Role of antibodies against Bordetella pertussis virulence factors in adherence of Bordetella pertussis and Bordetella parapertussis to human bronchial epithelial cells

Immunization with whole-cell pertussis vaccines (WCV) containing heat-killed Bordetella pertussis cells and with acellular vaccines containing genetically or chemically detoxified pertussis toxin (PT) in combination with filamentous hemagglutinin (FHA), pertactin (Prn), or fimbriae confers protection in humans and animals against B. pertussis infection. In an earlier study we demonstrated that FHA is involved in the adherence of these bacteria to human bronchial epithelial cells. In the present study we investigated whether mouse antibodies directed against B. pertussis FHA, PTg, Prn, and fimbriae, or against two other surface molecules, lipopolysaccharide (LPS) and the 40-kDa outer membrane porin protein (OMP), that are not involved in bacterial adherence, were able to block adherence of B. pertussis and B. parapertussis to human bronchial epithelial cells. All antibodies studied inhibited the adherence of B. pertussis to these epithelial cells and were equally effective in this respect. Only antibodies against LPS and 40-kDa OMP affected the adherence of B. parapertussis to epithelial cells. We conclude that antibodies which recognize surface structures on B. pertussis or on B. parapertussis can inhibit adherence of the bacteria to bronchial epithelial cells, irrespective whether these structures play a role in adherence of the bacteria to these cells.     

Development of a rapid diagnostic test for pertussis: direct detection of pertussis toxin in respiratory secretions.

Monoclonal antibodies (MAb) were produced against the specific Bordetella pertussis antigen pertussis toxin (PT). In preliminary studies, one MAb (IB12) was selected and used in an enzyme-linked dot blot immunoassay to evaluate the ability of the method to detect known amounts of PT in control experiments and to test its potential for direct detection of PT in nasopharyngeal secretion (NP) specimens from patients with confirmed cases of whooping cough. The dot blot assay was able to detect PT at levels as low as 10 ng per dot in either buffer or control NP specimens. The assay demonstrated specificity, reacting only with dot blots of whole B. pertussis and not Bordetella bronchiseptica, Bordetella parapertussis, or other bacterial strains. In preliminary studies, NP aspirate, swab, and wash specimens were compared. The specimen of choice was found to be the NP aspirate, for which 100% positive results were found in the assay. These initial studies suggest that the dot blot immunoassay in which a MAb is used for direct detection of PT in NP specimens may be useful as a rapid diagnostic test for pertussis.     

Immunization of teenagers with a fifth dose of reduced DTaP-IPV induces high levels of pertussis antibodies with a significant increase in opsonophagocytic activity

Waning vaccine-induced immunity against Bordetella pertussis is observed among adolescents and adults. A high incidence of pertussis has been reported in this population, which serves as a reservoir for B. pertussis. A fifth dose of reduced antigen of diphtheria-tetanus-acellular-pertussis and inactivated polio vaccine was given as a booster dose to healthy teenagers. The antibody activity against B. pertussis antigens was measured prior to and 4 to 8 weeks after the booster by different assays: enzyme-linked immunosorbent assays (ELISAs) of IgG and IgA against pertussis toxin (PT) and filamentous hemagglutinin (FHA), IgG against pertactin (PRN), opsonophagocytic activity (OPA), and IgG binding to live B. pertussis. There was a significant increase in the IgG activity against PT, FHA, and PRN following the booster immunization (P < 0.001). The prebooster sera showed a geometric mean OPA titer of 65.1 and IgG binding to live bacteria at a geometric mean concentration of 164.9 arbitrary units (AU)/ml. Following the fifth dose, the OPA increased to a titer of 360.4, and the IgG concentration against live bacteria increased to 833.4 AU/ml (P < 0.001 for both). The correlation analyses between the different assays suggest that antibodies against FHA and PRN contribute the most to the OPA and IgG binding.     

Immunoglobulin A antibodies to pertussis toxin and filamentous hemagglutinin in saliva from patients with pertussis.

