To investigate the fundamental nature of protective immunity to Bordetella pertussis, we studied intranasal immunization of adult mice with formalin-fixed B. pertussis (FFBP), followed by aerosol B. pertussis challenge. Mice given two doses of FFBP intranasally completely cleared a subsequent pertussis aerosol challenge from tracheae and lungs (defined as protection), but there was no correlation between levels of specific antibody and clearance of bacteria. Further, transfer of immune serum before aerosol challenge had minimal effects on bacterial burdens. However, pertussis-specific T cells producing interferon γ but not interleukin 4 or interleukin 10 were detected in draining lymph nodes of FFBP-immunized mice. Significantly, repeated immunization of B cell knockout (BKO) mice resulted in partial protection, and complete protection was reconstituted by transfer of pertussis-immune B cells; reconstituted BKO mice had little if any detectable antipertussis antibodies. Immunization of mice lacking all T cells or lacking CD4+ T cells did not lead to protection; in contrast, CD8− mice were protected. Mice depleted of CD4+ T cells after immunization but before aerosol challenge, which thus had normal amounts of specific antibodies, were not optimally protected. Taken together, these data indicate that protective immunity to pertussis is dependent on both CD4+ T cells and B cells, and both cell types provide significant functions other than specific antibody production.

Immunization with the 69-kD outer membrane protein (OMP) of Bordetella pertussis protected neonatal mice against lethal respiratory challenge with B. pertussis 18323. Active immunization elicited a serum IgG anti-69-kD OMP response at the time of challenge, with IgG anti-69-kD OMP antibodies detected in bronchoalveolar lavage fluid after challenge. Intravenous administration of BPE8, a monoclonal IgG1 anti-69-kD OMP, also protected young mice against B. pertussis challenge. Intravenously injected BPE8 was detected in the lungs of mice at the time of aerosol challenge, suggesting that the presence of specific antibody in the lungs may mediate protection. Thus the 69-kD OMP of B. pertussis is a protective antigen in mice that elicits specific serum antibody that can transude to the lung. The 69-kD OMP was detected in a preparation of a Takeda acellular vaccine by immunoblot analysis and a serum antibody response to the 69-kD OMP was observed in 18-mo-old children boosted with this preparation of Japanese acellular vaccine. Our results demonstrate that the B. pertussis 69-kD OMP is a protective antigen in animals, is immunogenic in humans, and is present in a preparation of acellular pertussis vaccine that is widely used in Japan. These findings indicate that the 69-kD OMP should be seriously considered as a candidate for inclusion in new formulations of antigenically defined acellular pertussis vaccines.

