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.     

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis . It was found that this homolog, named AfuA_Bpp, is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O‐antigen, a molecule that has been found to shield surface antigens on B. parapertussis , showed no influence on antibody recognition of AfuA_Bpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

     

Protective effects of in vivo‐expressed autotransporters against Bordetella pertussis infection

Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re‐emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine.

     

Immunogenicity and protective efficacy of recombinant iron superoxide dismutase protein from Bordetella pertussis in mice models

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. Although availability of effective pertussis vaccines reportedly decreases the incidence of the disease, B. pertussis circulation in populations has not been eliminated. Thus, it is necessary to find new protein candidates with greater immune protective capacities than the currently available acellular pertussis vaccines. In this study, iron superoxide dismutase (FeSOD) gene (sodB) was cloned, expressed in Escherichia coli and recombinant FeSOD protein thence purified. The recombinant protein (rFeSOD) was formulated with aluminum hydroxide (Alum) or monophosphoryl lipid A (MPLA) and injected intraperitoneally to immunize mice, after which IgG1, IgG2a and IFN‐γ titers were measured to assess humoral and cellular responses, respectively, to these immunizations. The extent of bacterial colonization in lungs of intranasally challenged mice was determined 5, 8 and 14 days post‐challenge. IgG1 and IgG2a responses were significantly stronger in mice that had been immunized with rFeSOD–MPLA than in those that had received rFeSOD‐Alum (P < 0.05). Additionally, IgG2a titers were higher in mice vaccinated with recombinant protein FeSOD (rFeSOD) formulated with MPLA, especially after the second immunization. Immunization with rFeSOD–MPLA also provided a modest, but significant decrease in bacterial counts in lungs of mice (P < 0.05). Antigen specific‐IFN‐γ responses were significantly stronger in the group vaccinated with rFeSOD–MPLA, which could account for the lower bacterial counts. These findings suggest that rFeSOD protein formulated with MPLA has potential as an acellular pertussis vaccine candidate component.     

Purification and Immunogenicity of a Recombinant Bordetella pertussis S1S3FHA Fusion Protein Expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.     

Biofilm forming potential and antimicrobial susceptibility of newly emerged Western Australian Bordetella pertussis clinical isolates

Whooping cough caused by Bordetella pertussis is increasing in several countries despite high vaccine coverage. One potential reason for the resurgence is the emergence of genetic variants of the bacterium. Biofilm formation has recently been associated with the pathogenesis of B. pertussis. Biofilm formation of 21 Western Australian B. pertussis clinical isolates was investigated. All isolates formed thicker biofilms than the reference vaccine strain Tohama I while retaining susceptibility to ampicillin, erythromycin, azithromycin and streptomycin. When two biofilm-forming clinical isolates were compared with Tohama I, minimum bactericidal concentrations of antimicrobial agents increased. Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis revealed significant differences in protein expression in B. pertussis biofilms, providing an opportunity for identification of novel biofilm-associated antigens for incorporation in current pertussis vaccines to improve their protective efficacy. The study also highlights the importance of determining antibiograms for biofilms to formulate improved antimicrobial therapeutic regimens.     

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.     

Investigation of an Aerosol Challenge Model as Alternative to the Intracerebral Mouse Protection Test for Potency Assay of Whole Cell Pertussis Vaccines

The current potency test for whole cell pertussis vaccines, the intracerebral mouse protection test, is still the only assay which has shown a correlation with protection in children. However, it has considerable disadvantages as it uses a severe challenge procedure and the results tend to show significant intra- and inter-laboratory variation. An alternative assay based on non-lethal aerosol challenge of mice has been investigated as a replacement for the current intracerebral mouse protection test. Evaluation of this indicated that the aerosol system allowed consistent inoculation of bacteria into mice and gave good reproducibility. The protective capacity of different vaccine preparations was distinguished by this assay. Furthermore, the viable counts of Bordetella pertussis in the lungs of challenged mice were immunisation dose-dependent, which allowed the relative potency of vaccines to be calculated. Comparison of potency of five batches of vaccine from different manufacturers assayed by both the intracerebral and the aerosol challenge methods ranked the vaccines in identical order. The results suggest that this method has potential for use as a potency test for whole cell pertussis vaccine which would result in a great reduction in the number of animals used. It would also replace the lethal challenge by a non-lethal procedure and thereby avoid the use of the severe intracerebral challenge procedure.     