Immunoglobulin A (IgA) antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) in 181 saliva samples obtained during various stages of pertussis from 112 patients were determined. Saliva samples obtained within 5 days after the onset of symptoms did not have detectable IgA antibodies against either of the two antigens. Of the samples obtained between 6 and 50 days after the onset of symptoms, 72% had antibodies against FHA but only 40% had antibodies against PT. With few exceptions, saliva samples obtained more than 50 days after the onset of symptoms contained antibodies against both antigens. In the 59 patients from whom paired saliva samples were obtained at intervals of 2 to 5 weeks, a significant increase in the geometric mean FHA antibody titers but not PT antibody titers occurred. However, increases that were fourfold or greater were observed against FHA in only 19 patients and against PT in 14 patients. Thus, IgA antibodies against FHA and PT in saliva develop during pertussis, and the importance of secretory IgA antibodies for protection against infection and disease should be investigated. Determination of these antibodies in paired saliva samples is, however, of little value for the laboratory diagnosis of pertussis.     

Protective activities in mice of monoclonal antibodies against pertussis toxin.

Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23 antibodies and also in the pathogenic mechanisms between aerosol and intracerebral infections. Furthermore, it was suggested that although not only FHA and agglutinogen 2 but also PT have roles as attachment factors, the processes of infection and protection are different between mice immunized with antibody against FHA or agglutinogen 2 and that against PT because the latter mice are also able to neutralize toxicity of PT diffused into the mice.     

Opsonophagocytic activity and other serological indications of Bordetella pertussis infection in military recruits in Norway

Bordetella pertussis is the causative agent of pertussis (whooping cough). Despite high vaccination coverage, pertussis remains a significant disease in many countries. Besides vaccination, transient carriage of Bordetella spp. or other cross-reacting organisms adds to the immunity against pertussis. However, the various immunological mechanisms conferring protection remain largely unknown. In this study, paired serum samples from 464 healthy Norwegian military recruits were collected, the first at enrolment and the second about 8 months later. The prevalence of pertussis during military service was examined by comparing the paired serum samples for immunoglobulin G (IgG) antibodies against pertussis toxin (PT) by enzyme-linked immunosorbent assay (ELISA). Seventy-eight percent of the recruits had low levels of IgG antibodies against PT in both samples. Conversely, 8.4% of the recruits demonstrated high anti-PT IgG levels in the first sample, indicative of recent pertussis prior to enrolment. One recruit experienced seroconversion, indicating pertussis during service. A subset of 248 serum samples with low, medium, and high anti-PT IgG titers were analyzed by a different ELISA kit for IgG and IgA antibodies against PT and filamentous hemagglutinin (FHA) and for opsonophagocytic activity (OPA), for induction of C3b deposition products, and for IgG binding with live B. pertussis as the antigen. We observed high correlations between OPA and IgG against live bacteria (r = 0.83), between OPA and IgG anti-FHA (r = 0.79), between OPA and anti-PT IgG (r = 0.68), and between OPA and C3b binding (r = 0.70) (P < 0.0001 for all). Anti-PT IgA did not correlate closely with the other assays.     

Recombinant Mycobacterium bovis BCG expressing pertussis toxin subunit S1 induces protection against an intracerebral challenge with live Bordetella pertussis in mice

The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries. Since Mycobacterium bovis BCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative. We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the beta-lactamase signal sequence or the whole beta-lactamase protein, under control of the upregulated M. fortuitum beta-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole beta-lactamase protein, and both were localized to the mycobacterial cell wall. The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance. Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-gamma) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response. The rBCG-S1PT strains induced a low humoral response against PT after 2 months. Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with live Bordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection against B. pertussis infection. Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.     

Bordetella pertussis infection in mice: correlation of specific antibodies against two antigens, pertussis toxin, and filamentous hemagglutinin with mouse protectivity in an intracerebral or aerosol challenge system

The correlation of titers of specific serum immunoglobulin G antibodies against two antigens, pertussis toxin (PT), and filamentous hemagglutinin (FHA), which are the main components of pertussis vaccine in Japan, with mouse protectivity was examined by both intracerebral and aerosol challenge systems with virulent Bordetella pertussis cells. Titer of the antibodies was calculated from the enzyme-linked immunosorbent assay (ELISA) unit given arbitrarily to reference antibodies. PT antibody titer which protected 50% of mice was indistinguishable in both active immunization followed by intracerebral challenge and passive immunization followed by aerosol challenge. The 50% effective dose was 23 ELISA U/ml in the former mice and 24 ELISA U/mouse in the latter. In the intracerebral challenge system, FHA did not elicit a protective response but was very helpful for PT as an immunizing antigen. When anti-FHA immunoglobulin G coexisted with anti-PT immunoglobulin G in mice, the 50% effective dose of PT antibody was 4.4 or 10 ELISA U/mouse in intracerebral or aerosol challenge systems, respectively. In this active immunization system, pertussis toxoid of 1 micrograms or 0.1 microgram/mouse produced PT antibody of ca. 20 or 5 ELISA U/ml, respectively. It was concluded that pertussis toxoid or its antibody was much more potent than Formalin-treated FHA or its antibody; Formalin-treated FHA or its antibody was helpful when it was administered with pertussis toxoid toxoid or its antibody, however.     