Using a mouse model of Bordetella pertussis infection, we have analyzed the role of gamma interferon (IFN-␥) in bacterial clearance from the respiratory tract. Adult BALB/c mice began to clear a respiratory infection within 3 weeks postinfection, with complete resolution of infection 6 to 8 weeks postinfection. In contrast, neither adult SCID mice (which lack mature B and T lymphocytes) nor adult nude mice (which lack mature T lymphocytes) controlled B. pertussis infection, and both strains died within 3 to 5 weeks postinfection. Short-term T-cell lines generated from the draining lymph nodes of the lungs of infected BALB/c mice were found to be CD4 ؉ and produced IFN-␥ but no detectable interleukin-4. Analyses of IFN-␥ mRNA induction in the lungs of mice following B. pertussis infection showed that in both BALB/c and C57BL/6 mice, IFN-␥ mRNA levels increased sharply by 1 week postinfection and then subsequently declined. Further exploration of a potential role for IFN-␥ demonstrated that infection of adult BALB/c mice depleted of IFN-␥ in vivo with anti-IFN-␥ monoclonal antibodies resulted in greater numbers of bacteria recovered from the lungs than in infected, control BALB/c mice, although IFN-␥-depleted mice could subsequently clear the infection. Infection of mice which have a disrupted IFN-␥ gene resulted in bacterial clearance with a time course similar to those seen with IFN-␥-depleted mice. These results indicate that IFN-␥ plays a role in controlling B. pertussis infection. Bordetella pertussis is a gram-negative bacterium that was first isolated by Bordet and Gengou in 1906. B. pertussis infects humans via inhalation of aerosol droplets and preferentially associates with the cilia of the respiratory epithelia lining the nasopharynx, trachea, and bronchial tree. During the course of disease, B. pertussis infection remains localized to the respiratory tract and does not progress to bacteremia or meningitis (4). The nature of protective immunity against B. pertussis infection and disease is poorly understood. In animal models of infection, antibody-mediated protection, to multiple antigen specificities, has been observed. Passive transfer of monoclonal antibodies to pertussis toxin, pertactin, and lipooligosaccharide, as well as polyclonal antibodies to filamentous hemagglutinin, has been shown to protect against B. pertussis infection (7, 10, 15, 17, 19). However, recent clinical trials of acellular pertussis vaccines have failed to show any correlation between protection and antibody level in postvaccination sera (1). Recently, it has been observed that pertussis-specific T-cell immunity can be detected following pertussis infection and following vaccination with whole-cell pertussis vaccine (9, 13). B. pertussis-specific T cells in humans have been demonstrated after immunization or infection (6). Transfer to mice of a murine CD4 ϩ T-cell clone specific for B. pertussis filamentous hemagglutinin, decreases peak B. pertussis infection by 2 log 10 CFU (9). Further, T cells derived from mice infected with...

Bordetella pertussis antigens, encapsulated in biodegradable poly(DL-lactide-co-glycolide) (DL-PLG) microspheres, were evaluated for their immunogenicity and ability to elicit a protective immune response against B. pertussis respiratory infection. Microencapsulated pertussis toxoid, filamentous hemagglutinin, and pertactin all retained their immunogenicity when administered parenterally. Intranasal immunization with a low dose (1 g) of encapsulated filamentous hemagglutinin, pertussis toxoid, or pertactin elicited strong specific immunoglobulin G and immunoglobulin A antibody responses in respiratory secretions that were greater in magnitude than the responses elicited by the same doses of unencapsulated antigen. Intranasal immunization with as little as 1 g of encapsulated pertussis antigen prior to infection reduced the bacterial recovery by 3 log 10 CFU. However, intranasal immunization with the same low doses of unencapsulated antigens did not reduce infection. Intranasal administration of a combination of 1 g of each of the microencapsulated pertussis antigens was more effective in reducing bacterial infection than administration of any single microencapsulated antigen. Intranasal administration of microencapsulated B. pertussis antigens elicits high levels of specific antibody coinciding with protection against infection when these microspheres are administered to the respiratory tract. These data provide evidence of the respiratory adjuvanticity of three different DL-PLG microsphere preparations, each of which contains a unique B. pertussis antigen.

Mucosal immunization of mice with purified BordeteUla pertussis filamentous hemagglutinin (FHA), by either the respiratory or the gut route, was found to protect against B. pertussis infection of the trachea and lungs. Intranasal immunization of BALB/c and (C57BL/6 x C3H/HeN)Fl adult female mice with FHA prior to B. pertussis aerosol challenge resulted in a 2 to 3 log reduction in number of bacteria recovered from the lungs and the tracheas of immunized mice in comparison to unimmunized controls. Intraduodenal immunization of adult mice with FEIA before infection also resulted in approximately a 2 log reduction in the recovery of bacteria from the lungs and the tracheas of immunized mice in comparison to unimmunized controls. Immunoglobulin A and immunoglobulin G anti-FHA were both detected in bronchoalveolar lavage fluids of mucosally immunized mice. Limiting dilution analysis revealed a 60-fold increase in the frequency of FHA-specific B cells isolated from the lungs of mice immunized intranasally with FHA in comparison to unimmunized control mice. These data suggest that both gut and respiratory mucosal immunization with a major adhesin ofB. pertussis generates a specific immune response in the respiratory tract that may serve as one means of mitigating subsequent B. pertussis respiratory infection.