Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis.

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.     

Cloning of the filamentous hemagglutinin of Bordetella pertussis and its expression in Escherichia coli.

Bordetella pertussis UT25 DNA was cloned into the kanamycin resistance gene of cosmid pCP13 to construct a genomic library in Escherichia coli LE392. One clone containing plasmid pDB441 expressed the filamentous hemagglutinin (FHA) as identified by protein immunoblots with the use of rabbit anti-B. pertussis antiserum, rabbit anti-FHA antiserum, and a monoclonal antibody to FHA. FHA is a protein of 220 to 210 kilodaltons, but the immunoreactive FHA, as expressed in E. coli, was larger than that expressed in B. pertussis, suggesting that there was a difference in the processing of this protein between these two bacteria. The fha gene was mapped to a 6.5-kilobase pair DNA fragment by the use of various restriction endonucleases. The kanamycin resistance gene of pCP13 was found to provide the promoter function but probably not the translation start signal for the fha gene. Conjugative transfer of pDB441 to B. pertussis BP353, a transposon Tn5-induced FHA mutant, increased the expression of the FHA over that seen with wild-type B. pertussis.     

Expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli using a two cistron system.

Expression of Bordetella pertussis filamentous hemagglutinin (FHA) has been achieved in Escherichia coli K-12. This involved the construction of a two cistron system where the first cistron was provided by the NH2-terminus (first 98 amino acids) of MS2 polymerase. When the FHA gene sequences were fused to the first cistron, higher levels of expression were obtained and the fusion protein aggregated in inclusion bodies. FHA expressed by the two cistron system, however, appeared to be diffusely dispersed in the cytoplasm.     

Murine antibody response to oral infection with live aroA recombinant Salmonella dublin vaccine strains expressing filamentous hemagglutinin antigen from Bordetella pertussis.

Two plasmids which express either nearly intact or truncated filamentous hemagglutinin (FHA) from Bordetella pertussis and which are marked with a tetracycline resistance (Tcr) gene were transformed into Salmonella dublin SL1438, an aroA deletion mutant intended for use as an attenuated oral vaccine against salmonellosis. These S. dublin recombinants, when fed to mice, induced serum immunoglobulin, immunoglobulin M (IgM), and sometimes IgA antibody responses to FHA and S. dublin. In addition, IgA antibodies against FHA were found in gut wash fluids. S. dublin carrying pDB2300, a multicopy plasmid encoding truncated FHA protein, induced a better antibody response than did S. dublin carrying pDB2000, a low-copy-number plasmid encoding full-sized FHA. Administration of tetracycline to mice enhanced the stability of recombinant plasmids, and tetracycline-treated mice developed higher anti-FHA titers. Although neither strain examined is suitable for use in a human oral vaccine, these data demonstrated that an immune response against B. pertussis FHA could be induced by oral administration of live attenuated recombinant strains of S. dublin and suggested that development of a live oral attenuated vaccine against pertussis may be possible.     

Cloning, partial sequence, expression, and antigenic analysis of the filamentous hemagglutinin gene of Bordetella pertussis.

The gene coding for the filamentous hemagglutinin (FHA), one of the main factors involved in mediating adherence of Bordetella pertussis to ciliated host cells, was cloned in Escherichia coli, and the 3,500-base-pair nucleotide sequence encoding the amino-terminal region was determined. Molecular cloning, together with the characterization of recombinant FHA-related proteins produced in E. coli, revealed that the primary translation product is a protein of about 370 kilodaltons (kDa). The mature 220-kDa FHA polypeptide secreted by B. pertussis is most probably generated by proteolytic processing that eliminates a carboxy-terminal portion of about 150 kDa. The 1,087 amino-terminal residues of the predicted FHA sequence showed a number of remarkable features. Extensive homology to the Serratia marcescens and Proteus mirabilis hemolysin proteins was found between amino acids 91 and 205 of the FHA sequence, suggesting involvement of this FHA domain in host cell binding or secretion of FHA from B. pertussis. In addition, two regions containing repetitive amino acid sequences were identified. One region, extending from residues 382 to 664, was formed by six repeats, and a second, extending from residues 701 to 912, contained three repeats. The reactivities of several recombinant FHA-derived proteins with a panel of monoclonal antibodies identified at least four epitopes composing an immunoreactive domain present in the carboxy-terminal moiety of the mature FHA.     