Synthesis of a conjugate vaccine composed of pneumococcus type 14 capsular polysaccharide bound to pertussis toxin.

Type 14 is one of the common types isolated from patients of all ages with infections caused by Streptococcus pneumoniae. Its capsular polysaccharide (Pn14) is composed of a neutrally charged tetrasaccharide repeat unit. Pn14 does not elicit protective levels of antibodies in infants and children and is a less than optimal immunogen of the 23-valent vaccine for adults. Pertussis toxin (PT) is both a virulence factor and protective antigen of Bordetella pertussis: it is not soluble at neutral pH and forms insoluble complexes with acidic polysaccharides. Both Pn14 and PT are potential components of vaccines for infants and children. Accordingly, a synthetic scheme was devised to prepare a conjugate of Pn14 and PT. An adipic acid hydrazide derivative of Pn14 was bound to PT at pH 3.9 by carbodiimide-mediated condensation. The conjugation procedure inactivated the PT as assayed by CHO cell and histamine-sensitizing activity. The Pn14-PT conjugate elicited antibodies in mice to Pn14 at levels estimated to be protective in humans and elicited neutralizing antibodies to PT. We plan to evaluate Pn14-PT clinically.     

Human antibody response to the B oligomer of pertussis toxin.

To determine whether antibodies to the B oligomer of pertussis toxin (PT) were present in patients diagnosed with pertussis or vaccinees who had received diphtheria-tetanus-whole-cell pertussis vaccine, we analyzed serum samples from 5 patients and 10 vaccinees by both enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting techniques. Antibodies to the B oligomer were detected by ELISA in all samples containing antibodies to holotoxin. Western immunoblotting procedures were less efficient than ELISA techniques for detecting antibodies to the B oligomer. Antibodies which inhibit the ability of the B oligomer to agglutinate erythrocytes were detected in purified human immunoglobulin preparations. In addition, serum samples containing antibodies to PT inhibited the binding of purified B oligomer and holotoxin to a 165-kDa glycoprotein which has been considered a potential PT receptor in Chinese hamster ovary (CHO) cells. These results suggest that antibodies to the B oligomer contribute to the human serologic response to PT, but their detection and characterization require appropriate methods.     

Evaluation of a tetraplex microsphere assay for Bordetella pertussis antibodies

To increase testing efficiency, a microsphere-based multianalyte immune detection (MAID) system was developed to measure serum immunoglobulin G (IgG) and IgA recognizing two Bordetella pertussis antigens, pertussis toxin (PT) and filamentous hemagglutinin antigen (FHA). The assay was performed as two separate duplexes. One duplex measured IgG to PT and FHA, and the other measured IgA to PT and FHA. The two duplexes were then combined and analyzed as a tetraplex. The MAID system and an in-house enzyme-linked immunosorbent assay (ELISA) system were used to evaluate 100 sera from blood donors and 220 consecutive sera submitted for B. pertussis antibody testing. For both the MAID and ELISA systems, antibody levels were defined as increased if greater than the blood donor group 95th percentile value. The qualitative concordance rates between MAID and ELISA results for the 220 consecutively submitted sera were as follows: PT IgG, 99%; PT IgA, 94%; FHA IgG, 93%; FHA IgA, 94%. The overall concordance rate was 95% (836 of 880 result sets). For 29 of 44 (66%) discordant result sets, the discordant MAID result was supported by the MAID and ELISA results for other B. pertussis antibodies. The MAID and in-house ELISA systems were also used to evaluate 20 sera previously tested for pertussis antibodies at a pertussis vaccine research laboratory; MAID results for all four analytes did not significantly differ from results obtained by the research laboratory. These findings show that antibodies to B. pertussis antigens can be measured easily and accurately using a tetraplex microsphere system.     

