine strain

Bordetella pertussis is the causative agent of pertussis. Here, we report the genome sequence of Bordetella pertussis strain CS, isolated from an infant patient in Beijing and widely used as a vaccine strain for production of an acellular pertussis vaccine in China.     

Antigenic similarity of the pertussis component of adsorbed DTP vaccine to human erythrocytes

The similarity of the heterogeneous antigens, types A and B, of human red blood cells to the most of B. pertussis strains constituting the pertussis component of commercial batches of adsorbed DPT vaccine has been established. This property makes the vaccine strains different from B. pertussis isolated from pertussis patients. One of the reasons of the insufficient effectiveness of immunization against pertussis has been determined: the intensity of immune response depends on the antigenic heterogeneity of the pertussis component of the vaccine and the AB0 group factors in the blood of the vaccinees. For the first time the accumulation of immune alpha- and beta-isoagglutinins in the blood of persons immunized with absorbed DPT vaccine has been established. This accumulation shows the medium degree of direct correlation with the manifestations of the clinical reaction to the injection of the vaccine. The data obtained in this study indicate the necessity to revise the existing method of obtaining the pertussis component of adsorbed DPT vaccine on solid culture media with human red blood cells added and to develop the technique of the additional purification of this component from heterogeneous antigens.     

Role of whole-cell pertussis vaccine in severe local reactions to the preschool (fifth) dose of diphtheria-pertussis-tetanus vaccine.

OBJECTIVE: To estimate the contribution of whole-cell pertussis vaccine to severe local reactions after the preschool (fifth) dose of adsorbed diphtheria toxoid-pertussis vaccine-tetanus toxoid (DPT) vaccine. DESIGN: Double-blind randomized controlled trial. SETTING: Urban community. PARTICIPANTS: Volunteer sample of 200 healthy children 4 to 6 years old who were eligible for the fifth dose of DPT vaccine. INTERVENTIONS: Children received, in both arms, either diphtheria toxoid-tetanus toxoid (DT) and monovalent pertussis vaccines (group A, 99 children) or DPT and meningococcal vaccines (group B, 101 children). All were licensed products from single lots. The children were assessed 24 hours later by a trained observer. Serum samples obtained before vaccination were tested for antibodies to tetanus and diphtheria toxins and five pertussis antigens by means of enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Rates of severe local reactions (an area of redness or swelling or both of 50 mm or greater) 24 hours after vaccination. Relation between serum antibody levels before vaccination and rates of severe local reactions to corresponding vaccines. RESULTS: All of the subjects were followed up 24 hours after vaccination. Severe redness was present in 38% given DPT vaccine, 29% given intramuscular pertussis vaccine and 9% given DT vaccine (p < or = 0.002, three-way comparison). Severe swelling was common after vaccination with all three products. After intramuscular pertussis vaccination a relation was evident between the prevaccination levels of antibody to whole-cell pertussis bacteria and the rates of redness (p < 0.02) but not between the prevaccination subcellular antibody levels and the rates of redness. CONCLUSION: That pertussis vaccine resembled the DPT vaccine in causing severe redness suggests that it is the principal cause of such reactions after DPT vaccination. The DT vaccine was also reactogenic; thus, cumulative sensitization to one or more of its constituents may be a factor.     

中国百日咳疫苗的现状及研发趋势初探

百日咳是许多国家的严重公共卫生问题,目前预防百日咳最有效和最经济的方法是接种百日咳疫苗,百日咳组分疫苗由于其保护效果好、不良反应低、质量稳定可控而成为新一代无细胞百日咳疫苗的首选。本文简要介绍百日咳的病原学、传播途径及流行病学特点,并对中国的百日咳疫苗研发趋势进行初步探讨。     

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.     

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.     

Lipopolysaccharides in a traditional pertussis vaccine

Analysis of the lipopolysaccharide (LPS, endotoxin) in cell sonicates of four Danish vaccine strains of Bordetella pertussis (3803, 3825, 3843 and 3860) and of purified strain 3803 LPS in sodium dodecyl sulphate-polyacrylamide gel electrophoresis by silver staining, showed identical profiles. The LPS profile revealed a dominant, brownish LPS II band and a minor, faster-migrating, black-stained LPS I band. However, the ratio of LPS I to LPS II in the preparation of purified LPS differed slightly from the cell sonicates. Using marker LPS, the molecular weights of LPS I and LPS II were estimated at 5.4 and 6.0 kD, respectively. Seven different lots of whole cell pertussis vaccine were assayed for LPS in the Limulus Amoebocyte Lysate test and were found to contain 0.9-2.8 micrograms LPS/ml. No significant difference in the content of LPS in similar dilutions of the individual strains was observed. In addition, the distribution of free and cell-bound LPS in four pertussis vaccines was investigated. Most of the LPS was found to exist as free LPS. During several months, the course of both LPS and pertussis toxin (Pt) release in freshly killed B. pertussis preparations was followed. In the first few weeks, 35-50% of the LPS was released and after 5-6 months of storage 60-80% had been released. In contrast, less than 10% of the biologically active pertussis toxin was released during the experimental period. The possibility of producing a safer whole cell pertussis vaccine by reducing the amount of free LPS without reducing the protective value correspondingly is discussed.     