The primary and secondary cellular immune responses to whole cell Bordetella pertussis vaccine and its components.

The cellular immune responses of Balb/c mice and Wistar rats immunized in hind footpads with intact killed Bordetella pertussis were found to differ from those of similar animals immunized with other bacteria including Bordetella bronchiseptica, Salmonella typhimurium and Escherichia coli. All the bacteria stimulated increases in cell number, proliferation and interleukin 2 (IL-2) production in popliteal lymph nodes which peaked 3-5 days after injection and decreased to resting levels by day 7. However, B. pertussis also caused a second peak in all three parameters at 11 days after immunization. This peak was not seen following injection with any of the other bacteria. Bordetella pertussis also caused systemic effects, increased cellular proliferation in bone marrow and thymus, with similar biphasic kinetics. It possesses a potent toxin, distinguishing it from the closely related B. bronchiseptica. The use of purified materials confirmed that the presence of this pertussis toxin (PT) was responsible for the later peak in stimulation, whereas lipopolysaccharide (LPS) in combination with PT and also the filamentous haemagglutinin (FHA) could mimic the early peak of stimulation. Primary immunization with B. pertussis was also shown to generate lymph node cells which responded in vitro to secondary challenge with B. pertussis cells, FHA or PT. Both proliferation and IL-2 production were enhanced, except with FHA which only increased IL-2 production. Lymph node cells from mice immunized with E. coli showed no such responses.     

Construction of stable LamB-Shiga toxin B subunit hybrids: analysis of expression in Salmonella typhimurium aroA strains and stimulation of B subunit-specific mucosal and serum antibody responses.

The complete Shiga toxin B subunit and two N-terminal segments of the B subunit have been inserted into a cell surface exposed loop of the LamB protein, and expression of the hybrid proteins from three different promoter systems, i.e., (i) an in vitro-inducible tac promoter that provides high-level expression, (ii) the iron-regulated aerobactin promoter presumably induced in vivo under the iron-limiting conditions of the intestinal mucosal environment, and (iii) a synthetic, modified beta-lactamase promoter providing moderate level constitutive expression, has been analyzed in Escherichia coli, Salmonella typhimurium, and attenuated antigen carrier strains of S. typhimurium (aroA mutants). The hybrid vaccine strains were used to immunize mice by the oral and intraperitoneal routes. S. typhimurium aroA mutants apparently have a membrane export defect which prevents the transport of LamB and its derivatives across the cytoplasmic membrane. High-level expression of hybrid proteins through use of the tac promoter proved deleterious to the vaccine strains and prevented the production of viable cells at reasonable cell densities. The lower levels of gene expression observed with the beta-lactamase and aerobactin promoters did not have this effect. Immunization of mice with S. typhimurium aroA strains carrying the hybrid genes expressed from these two promoters resulted in significant B subunit-specific mucosal and serum antibody responses. This suggests that such expression systems may be useful when incorporated into candidate antidysentery live oral vaccines for inducing protection against the effect of Shiga toxin in infections caused by Shigella dysenteriae 1 and other Shiga toxin-or Shiga-like toxin-producing pathogens.     

Construction and characterization in vivo of Bordetella pertussis aroA mutants.

A DNA fragment encoding a kanamycin resistance determinant was used to insertionally inactivate the cloned aroA gene of Bordetella pertussis in Escherichia coli K-12, and a conjugative shuttle vector system based on the suicide vector pRTP1 was used to deliver the mutations from E. coli back into B. pertussis CN2992FS and BP1. The aroA mutation was introduced by allelic exchange into the chromosome of B. pertussis, resulting in otherwise isogenic parental and aroA mutant pairs. The B. pertussis aroA mutants grew well on laboratory medium supplemented with aromatic compounds but failed to grow on unsupplemented medium. The B. pertussis aroA mutants expressed the normal B. pertussis extracellular, virulence-associated proteins; inactivated, whole-cell vaccines prepared from the mutants protected mice as efficiently as vaccines made from the parent strains against intracerebral challenge with the virulent B. pertussis 18323. Live B. pertussis aroA bacteria inefficiently colonized the lungs of NIH/S mice after they were challenged with aerosol, unlike the wild-type B. pertussis organism. Mice exposed to three separate aerosols of live B. pertussis aroA bacteria were protected against lung colonization after being exposed to an aerosol containing the virulent parental B. pertussis strain. High-level antibodies against B. pertussis rapidly appeared in the sera of mice immunized by aerosol with the B. pertussis aroA strains and challenged with the virulent parent.     