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.     

Quantitative priming with inactivated pertussis toxoid vaccine in the aerosol challenge model

Serum antibodies to pertussis toxin (PT) have been shown to be protective against severe pertussis disease, although a specific level of anti-PT antibody that correlates with protection has not been demonstrated. Current animal models such as the intracerebral challenge model have significant limitations in correlating protection to a specific level of anti-PT antibody. This study examines the protective effects of priming with tetranitromethane-inactivated pertussis toxoid (PTx) vaccine in the aerosol challenge model and whether a measurable response to a priming dose of PTx is enough to initiate a protective secondary response when challenged with infection. The correlation of priming with markers of illness such as leukocytosis, weight loss, bacterial proliferation, and mortality after established infection with Bordetella pertussis was explored. BALB/c mice were immunized with PTx vaccine on day 6 of life and then challenged with B. pertussis using the aerosol challenge model. Data were analyzed according to the primary immunologic response, differentiating responders (anti-PT immunoglobulin G [IgG] > or =1 microg/ml) from nonresponders (anti-PT IgG <1 microg/ml). Mice that showed evidence of priming on the day of aerosol challenge were able to mount a secondary response to the challenge with a > or =2-fold rise in anti-PT IgG antibody by day 7 and a > or =10-fold rise by day 14 post-aerosol challenge. These primed mice were significantly better protected against leukocytosis, weight loss, and proliferation of B. pertussis in the lungs following aerosol challenge than the nonprimed group. This protection correlated with levels of anti-PT antibody in serum present on the day of aerosol challenge.     

Recognition of pertussis toxin by antibodies to synthetic peptides

Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-terminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237-255), could recognize the conformation of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.     

Description of a hybridoma bank towards Bordetella pertussis toxin and surface antigens

This paper describes the development of a murine bank of monoclonal antibodies against Bordetella pertussis toxin, filamentous hemagglutinin (FHA), pili, lipopolysaccharide (LPS), or outer membrane proteins (OMPs). Subunits S1, S2, S3 of pertussis toxin (PT) bound immunoglobulins and glycoproteins such as fetuin and haptoglobin in an unspecific manner. The specificity of monoclonal antibodies towards subunits S1, S2, S3 or S4 of PT could be demonstrated by using purified immunoglobulins or their Fab2 fragments. A set of FHA-specific monoclonal antibodies could be differentiated on the basis of their binding to the various breakdown products present in FHA preparations. Pili-specific monoclonal antibodies reacted with either native pili or denatured pilin, and both demonstrated serotype specificity. Monoclonal antibodies to Bordetella pertussis OMPs were directed to either the virulent phase-regulated trypsin-sensitive, detergent-extractable OMPs 92 kDa, 32 kDa, and 30 kDa or the non-virulent phase-expressed, not-trypsin sensitive OMPs 38 kDa, 33kDa, and 18 kDa.     

Reciprocal protective immunity against Bordetella pertussis and Bordetella parapertussis in a murine model of respiratory infection

The protective immunity induced by infection with Bordetella pertussis and with Bordetella parapertussis was examined in a murine model of respiratory infection. Convalescent mice that had been infected by aerosol with B. pertussis or with B. parapertussis exhibited a protective immune response against B. pertussis and also against B. parapertussis. Anti-filamentous hemagglutinin (anti-FHA) serum immunoglobulin G (IgG) and anti-FHA lung IgA antibodies were detected in both mice infected with B. pertussis and those infected with B. parapertussis. Antibodies against pertussis toxin (anti-PT) and against killed B. pertussis cells were detected in mice infected with B. pertussis. Pertactin-specific antibodies and antibodies against killed B. parapertussis cells were detected in mice infected with B. parapertussis. Spleen cells from mice infected with B. pertussis secreted interferon-gamma (IFN-gamma) in response to stimulation by FHA or PT. Spleen cells from mice infected with B. parapertussis also secreted IFN-gamma in response to FHA. Interleukin-4 was not produced in response to any of the antigens tested. The profiles of cytokine secretion in vitro revealed induction of a Th1-biased immune response during convalescence from infection by B. pertussis and by B. parapertussis. It is possible that Th1 and Th2 responses against FHA might be related to the reciprocal protection achieved in our murine model.     