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.     

Protective immunity induced by an intranasal multivalent vaccine comprising 10 Lactococcus lactis strains expressing highly prevalent M‐protein antigens derived from Group A Streptococcus

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N‐terminal fragment of streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N‐ terminal fragment is hypervariable: there are more than 200 different M types. In this study, an intranasal live bacterial vaccine comprising 10 strains of Lactococcus lactis, each expressing one N‐terminal fragment of M protein, has been developed. Live bacterial‐vectored vaccines cost less to manufacture because the processes involved are less complex than those required for production of protein subunit vaccines. Moreover, intranasal administration does not require syringes or specialized personnel. Evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All 10 strains combined in a 10‐valent vaccine (M×10) induced serum and bronchoalveolar lavage IgG titers that ranged from three‐ to 10‐fold those of unimmunized mice. After intranasal challenge with M28 streptococci, survival of M×10‐immunized mice was significantly higher than that of unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of M×10‐immunized mice did not differ significantly from that of unimmunized mice. Mx‐10 immunized mice had significantly less S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge than did unimmunized mice. Our L. lactis‐based vaccine may provide an alternative solution to development of broadly protective group A streptococcal vaccines.

     

Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis

The filamentous hemagglutinin (FHA) of Bordetella pertussis is a principal adhesin, which plays a key role in the colonization of the upper respiratory tract. FHA is also a protective antigen, which has been incorporated in the new generation of acellular vaccines against whooping cough. The protein is synthesized as a large 367-kDa precursor, which is then processed into a 220-kDa secreted polypeptide. To optimize the use of this protein for vaccine purposes it would be helpful to define the regions encompassing immunodominant epitopes. Twelve recombinant plasmids have been generated encoding fusion proteins between fragments of the matured-secreted 220-kDa form of FHA and the vector-encoded phage MS2 polymerase. Protein extracts of the resulting recombinant clones have been tested for reactivity with sera from 20 patients convalescent from whooping cough, and two human standard sera. The results indicate the presence of an immunodominant B cell epitope in the polypeptide coded by a 1-kb DNA fragment encompassing positions 5781-6800 of the published sequence. These results suggest that the identified fragment should be conserved in the formulation of vaccines against pertussis.     

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.     

Evaluation of efficacy in terms of antibody levels and cell-mediated immunity of acellular pertussis vaccines in a murine model of respiratory infection

The efficacy of six acellular pertussis vaccines, prepared by various manufacturers in Japan, was investigated in a murine model of respiratory infection (aerosol challenge model) and a murine intracerebral (i.c.) challenge model. There was a good correlation between bacterial clearance from the lungs after aerosol challenge and the potency of vaccines as determined by i.c. challenge. The levels of antibodies against filamentous hemagglutinin were higher after immunizations with all tested vaccines than the levels of antibodies against pertussis toxin and pertactin. Spleen cells from mice immunized with each individual vaccine secreted interferon gamma (IFN-gamma) in response to stimulation by pertussis toxin, filamentous hemagglutinin and fimbriae. The production of interleukin-4 in response to each of the antigens tested was detected but was lower than that of IFN-gamma. However, antibody levels and cell-mediated immune responses were not correlated with the protective effects of the vaccines after aerosol challenge and after i.c. challenge.     