Direct anti-inflammatory effect of a bacterial virulence factor: IL-10-dependent suppression of IL-12 production by filamentous hemagglutinin from Bordetella pertussis

IL-12 plays a critical role in protective immunity against intracellular pathogens by promoting the development of Th1 cells. Here we demonstrate that filamentous hemagglutinin (FHA), a virulence factor of Bordetella pertussis, is capable of suppressing IL-12 production by macrophages. FHA inhibited IL-12 secretion by a macrophage cell line or ex vivo alveolar macrophages in response to Escherichia coli or B. pertussis lipopolysaccharide (LPS) and IFN-gamma. Antibodies to FHA or denaturation of FHA abrogated the inhibitory effect. Injection of mice with FHA suppressed IL-12 and IFN-gamma levels in the serum in response to i. v. injection of LPS in a model of septic shock. The suppressive effect of FHA was specific for IL-12, since the production of TNF-alpha, IL-6 and IL-10 was not suppressed, and production of IL-6 and IL-10 was up-regulated. Antibody blocking studies revealed that the inhibitory effect of FHA on IL-12 production was dependent on IL-10. Since FHA is secreted at high levels and local T cell responses are suppressed during B. pertussis infection, the findings suggest that FHA may be a critical virulence factor in facilitating pathogen persistence in the respiratory tract by suppressing or delaying the development of cell-mediated immunity.     

Characterization of a recombinant fragment that contains a carbohydrate recognition domain of the filamentous hemagglutinin.

The filamentous hemagglutinin (FHA) of Bordetella pertussis plays an important role in establishing infection by attaching the bacteria to the ciliated respiratory epithelial cells. Expression of DNA encoding residues 1141 to 1279 of FHA in Escherichia coli yields a protein of 18,000 Da that exhibits some of the carbohydrate recognition properties of FHA (S. M. Prasad, Y. Yin, E. Rodzinski, E. I. Tuomanen, and H. R. Masure, Infect. Immun. 61:2780-2785, 1993). We have constructed an E. coli strain that expresses this protein, designated fragment A, in a soluble form at markedly elevated levels. Fragment A could be purified with high purity and yields and was immunogenic in mice. Both fragment A and anti-fragment A sera inhibited the binding of B. pertussis to asialo-GM2 and to rabbit ciliated cells. These observations demonstrate that this fragment of FHA contains a cellular binding domain capable of eliciting functional antibodies.     

Immunogenicity and protective efficacy of a recombinant filamentous haemagglutinin from Bordetella pertussis

Bordetella pertussis is the causative agent of whooping cough, a major childhood pathogen; acellular vaccines consisting of purified B. pertussis antigens such as filamentous haemagglutinin (FHA) are commonly used to prevent pertussis. Despite the importance of FHA in B. pertussis pathogenesis and its inclusion in most acellular vaccines, the functional importance of individual domains in the induction of protective immunity is largely unknown. In this study, we have purified a recombinant FHA protein from Escherichia coli consisting of a 42 kDa maltose binding domain of E. coli and the 43 kDa type I immunodominant domain of FHA. The fusion protein (Mal85) was purified from E. coli cell lysates via affinity chromatography with an amylose column. Mal85 was then delivered to BALB/c mice intranasally encapsulated in liposomes, formulated with Protollin(TM) or in conjunction with an immunostimulatory CpG oligonucleotide. Mice were also vaccinated intraperitoneally with alum-adsorbed Mal85. Sera from all treatment groups showed strong IgG responses to Mal85 and recognized native FHA. Specific salivary IgA was induced in mice vaccinated with Mal85 in liposomes, Protollin(TM) and delivered with CpG. Vaccination with Mal85 encapsulated in liposomes or formulated with Protollin(TM) provided protection against aerosol challenge with B. pertussis in BALB/c mice. These data indicate that the type I domain of FHA is a protective antigen in mice and may serve as a candidate for inclusion in new acellular pertussis vaccines.     