Serum antibody response to B. pertussis Tn5 mutants, purified PT and FHA in two different mouse strains and passive protection in the murine intranasal infection model

The protective capacities of antibodies to pertussis toxin (PT) were compared with antibodies to several other pertussis antigens in an experimental murine model of intranasal infection with Bordetella pertussis. Protection from lethal challenge was achieved by passive immunization with mouse antisera to whole cells of the virulent B. pertussis BP338(Vir+) and its Tn5-generated mutants, BP353(Fha-), BP348(Adc-Hly-) and to a lesser extent of BP347(Vir-). The immune sera were produced in two different mouse strains, a good PT antibody responder (NIH) and a poor responder (F1 of CBA x C57BL/6). The antisera produced in the F1 mice contained no detectable neutralizing antibodies to PT as measured by the CHO cell assay. In spite of this the anti-BP353(Fha-) and BP348(Adc-Hly-) sera of the F1 mice seemed as protective as those of the NIH mice. A strong dependence between PT neutralizing antibody and protection was seen only when comparing sera of NIH and F1 mice immunized with purified active PT. The protective capacity of sera of both mouse strains immunized with purified filamentous hemagglutinin (FHA) correlated with their anti-FHA titers measured by enzyme immunoassay. The data thus confirm the protective capacity of anti-PT and anti-FHA, but also show that antibodies of other specificities can confer protection.     

Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay

Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of <0.0001 for the 100 individual samples. We, therefore, conclude that the improved IgG anti-PT ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT.     

Production of Neisseria meningitidis transferrin-binding protein B by recombinant Bordetella pertussis

Neisseria meningitidis serogroup B infections are among the major causes of fulminant septicemia and meningitis, especially severe in young children, and no broad vaccine is available yet. Because of poor immunogenicity of the serogroup B capsule, many efforts are now devoted to the identification of protective protein antigens. Among those are PorA and, more recently, transferrin-binding protein B (TbpB). In this study, TbpB of N. meningitidis was genetically fused to the N-terminal domain of the Bordetella pertussis filamentous hemagglutinin (FHA), and the fha-tbpB hybrid gene was expressed in B. pertussis either as a plasmid-borne gene or as a single copy inserted into the chromosome. The hybrid protein was efficiently secreted by the recombinant strains, despite its large size, and was recognized by both anti-FHA and anti-TbpB antibodies. A single intranasal administration of recombinant virulent or pertussis-toxin-deficient, attenuated B. pertussis to mice resulted in the production of antigen-specific systemic immunoglobulin G (IgG), as well as local IgG and IgA. The anti-TbpB serum antibodies were of the IgG1, IgG2a, and IgG2b isotypes and were found to express complement-mediated bactericidal activity against N. meningitidis. These observations indicate that recombinant B. pertussis may be a promising vector for the development of a mucosal vaccine against serogroup B meningococci.     

Interaction of pertussis toxin with human T lymphocytes.

The binding of pertussis toxin (PT) to the human T-cell line Jurkat was examined by using flow cytometry. Fluorescein isothiocyanate (FITC)-labeled PT bound rapidly to the cells in a specific manner as determined by blocking experiments with unlabeled toxin, B oligomer, and the S2-S4 and S3-S4 dimers. Monoclonal antibodies against the S3 subunit of the toxin also significantly inhibited the binding of FITC-PT. Sialidase treatment of the cells resulted in decreased binding of FITC-PT, indicating that sialic acid residues are involved in the binding process. In addition, we studied the effect of PT binding on the expression of cell surface molecules. On binding of PT to the cell surface, a rapid down-regulation of the T-cell receptor (TCR)-CD3 complex was observed. The modulation of the TCR-CD3 complex was independent of the toxin's enzymatic activity, as the B oligomer and a nonenzymatic toxin mutant induced modulation comparable to that caused by the native holotoxin. Isolated dimers did not cause down-regulation. Stimulation of the TCR-CD3 complex, leading to reduced cell surface expression of this complex, provides a possible explanation for the second messenger production associated with the interaction of PT or B oligomer with T lymphocytes. We therefore conclude that PT activates T cells by divalent binding to the TCR-CD3 complex itself or by binding a structure closely associated with it.