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.     

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.     

Verification of components of acellular pertussis vaccines that have been distributed solely, been in routine use for the last two decades and contributed greatly to control of pertussis in Japan.

An ideal acellular pertussis vaccine is now under investigation worldwide. We have had acellular pertussis vaccines available for the last 22 years, which contributed greatly to the control of pertussis in Japan, although it has not been known whether they are one of ideal acellular pertussis vaccines or not. Moreover, the formulations of acellular pertussis vaccines that we have been using have not been widely recognized. Serum samples were taken from recipients of the T type, B type, and two-component acellular pertussis vaccine and assayed by ELISA for anti-PT, anti-FHA, and anti-69 kD OMP antibody levels and by the agglutination test. Although it was shown that T type vaccine contained four components (PT, FHA, 69 kD OMP, agglutingen), B type vaccine contained three components (PT, FHA, 69 kD OMP) and the two-component vaccine contained PT and FHA, it was concluded that PT and FHA were essential and common antigens contained in all three acellular pertussis vaccines in Japan. The national monitoring system for adverse effects of routine immunization demonstrated low reactogenicity of DTaP in Japan. This resulted in high acceptance rates of DTaP and in virtual control of pertussis.     

百日咳杆菌69KDa外膜蛋白的分离纯化及生物学特性研究

本文发展了一种从百日咳杆菌Ⅰ相菌株中纯化６９ＫＤａ外膜蛋白的简易方法,将细菌体经加热浸提、乙醇沉淀蛋白、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析精制而成。用ＳＤＳ－ＰＡＧＥ、免疫印迹、光密度仪扫描分析,证明纯化制剂为均一的、特异性的６９ＫＤａ外膜蛋白,其收率为５４．２％,纯度达９９．２％,每微克６９ＫＤａ蛋白制剂中的内毒素含量低于０．８５ＥＵ;ＰＴ残留量小于０．１０５ｎｇ。抗６９ＫＤａ蛋白抗血清能     

Composition of acellular pertussis and combination vaccines: a general review.

Since the development and introduction of the acellular pertussis vaccine in Japan in the early eighties, we have come a long way in using this component in combination with other vaccines. However, the basic problem in development of an effective and safe pertussis vaccine is that the antigens to induce complete protection against clinical pertussis and the precise mechanism by which pertussis vaccine confers immunity is yet unknown. Hence, the composition of future acellular pertussis vaccine remains an open issue. Recently, acellular pertussis vaccine has been licensed for the booster doses in the U.S.A. and for primary immunization of infants in Italy and Germany. A multicentric trial has been carried out to compare the serological response and adverse reactions of 13 acellular pertussis vaccines. These vaccines contained one or more of the four components, i.e. FHA, PT, 69 kDa OMP and fimbriae. All vaccines were associated with substantially fewer and less adverse reactions and were more immunogenic with respect to antibodies against the added antigens. DTP vaccines in the near future will have combinations of other components and the key antigen for combination will be acellular pertussis component which is going to replace whole cell pertussis component in DTP vaccines. In view of this, manufacturers like ourselves from the developing countries are still groping in the dark, uncertain whether we should have a single component acellular pertussis vaccine or multicomponent one. This will have a major impact on the cost of production, the final cost of the combination vaccines and the regulatory issues that we will have to tackle in view of the recent thinking on harmonization in the pharmaceutical industry.     

