Search

Pertussis toxin (PT) is a major virulence factor of Bordetella pertussis which exerts a range of effects on the immune system, including the enhancement of IgE, IgA and IgG production, delayed-type hypersensitivity reactions, and the induction of experimental autoimmune diseases. However, the mechanism by which PT mediates adjuvanticity remains to be defined. In this investigation we have shown that PT can potentiate antigen-specific T cell proliferation and the secretion of IFN-gamma, IL-2, IL-4 and IL-5 when injected with foreign antigens. A chemically detoxified PT and a genetic mutant with substitutions/deletions in the S-1 and B oligomer components that abrogate enzymatic and binding activity displayed no adjuvant properties. In contrast, a non-toxic S-1 mutant devoid of enzymatic activity but still capable of receptor binding retained its adjuvanticity, augmenting the activation of both Th1 and Th2 subpopulations of T cells. In an attempt to address the mechanism of T cell activation, we found that PT stimulated the production of IFN-gamma and IL-2 by naive T cells and IL-1 by macrophages. Therefore potentiation of distinct T cell subpopulations may have resulted in part from the positive influence of IFN-gamma on the development of Th1 cells and the co-stimulatory role of IL-1 for Th2 cells. Furthermore, PT augmented expression of the co-stimulatory molecules B7-1 and B7-2 on macrophages and B cells, and CD28 on T cells, suggesting that the adjuvant effect may also be associated with facilitation of the second signal required for maximal T cell activation. This study demonstrates that the immunopotentiating properties of PT are largely independent of ADP-ribosyltransferase activity, but are dependent on receptor binding activity and appear to involve enhanced activation of T cells.

The results of phase 3 efficacy trials have shown that acellular and whole-cell pertussis vaccines can confer protection against whooping cough. However, despite the advances in vaccine development, clinical trials have not provided significant new information on the mechanism of protective immunity against Bordetella pertussis . Classical approaches based on measurement of antibody responses to individual antigens failed to define an immunological correlate of protection. A reliable animal model, predictive of acellular and whole-cell pertussis vaccine potency in children, would facilitate an elucidation of the mechanism of immune protection against B. pertussis and would assist in the regulatory control and future development of pertussis vaccines. In this study, we have shown that the rate of B. pertussis clearance following respiratory challenge of immunized mice correlated with vaccine efficacy in children. Using this model together with mice with targeted disruptions of the gamma interferon (IFN-γ) receptor, interleukin-4 or immunoglobulin heavy-chain genes, we have demonstrated an absolute requirement for B cells or their products in bacterial clearance and a role for IFN-γ in immunity generated by previous infection or immunization with the whole-cell pertussis vaccine. The results of passive immunization experiments suggested that protection early after immunization with acellular pertussis vaccines is mediated by antibody against multiple protective antigens. In contrast, more complete protection conferred by previous infection or immunization with whole-cell pertussis vaccines reflected the induction of Th1 cells. Our findings suggest that the mechanism of immunity against B. pertussis involves humoral and cellular immune responses which are not directed against a single protective antigen and thus provide an explanation for previous failures to define an immunological correlate of protection.

The mechanism of protective immunity against Bordetella pertussis generated following recovery from whooping cough in childhood has not yet been elucidated. Studies with a murine respiratory infection model have indicated that cellular immunity, mediated by Th1 cells, plays a role in the clearance of a primary infection with B. pertussis and in protection against subsequent challenge. In the present study, the induction of B. pertussis-specific Th cell subsets in children was examined. Peripheral blood mononuclear cells from B. pertussis-infected or convalescent children proliferated and secreted cytokines following antigen stimulation in vitro. In contrast, responses were weak or undetectable in the majority of children who had not been infected or vaccinated. In all cases, responding T cells produced interferon-gamma but low or undetectable interleukin-5. The findings suggest that Th1 cells may play a role in protective immunity generated following infection with B. pertussis in children.