Influence of CR3 (CD11b/CD18) expression on phagocytosis of Bordetella pertussis by human neutrophils

CR3 (CD11b/CD18) is expressed on neutrophils, and the engagement of CR3 can promote phagocytosis. CR3 serves as the receptor for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and for the adenylate cyclase toxin (ACT), which blocks neutrophil function. The influence of CR3, FHA, and ACT on the phagocytosis of B. pertussis by human neutrophils was examined. The surface expression and function of CR3 are regulated. Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) increased CR3 surface expression, but only TNF-alpha increased the ability of neutrophils to phagocytose B. pertussis, suggesting that elevated CR3 expression alone is not sufficient to promote phagocytosis. Purified FHA and pertussis toxin also increased the surface expression of CR3 on neutrophils, while ACT and the B subunit of pertussis toxin did not affect CR3 expression. FHA-mediated attachment to CR3 can lead to phagocytosis, especially in the absence of ACT. FHA mutants failed to attach and were not phagocytosed by neutrophils. Similarly, an antibody to CR3 blocked both attachment and phagocytosis. The addition of exogenous FHA enhanced the attachment and phagocytosis of wild-type B. pertussis and FHA mutants. Mutants lacking the SphB1 protease, which cleaves FHA and allows the release of FHA from the bacterial surface, were phagocytosed more efficiently than wild-type bacteria. ACT mutants were efficiently phagocytosed, but wild-type B. pertussis or ACT mutants plus exogenous ACT resisted phagocytosis. These studies suggest that the activation and surface expression of CR3, FHA expression, and the efficiency of ACT internalization all influence whether B. pertussis will be phagocytosed and ultimately killed by neutrophils.     

Genital antibody responses in mice after intranasal infection with an attenuated candidate vector strain of Bordetella pertussis

Intranasal administration of live attenuated Bordetella pertussis, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of B. pertussis. By comparing the immune responses induced after infection with wild-type virulent B. pertussis with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of B. pertussis-infected mice. The intranasal infection with either the attenuated or the virulent B. pertussis strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuated B. pertussis could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.     

Serum antibody response to filamentous hemagglutinin in patients with clinical pertussis measured by an enzyme-linked immunosorbent assay

Titers of antibodies to filamentous hemagglutinin (FHA) were determined by enzymelinked immunosorbent assay in acute and convalescent phase serum samples from 158 patients with clinical symptoms typical of whooping-cough. In 96 of the patients the diagnosis was verified by culture. Significant changes in serum levels of IgG, IgM and/or IgA antibodies against FHA were demonstrated in 126 patients (80%). Thus, demonstration of significant changes in FHA antibody titers in serum can be used for serological diagnosis of pertussis. The results also show that high levels of IgG, IgM and/or IgA antibodies in a single serum sample suggest current pertussis infection, but if the diagnosis is based on determinations of FHA antibody titers in a single serum sample the sensitivity is low. The levels of antibody to FHA were compared with previously determined levels of antibodies to pertussis toxin. A significant antibody response against both FHA and pertussis toxin was seen in 111 patients (70 %) while 147 patients (93 %) developed a significant increase in antibodies against one or both antigens.     

Preparation of filamentous hemagglutinin from Bordetella pertussis and assay for serum antibodies to filamentous hemagglutinin and pertussis toxin for clinical and public health laboratories.

A procedure that is sufficiently simple and economical for use in clinical and public health laboratories for producing and purifying filamentous hemagglutinin (FHA) and determining antibodies to this major antigen of Bordetella pertussis in serum is described. High yields of FHA (40 to 80 mg/liter) were obtained in the supernatant by cultivating B. pertussis in modified CL medium. The FHA antigen was separated from pertussis toxin (PT) and other antigens by chromatography on hydroxylapatite. Removal of residual PT activity in the FHA fraction was effected by affinity absorption of PT with Fetuin immobilized to Sepharose 4B. The FHA was used as the antigen for determining titers of immunoglobulin G (IgG), IgA, and IgM to FHA in sera of patients with pertussis by an improved enzyme-linked immunosorbent assay. Development of the interfering background color commonly observed in conventional FHA enzyme-linked immunosorbent assay procedures was eliminated by washing the reaction wells with a buffer of high ionic strength before adding the peroxidase conjugates. In the absence of nonspecific background color, the reaction endpoints were easy to read. The FHA prepared by the procedure described was identical to a reference preparation of purified FHA in sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and serological specificity assays. High yields of FHA were obtained from all four strains of B. pertussis tested in this study, indicating that the procedure for enhanced production of FHA may be generally applicable to other strains of B. pertussis. Results from tests of 50 serum specimens with clinical information on pertussis for FHA and PT antibodies by the assay procedures described exemplified the usefulness and caveats of serodiagnosis for pertussis.     