Expression of a pertussis toxin S1 fragment by inducible promoters in oral Streptococcus and the induction of immune responses during oral colonization in mice

Previous work aimed at developing a live oral vaccine expressing pertussis toxin S1 fragment on the surface of the bacterium Streptococcus gordonii elicited a lower than expected antibody response, perhaps because of low antigen expression. In this study, in-frame promoter fusions were constructed to investigate whether an increase in antigen production by the streptococcal vaccine strain results in a better antibody response. The promoters tested were (i) the Streptococcus mutans sucrose-inducible fructosyltransferase (ftf) promoter and (ii) the Bacillus subtilis/Escherichia coli chimeric tetracycline-inducible xyl/tetO promoter. Each of these two promoters was placed upstream of the spaP/s1 fusion gene to drive its expression. The constructs were introduced into S. gordonii DL1 and S. mutans 834. The inducibility of the promoters was confirmed through the determination of SpaP/S1 production via Western blottings. Induced production of SpaP/S1 was observed in S. gordonii and S. mutans with each of the promoters, but the level of expression was the highest in S. mutans, using the xyl/tetO promoter. Thus, S. mutans carrying the xyl/tetO/spaP/s1 construct (S. mutans PM14) was used in oral colonization studies in BALB/c mice. Streptococccus mutans PM14 was able to colonize the animals for the 14-week duration of experimentation. A mucosal IgA response was observed in all the treatment groups but was highest in mice receiving tetracycline induction. In the mouse model of Bordetella pertussis respiratory infection, animals colonized with S. mutans PM14 showed a decreased in B. pertussis lung colony count (P = 0.03) on day 3 compared with control mice colonized by the parent S. mutans 834.     

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.     

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.     

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.     

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)     

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.     

A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein

The emerging SARS-CoV-2 variants, commonly with many mutations in S1 subunit of spike (S) protein are weakening the efficacy of the current vaccines and antibody therapeutics. This calls for the variant-proof SARS-CoV-2 vaccines targeting the more conserved regions in S protein. Here, we designed a recombinant subunit vaccine, HR121, targeting the conserved HR1 domain in S2 subunit of S protein. HR121 consisting of HR1–linker1–HR2–linker2–HR1, is conformationally and functionally analogous to the HR1 domain present in the fusion intermediate conformation of S2 subunit. Immunization with HR121 in rabbits and rhesus macaques elicited highly potent cross-neutralizing antibodies against SARS-CoV-2 and its variants, particularly Omicron sublineages. Vaccination with HR121 achieved near-full protections against prototype SARS-CoV-2 infection in hACE2 transgenic mice, Syrian golden hamsters and rhesus macaques, and effective protection against Omicron BA.2 infection in Syrian golden hamsters. This study demonstrates that HR121 is a promising candidate of variant-proof SARS-CoV-2 vaccine with a novel conserved target in the S2 subunit for application against current and future SARS-CoV-2 variants.Subject terms: Mechanisms of disease, Molecular modelling     

The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults.     

Multiple T and B cell epitopes in the S1 subunit ("A"-monomer) of the pertussis toxin molecule

The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT.     

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.     

DTaP vaccines from north american vaccine (NAVA): composition and critical parameters.

NAVA's acellular pertussis vaccine is based on highly purified pertussis toxin (PT) inactivated with H(2)O(2). PT was analysed using advanced biochemical methodology including mass spectroscopy (LC/MS), yielding mass and peptide mapping information on the subunits. Pertactin, adenylate cyclase, and Fim 1, 2 were below detection levels and only trace amounts of filamentous haemagglutinin (FHA) have been identified as a minor impurity. The vaccine does not induce anti-FHA antibodies during the course of a 3-dose primary immunization series in infants. B and T cell epitopes are preserved to a higher extent after H(2)O(2)detoxification when compared with chemical inactivation with formaldehyde, thus providing new information explaining why vaccines employing formaldehyde detoxified PT may need additional pertussis components added to induce high levels of protection. Anti-PT antibodies generated by NAVA diphtheria, tetanus, and acellular pertussis vaccine (DTaP) showed a positive correlation with protection against WHO-defined pertussis. The safety profiles for these vaccines showed low reactogenicity with no serious adverse events due to the vaccines.     

Overview of recent clinical trials of acellular pertussis vaccines.