Opsonizing activity of sera from animals immunized with pertussis vaccine

Opsonizing activity of guinea pig blood serum containing mercaptoethanol-resistant pertussis antibodies was studied in vitro on a model of microorganism ingestion by the mononuclears of the guinea pig peritoneal exudate. There were revealed distinct differences in the serum activity depending on the phagocytosis object. The blood serum of hyperimmunized rabbits stimulated the ingestion of Bordetella pertussis by mononuclears of guinea pigs--normal and immunized with pertussis vaccine. The blood sera of hyperimmunized guinea pigs and of mice immunized with pertussis vaccine twice displayed opsonins to B. pertussis. The blood sera of animals immunized with pertussis vaccine inhibited the staphylococcus ingestion by the peritoneal exudate mononuclears of guinea pigs, both normal and those immunized with pertussis vaccine.     

Importance of the degree of antigen polymerization by detoxification in modulating the immunogenicity of acellular pertussis vaccine

For the acellular pertussis vaccine with a high immunogenicity, the concentration, composition and characteristics of acellular pertussis antigens are the crucial points to be considered. Nevertheless, it has not been proved yet whether or not the polymerization degree, one of the characteristics of formalin-detoxified acellular pertussis antigens, has an influence on vaccine potency. Thus, in the present study, the correlations among detoxification conditions of acellular pertussis bulks, their polymerization degrees and their immunogenicities were examined. In addition, the relative importance of pertussis toxoid in vaccine immunogenicity was also investigated. Results show that a lower lysine concentration during detoxification induces highly-polymerized antigens, the immunogenicity has a great dependency on the polymerization degree of antigens, and also pertussis toxoid has a relatively stronger influence on the immunogenicity than other antigens. Accordingly, in the aspect of the potency of detoxified acellular pertussis vaccine, it can be demonstrated that the polymerization of antigens and its degree are the major factors affecting the immunogenicity along with a relatively high content of pertussis toxoid.     

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.     

Immunomodulation activity of new vaccines for pertussis prophylaxis--acellular pertussis vaccine and adsorbed DPT vaccine with acellular component

The immunomodulating activity of acellular pertussis vaccine (APV) and adsorbed DPT vaccine with acellular pertussis component (DPTA vaccine) was studied. The study revealed that only large doses of APV, 10 immunizing doses (ID), suppressed humoral and cell-mediated response to sheep red blood cells (SRBC). 1 ID produced no influence on the formation of antibody producing cells, but increased the development of delayed hypersensitivity (DH) to SRBC. The modulation of cell-mediated immune response, induced by APV, returned to normal after the injection of purified staphylococcal toxoid, used as immunomodulator, in doses of 0.15 BU per mouse and 1.5 BU per mouse. DPTA vaccine containing 1 ID, as well as 10 ID, produced no immunomodulating effect. This was established by the evaluation of humoral response to SRBC in CBA mice and the study of the formation of DH to SRBC in BALB/c mice. As indicated by the total of the presented data, the inclusion of APV into DPTA vaccine enhanced the immunological safety of its pertussis component.     

The purification and characterization of an acellular pertussis vaccine

An acellular pertussis vaccine manufactured by Biken was investigated for purity, potency and toxicity. The vaccine was composed of almost equal proportions of pertussis toxin (PT) and filamentous hemagglutinin (FHA). The purity of the vaccine was 97-99%. The protective effects of component vaccines containing various ratios of PT and FHA were tested and it was found that the ratio of 1:1 provided the most effective vaccine.     

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.     

Comparative pathomorphologic study of the toxic properties of a corpuscular pertussis vaccine and of a cell-free pertussis preparation

The comparative study of morphological changes in the body of outbred mice under the action of corpuscular pertussis vaccine and acellular pertussis preparation has been made. The corpuscular vaccine has been shown to produce a pronounced, dynamically increasing toxic effect, thus causing the damage of lymphoid thymic and spleen cells, prolonged interstitial reaction in the lungs, destructive inflammatory process at the site of injection. The acellular pertussis preparation is less toxic, induces less pronounced changes in these organs at the early period of the experiment, stimulates the proliferation of lymphoid cells and lymphoblast transformation. As noted in this study, the damaging action of pertussis vaccine is mainly indicated by pathological phenomena appearing in the organs of the immune system, pulmonary parenchyma and muscular tissue (in the inoculation zone).     

Evaluation of the stability of pertussis vaccine OCO-3 standard

The evaluation of the immunogenic activity and residual toxicity of the National Standard of pertussis vaccine (OCO-3) was carried out. As shown by observations lasting for a period of 25 years, the preparation possesses stable immunogenicity and its toxicity remained unchanged, which makes it possible to use OCO-3 for the routine control of the pertussis component of commercial lots of adsorbed DPT vaccine.