Surface-associated filamentous hemagglutinin induces autoagglutination of Bordetella pertussis.

Filamentous hemagglutinin (FHA) is a major adhesin produced by Bordetella pertussis, the etiologic agent of whooping cough. FHA has been shown to be surface associated but is also secreted by virulent bacteria. Microscopic observations of lungs of mice infected with B. pertussis showed that the bacteria grow as clusters within the alveolar lumen. When B. pertussis was cultivated in vitro with chemically defined medium, bacteria grew as aggregates, mimicking growth observed in vivo. This aggregation was abolished by the addition of cyclodextrin (CDX) to the growth medium and depended on the production of FHA, because a mutant lacking the FHA structural gene failed to form aggregates in a CDX-free medium. Western blot (immunoblot) analyses revealed that, in the absence of CDX, FHA was attached to the bacterial surface and was not efficiently released into the growth medium. Hydrophobic chromatography of FHA showed that CDX drastically reduced the hydrophobicity of FHA, suggesting a direct binding of CDX to FHA, which was further supported by the partial protection of FHA from trypsin digestion in the presence of CDX. In addition, free FHA can interact in a CDX-inhibitable manner with solid phase-immobilized FHA. It can therefore be postulated that the B. pertussis aggregates are most likely due to direct FHA-FHA interaction.     

Salmonella typhimurium aroA mutants as carriers of the Escherichia coli heat-labile enterotoxin B subunit to the murine secretory and systemic immune systems

We investigated the ability of Salmonella typhimurium vaccines to deliver heterologous antigens to the systemic and secretory immune systems of the mouse, while retaining their immunogenicity against salmonellosis. S. typhimurium SL3261, an avirulent aroA mutant, or SL3261 carrying plasmid pBRD026, a pBR322 derivative encoding the gene for Escherichia coli LT-B were used to immunize BALB/c mice orally. Both immunizing strains invaded the mononuclear phagocyte system of the mice, grew slowly until approximately day 14 post-infection, and then were rapidly cleared. No salmonellae were detected in livers, spleens, mesenteric lymph nodes or Peyer's patches by day 42. Mice immunized with either strain and challenged orally with the virulent parent strain, SL1344, several weeks after clearing the immunizing organism, were protected against the lethal S. typhimurium infection. Mice infected with SL3261 (pBRD026) developed substantial levels of IgG and IgA anti-LT-B antibodies 14 days post-infection in both serum and gut samples. The sera neutralized the effects of LT in an in vitro Vero cell assay. Thus, aroA mutants of S. typhimurium can deliver a heterologous antigen from a different enteric pathogen to the murine systemic and secretory immune systems without altering their efficacy against salmonellosis.     

Expression of the envelope antigen of dengue virus in vaccine strains of Salmonella

The envelope gene of dengue 4 virus (DEN) was cloned in a plasmid under the control of Escherichia coli expression signals. A clone that expressed 93% of the gene was found to be detrimental to the bacterial host. Another clone which carried only 76% of the E gene was found to be quite stable in vitro as well as in vivo. The killed recombinant bacteria induced antibodies in mice which recognized native DEN virus. Attenuated Salmonella typhimurium (SAL) strains carrying the DEN-E plasmid were tested for their efficacy as orally administered live vaccines. Protective immunization was assessed in a mouse model by immunizing three-week old BALB/c mice followed by challenge with DEN virus. It was found that these young mice were highly susceptible to the carrier SAL strains (M206 and aroA SL3261). Moreover, the SAL-infected mice were more susceptible to DEN virus challenge than control mice, suggesting that the SAL infection caused immunosuppression in these young mice.     

口服基因疫苗预防柯萨奇B组病毒性心肌炎的实验研究

为研制防治病毒性心肌炎的口服基因疫苗 ,本文以减毒鼠伤寒杆菌SL72 0 7作载体 ,将表达CVB3VP1基因的pcDNA3 VP1重组质粒转化入该细菌中 ,制备成口服基因疫苗。经口服免疫小鼠后 ,检测其免疫效果 ,并用CVB3攻击小鼠 ,观察其免疫保护作用 ,结果表明小鼠经 1次口服免疫后 ,血液中即产生了高滴度的抗体 ;病毒攻击后 ,免疫小鼠产生了明显的保护作用。