Analysis of chosen parameters of immuno response in mice immunized with whole-cell or acellular pertussis vaccines and challenged with B. pertussis strains harbouring different ptxS1/prn allele genes combinations

Studies concerned evaluation of differences between parameters of cell-mediated immunity in mice, induced with whole-cell and acellular pertussis vaccines with subsequent challenge with B. pertussis strains harbouring different ptxS1/prn allele genes. In the study, concentrations of IFN-gamma/Il-2 and 1l-4/Il-5 in supernatants of cultured mice splenocytes have been determined to evaluate differences in Th1 or Th2 lymphocytes subpopulation response. Simultaneously, studies of intracellular expression of genes encoding of Il-2, Il-12, IFN-gamma and Il-4, Il-5, Il-10, Il-13 in mice splenocytes, and genes encoding factors involved in inflammatory process in the lung tissue (GM-CSF, TNF-alpha, Il-1beta, Il-6 i TGF-beta) have been performed on RNA level. The obtained results, confirmed high polarization of immunological response toward Th1 in mice immunized with DTP vaccine with whole-cell pertussis component, and toward Th2 in mice immunized with acellular pertussis vaccine. Inflammatory process in the lung tissue was more pronounced in animals immunized with whole-cell pertussis vaccine. There were no quantitative differences of analysed factors involved in the immune response among mice challenged B. pertussis strains containing different ptxS1/prn composition.     

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.     

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)     

Induction of humoral immunity in response to immunization with recombinant Mycobacterium bovis BCG expressing the S1 subunit of Bordetella pertussis toxin

Two recombinant Mycobacterium bovis BCG (rBCG) vaccine strains were developed for the expression of cytoplasmically located S1 subunit of pertussis toxin, with expression driven by the hsp60 promoter of M. bovis (rBCG/pPB10) or the pAN promoter of Mycobacterium paratuberculosis (rBCG/pPB12). Both strains showed stable expression of equivalent levels of recombinant S1 in vitro and induced long-term (up to 8 months) humoral immune responses in BALB/c mice, although these responses differed quantitatively and qualitatively. Specifically, rBCG/pPB12 induced markedly higher levels of IgG1 than did rBCG/pPB10, and mice immunized with the former strain developed specific long-term memory to S1, as indicated by the production of high levels of S1-specific IgG in response to a sublethal challenge with pertussis toxin 15 months after initial immunization. When considered in combination with previous studies, our data encourage further evaluation of rBCG as a potential means of developing a low-cost whooping cough vaccine based on defined antigens.     

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.     

Pertussis antibody levels in infants immunized with an acellular pertussis component vaccine, measured using whole-cell pertussis ELISA

A commercially available whole-cell pertussis IgG ELISA was used to test the response of 137 2-month-old infants to immunization with a trivalent acellular pertussis vaccine. The pre-immunization geometric mean (GM) IgG index was 6.96 (95% confidence interval (CI) 5.88-8.04) and the postimmunization GM index was 13.16 (95% CI 12. 20-14.11), P < 0.001. Eighty percent of subjects (110/137) had a significant 1.5-fold increase of pertussis IgG index (97/137, 71%) or a postimmunization IgG index > 10 (93/137, 68%). In single antigen ELISA, 83% showed at least a fourfold increase in pertussis toxin-specific IgG (PT-IgG) and 91% showed an increase in IgG specific for filamentous haemagglutinin (FHA-IgG). Four percent had high pre- immunization antibody levels (index > 20), likely to reflect recent maternal exposure to pertussis. This correlated with a smaller increase in pertussis IgG index. A decline in pertussis IgG index postimmunization occurred in 17/24 infants (71%) whose pre-immunization IgG index was > 10. This postimmunization pertussis IgG index was not significantly different to that of infants with a low pre-immunization index. A similar trend was noted with PT-IgG and FHA-IgG results. The whole-cell ELISA can detect a response to acellular pertussis vaccination in most infants if both antibody index and degree of seroconversion are calculated and at least one criterion is satisfied.     

Role of pertussigen (pertussis toxin) on the mouse protective activity of vaccines made from Bordetella species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.     

Proteomics-Identified Bvg-Activated Autotransporters Protect against Bordetella pertussis in a Mouse Model

Pertussis is a highly infectious respiratory disease of humans caused by the bacterium Bordetella pertussis. Despite high vaccination coverage, pertussis has re-emerged globally. Causes for the re-emergence of pertussis include limited duration of protection conferred by acellular pertussis vaccines (aP) and pathogen adaptation. Pathogen adaptations involve antigenic divergence with vaccine strains, the emergence of strains which show enhanced in vitro expression of a number of virulence-associated genes and of strains that do not express pertactin, an important aP component. Clearly, the identification of more effective B. pertussis vaccine antigens is of utmost importance. To identify novel antigens, we used proteomics to identify B. pertussis proteins regulated by the master virulence regulatory system BvgAS in vitro. Five candidates proteins were selected and it was confirmed that they were also expressed in the lungs of naïve mice seven days after infection. The five proteins were expressed in recombinant form, adjuvanted with alum and used to immunize mice as stand-alone antigens. Subsequent respiratory challenge showed that immunization with the autotransporters Vag8 and SphB1 significantly reduced bacterial load in the lungs. Whilst these antigens induced strong opsonizing antibody responses, we found that none of the tested alum-adjuvanted vaccines - including a three-component aP - reduced bacterial load in the nasopharynx, suggesting that alternative immunological responses may be required for efficient bacterial clearance from the nasopharynx.     