Using a murine respiratory infection model, we have demonstrated previously that infection with Bordetella pertussis or immunization with a whole-cell pertussis vaccine induced antigen-specific Th1 cells, which conferred a high level of protection against aerosol challenge. In contrast, immunization with an acellular vaccine, consisting of the B. pertussis components detoxified pertussis toxin, filamentous hemagglutinin, and pertactin adsorbed to alum, generated Th2 cells and was associated with delayed bacterial clearance following challenge. In this study, we demonstrated that addition of interleukin-12 (IL-12) either in vitro or in vivo enhanced type 1 T-cell cytokine responses induced with an acellular vaccine. Furthermore, the rate of bacterial clearance in mice coinjected with IL-12 and the acellular vaccine was similar to that observed following immunization with a potent whole-cell vaccine. Analysis of IL-12 secretion by murine macrophages suggested that this cytokine is produced in vivo following B. pertussis infection or immunization with the whole-cell vaccine. IL-12 was detected in the supernatants of lung, splenic, and peritoneal macrophages infected with live B. pertussis or stimulated with heat-killed whole B. pertussis or B. pertussis lipopolysaccharide. In contrast, IL-12 could not be detected following stimulation of macrophages with the bacterial antigens filamentous hemagglutinin, detoxified pertussis toxin, and pertactin, the components of acellular vaccines. Our findings suggest that induction of endogenous IL-12 may contribute to the high efficacy of pertussis whole-cell vaccines and also demonstrate that it is possible to attain these high levels of protection with a less reactogenic acellular vaccine incorporating IL-12 as an adjuvant.

Pertussis toxin (PT) is a major virulence factor of Bordetelfa pertussis which exerts a range of biological effects on the immune system. The toxin is a heterohexameric protein with an A-B toxin structure [ 11. The A-promoter comprises a single S1 subunit with ADP-ribosyltransferase activity which regulates signal transduction in mammalian cell membranes. The Boligomer is composed of five subunits S2, S3, S4 and S5 and mediates binding of the toxin to eukaryotic cells as well as inducing mitogenic T cell activation. It has been reported that PT, like other A-B toxins, such as cholera toxin and E. coli enterotoxin, possesses adjuvant properties. I t has been demonstrated that co-injection of antigens with PT enhances IgE production, DTH reactions and experimental autoimmune disease in mice [ I , 31. Paradoxically PT appears to influence immune responses mediated by reciprocally regulated subpopulations of CD4+ Th cells that secreted IFNy. IL-2 or IL-4 and IL-5, termed Thl and Th2 cells respectively. However the mechanisms by which PT exerts its adjuvanticity remain to be defined. In this study we have examined the capacity of PT to augment cytokine secretion by antigen-specific T cells when coinjected with a foreign antigen. Mice were immunized with 20pg of KLH alone or with 1.Opg of active PT at week 0 and week 4. Spleen cells were isolated on week six and stimulated with a range of concentrations of KLH to determine Th cell responses. We found that PT can potentiate antigen-specific CD4' T cell proliferation and the secretion of IFNy, IL-2, IL-4 and IL-5 when injected with the antigen (Fig 1 A & B and data not shown). A chemically detoxified PT (PTd), or a genetic mutant with substitutions/de\etions in the S1 and B-oligomer components, that abrogate enzymatic activity and receptor binding [3], were found to be devoid of the adjuvant properties. In contrast, a non-toxic S-1 mutant devoid of enzymatic activity, but still capable of receptor binding, had a modest effect on IFN-y and IL-2 secretion, but lost its ability to enhance IL-4 or IL-5 production. These findings demonstrate that PT can potentiate the activation of both Thl and Th2 subpopulations of T cells. Although we have evidence that enhanced T cell responses may be associated with increased expression of the co-stimulatory molecules B7-1 and B7-2, the adjuvant effect of PT also appears to reflect distinct effects of the B-oligomer and the S-1 subunit on T cell cytokine secretion, through their stimulation of alternative signal transduction pathways. In recent studies it has been shown that the activation of the adenylate cyclase pathway upregulates IL-4 and IL-5 production by activated CD4+ T cells, the ADPribosyltransferase activity of the SI subunit may allow it to selectively induce the secretion of Th2 type cytokines by T cells [4]. Conversely activation of the inositol phosphate pathway, the signal transduction pathway of the B-oligomer, induces the production of new mRNA for IFNy and IL-2 [5 ] , suggesting that the B-oligomer may selectively potentiate Thl responses. Recent evidence suggests that Th 1 cells play a key role in protection against B. pertussis infection. Using a murine aerosol challenge model we have shown that infection with B. pertussis