The evidence from pre-licensure studies does not suggest that there are clinically important differences in reactogenicity between acellular vaccines. The merits of different acellular products will therefore have to be compared on efficacy criteria. Ideally, acellular vaccines with the minimum antigen content necessary to ensure optimum protection should be used in order to avoid administration of superfluous antigens to children and to simplify licensing and batch release procedures.On the basis of the evidence so far available it seems unlikely that monocomponent pertussis toxin (PT) vaccines provide optimal protection and that multicomponent vaccines are needed to achieve a level of disease control that approaches that of a good whole-cell vaccine. It is unclear whether all two component vaccines containing PT and filamentous haemagglutinin (FHA) have similar efficacy but on the available evidence the safest option for policy makers would seem to be to use a vaccine with at least three components, PT+FHA+pertactin. There is now good evidence that the five component vaccine which contains agglutinogens 2 and 3 in addition to PT/FHA and pertactin provides the best protection and is the only acellular vaccine whose efficacy matches that of a good whole cell vaccine. However, the public health advantage of the five component vaccine over other acellular vaccines may not become apparent until they have been in routine use for some decades and their ability to protect against transmission as well as clinical pertussis has emerged.The decision to replace an effective whole-cell vaccine by an acellular vaccine for primary immunisation needs careful consideration. Apart from the probable sacrifice of efficacy for reduced reactogenicity (at least for vaccines which do not contain agglutinogens 2 and 3) there is the question of value for money and the ease with which acellular DTP vaccines can be combined with conjugate polysaccharide vaccines such as Haemophilus influenzae type b.Whatever the decision of policy makers, the need for continued follow up of trial cohorts and active surveillance of the efficacy and safety of those acellular vaccines that are introduced into routine use must be accorded a high priority.     

Approaches to the control of acellular pertussis vaccines.

The quality control of acellular pertussis vaccines presents particular problems related to the differences in composition and method of detoxification used in the various type of preparation. These vaccines are not amenable to potency assay by the active mouse protection test used for whole-cell pertussis vaccines and assurance of protective activity is problematic.In contrast, monitoring of these vaccines for safety is relatively straightforward and is centred on assays for the lipooligosaccharide endotoxin, active pertussis toxin and absence of reversion to toxicity of detoxified product. The absence of heat-labile toxin, tracheal cytotoxin and adenyl cyclase toxin is assumed provided that adequate validation of the process has been performed.Confirmation of the antigenic content of the detoxified bulk components is difficult to achieve by conventional binding assays based on monoclonal antibodies because of changes in accessibility of reactive sites post-toxoiding. However, single radial diffusion assay using polyclonal antisera permits estimation of pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (P69). Dot blot immunoassay can be used for the fimbrial agglutinogens 2 and 3 (Fim 2 and 3) and potentially could also be used to check the composition of final filling lots for PT, FHA, P69 and Fim 2 and 3.Gel electrophoresis and immunoblotting can be applied to monitor purity of purified bulk components and the characteristics of these change after chemical detoxification. Electron microscopy provides a useful semi-quantitative supporting method for checking purity of bulk components. Physico-chemical examination, particularly CD and fluorescence spectroscopy, offer a means of monitoring the consistency of detoxified bulk components.No completely satisfactory method is available for monitoring potency. Immunogenicity assays may be useful for checking consistency but do not necessarily correlate with protection. At present, active protection against aerosol challenge offers the best prospect of a functional assay.     

A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.     

The effects of purified components of Bordetella pertussis in the weight gain test for the toxicity testing of pertussis vaccines

The effects of highly purified preparations of three Bordetella pertussis components--pertussis toxin (PT), lipopolysaccharide (LPS) and filamentous haemagglutinin (FHA)--were examined in the mouse weight gain test, a toxicity test for pertussis vaccine. When these components were administered alone, PT enhanced initial weight gains of the mice, LPS produced an initial weight loss and FHA had no detectable effect on the weights of the mice. However, testing the components in combinations revealed that the effect of PT and LPS together was not simply the sum of their individual effects. This combination generally produced lower weights than LPS alone, particularly in the later stages of the test.