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.     

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.     

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.     

Neonatal immunization with a single dose of recombinant BCG expressing subunit S1 from pertussis toxin induces complete protection against Bordetella pertussis intracerebral challenge

The currently used pertussis vaccines are highly efficacious; however, neonates are susceptible to whooping cough up to the sixth month. In agreement, DTP-immunized neonate mice were not protected against intracerebral challenge with Bordetella pertussis. Neonate mice immunized with either DTP or a recombinant-BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin do not show a humoral immune response against PT. On the other hand, rBCG-Pertussis induces higher PT-specific IFN-gamma production and an increase in both IFN-gamma(+) and TNF-alpha(+)-CD4(+)-T cells than the whole cell pertussis vaccine and confers protection against a lethal intracerebral challenge with B. pertussis.     

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.     

Potent CTL induction by a whole cell pertussis vaccine in anti-tumor peptide immunotherapy

Promising yet limited clinical responses have been reported for peptide based immunotherapy against tumors. In order to induce more potent cytolytic CD8 T cell responses, we investigated the use of Bordetella pertussis vaccine as an adjuvant for peptide immunization. A whole cell (Wc) vaccine has been known to induce a Th1 biased immune response while an acellular (Ac) vaccine tends to induce that of the Th2 type. Natural infection by B. pertussis helps to maintain a robust Th1 memory in the host population. To examine the adjuvant activity of the pertussis vaccine, we immunized mice with an ovalbumin peptide as a model tumor antigen, and monitored the development of anti-tumor activities. The addition of either the Ac or the Wc vaccine helped expand the specific CD8 T cells. However, there was a marked difference in the induced cytolytic activity where the Wc vaccine was superior to the Ac. The Wc vaccine was also more effective in inducing in vivo tumor rejection. The adjuvant activity was not only effective against ovalbumin, but was also evident when an endogenous tumor antigen, Wilms' tumor 1 gene product, was targeted. These results indicate that, although the Wc vaccine does not share the same antigen specificity with tumor cells, it can aid in the development of highly cytolytic CD8 T cells as an adjuvant at the site of peptide immunization.     

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.     

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.     

Inoculation of plasmids encoding Japanese encephalitis virus PrM-E proteins with colloidal gold elicits a protective immune response in BALB/c mice

We established a simple and effective method for DNA immunization against Japanese encephalitis virus (JEV) infection with plasmids encoding the viral PrM and E proteins and colloidal gold. Inoculation of plasmids mixed with colloidal gold induced the production of specific anti-JEV antibodies and a protective response against JEV challenge in BALB/c mice. When we compared the efficacy of different inoculation routes, the intravenous and intradermal inoculation routes were found to elicit stronger and more sustained neutralizing immune responses than intramuscular or intraperitoneal injection. After being inoculated twice, mice were found to resist challenge with 100,000 times the 50% lethal dose (LD(50)) of JEV (Beijing-1 strain) even when immunized with a relatively small dose of 0.5 micro g of plasmid DNA. Protective passive immunity was also observed in SCID mice following transfer of splenocytes or serum from plasmid DNA- and colloidal gold-immunized BALB/c mice. The SCID mice resisted challenge with 100 times the LD(50) of JEV. Analysis of histological sections detected expression of proteins encoded by plasmid DNA in the tissues of intravenously, intradermally, and intramuscularly inoculated mice 3 days after inoculation. DNA immunization with colloidal gold elicited encoded protein expression in splenocytes and might enhance immune responses in intravenously inoculated mice. This approach could be exploited to develop a novel DNA vaccine.     

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.     

Whole-cell Bordetella pertussis vaccine component modulates the mouse immune response to an unrelated soluble antigen

Several factors are involved in the selective activation of Th1 or Th2 cells, such as different physical characteristics of antigens and the type of antigen-presenting cells involved in the immune response, among others. To study the influence of a particulate antigen on Th1/Th2 cell differentiation during the immune response to another antigen, we analysed the immune response to tetanus toxoid (soluble antigen) in BALB/c mice immunized with one of the three following vaccines: tetanus and diphtheria toxoids (DT), or DT associated with whole-cell Bordetella pertussis or its soluble antigens (DTPw and DTPa, respectively). Similar total antibody levels were observed for all vaccines. DT vaccine showed a higher IgG1/IgG2a ratio than the similar values observed for DTPw and DTPa vaccines. DT- and DTPa-primed spleen cells showed a Th2 (IL-5) profile while a Th1/Th2 (IFN gamma, IL-5) profile was observed for DTPw. IL-6 was only produced by DTPw-primed cells. Besides, IL-12 levels induced by DTPw were three times higher than the ones induced by both DT and DTPa. Our findings indicate that whole-cell B. pertussis priming modifies the tetanus immune response from Th2 to Th1/Th2 type probably via inflammatory mechanisms. In addition, in the light of conflicting reports regarding the mechanisms of protection induced by DTP vaccines, we studied the pertussis immune response. Only DTPw immunization generated memory T cells capable of proliferating with B. pertussis as an in vitro stimulus. Results might indicate that these cells may not play a key role in protecting against B. pertussis when the host is vaccinated with DTPa.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.