Fusion expression and immunogenicity of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> PTS1-FHA protein: implications for the vaccine development

Mutants of pertussis toxin (PT) S1 subunit and filamentous hemagglutinin (FHA) type I immunodominant domain from Bordetella pertussis (B. pertussis) are considered to be effective candidate antigens for acellular pertussis vaccines; however, the substantial progress is hampered in part for the lack of a suitable in vitro expression system. In this paper, the gene sequences of a S1 mutant C180-R9K/E129G (mS1) and a truncated peptide named Fs from FHA type I immunodominant domain were linked together and constructed to pET22b expression vector as a fusion gene; after inducing with IPTG, it was highly expressed in E. coli BL21 (DE3) as inclusion body. The fusion protein FsmS1 was purified from cell lysates and refolded successfully. The result of Western blotting indicate that it was able to react with both anti-S1 and anti-FHA McAbs; antiserum produced from New Zealand white rabbits immunized with this protein was able to recognize both native PT and FHA antigens as determined by western blotting. These data have provided a novel feasible method to produce PT S1 subunit and FHA type I immunodominant domain in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against B. pertussis infection.     

炭疽活疫苗家兔免疫力与血清抗芽胞IgG关系的研究

炭疽疫苗是预防炭疽流行和炭疽生物恐怖的重要手段。已有动物实验表明,炭疽活疫苗的保护力优于以保护性抗原为主要成份的无细胞疫苗,但两类现行疫苗都有待重新评价和改进。炭疽疫苗的效力必须用适当的实验室方法进行检测与分析才能了解其性质和细节。试验中力图探寻炭疽活疫苗家兔免疫力与血清抗芽胞抗体水平的关系。用“皮上划痕人用炭疽活疫苗”免疫家兔,以特定制备的炭疽芽胞抗原用ELISA法检测血清抗炭疽芽胞IgG抗体水平,并用强毒炭疽杆菌攻击进行效力试验。免疫家兔血清几何平均抗芽胞IgG滴度在免疫后一个月内持续升高,14d达到206,28d时达到776,这时其抵抗20MLD毒菌攻击的保护率为80％,符合中国生物制品规程要求的保护力。一个月后抗体水平开始下降,42d时滴度降至223。实验所揭示的炭疽减毒活疫苗诱导的家兔抗芽胞IgG抗体与抗炭疽保护力之间的关系,既为评价现行疫苗提供了资料,也为研制新型疫苗建立了参考性指标。     

T- and B-Cell-Mediated Protection Induced by Novel,Live Attenuated Pertussis Vaccine in Mice. Cross Protection against Parapertussis

Background

Despite the extensive use of efficacious vaccines, pertussis still ranks among the major causes of childhood mortality worldwide. Two types of pertussis vaccines are currently available, whole-cell, and the more recent acellular vaccines. Because of reduced reactogenicity and comparable efficacy acellular vaccines progressively replace whole-cell vaccines. However, both types require repeated administrations for optimal efficacy. We have recently developed a live attenuated vaccine candidate, named BPZE1, able to protect infant mice after a single nasal administration.

Methodology/Principal Findings

We determined the protective mechanism of BPZE1-mediated immunity by using passive transfer of T cells and antibodies from BPZE1-immunized mice to SCID mice. Clearance of Bordetella pertussis from the lungs was mediated by both BPZE1-induced antibodies and CD4⁺, but not by CD8⁺ T cells. The protective CD4⁺ T cells comprised IFN-γ-producing and IL-17-producing subsets, indicating that BPZE1 induces both Th1 and Th17 CD4⁺ T cells. In addition, and in contrast to acellular pertussis vaccines, BPZE1 also cross-protected against Bordetella parapertussis infection, but in this case only the transfer of CD4⁺ T cells conferred protection. Serum from BPZE1-immunized mice was not able to kill B. parapertussis and did not protect SCID mice against B. parapertussis infection.

Conclusions/Significance

The novel live attenuated pertussis vaccine BPZE1 protects in a pre-clinical mouse model against B. pertussis challenge by both BPZE1-induced antibodies and CD4⁺ T cell responses. It also protects against B. parapertussis infection. However, in this case protection is only T cell mediated.     

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)     

Purification and immunogenicity of a recombinant Bordetella pertussis S1S3FHA fusion protein expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.