Modeling the catarrhal stage of Bordetella pertussis upper respiratory tract infections in mice.

Pertussis (whooping cough) is a highly transmissible human respiratory disease caused by Bordetella pertussis, a human-restricted pathogen. Animal models generally involve pneumonic infections induced by depositing large numbers of bacteria in the lungs of mice. These models have informed us about the molecular pathogenesis of pertussis and guided development of vaccines that successfully protect against severe disease. However, they bypass the catarrhal stage of the disease, when bacteria first colonize and initially grow in the upper respiratory tract. This is a critical and highly transmissible stage of the infection that current vaccines do not prevent. Here, we demonstrate a model system in which B. pertussis robustly and persistently infects the nasopharynx of TLR4-deficient mice, inducing localized inflammation, neutrophil recruitment and mucus production as well as persistent shedding and occasional transmission to cage mates. This novel experimental system will allow the study of the contributions of bacterial factors to colonization of and shedding from the nasopharynx, as occurs during the catarrhal stage of pertussis, and interventions that might better control the ongoing circulation of pertussis.

Bbvac: A Live Vaccine Candidate That Provides Long-Lasting Anamnestic and Th17-Mediated Immunity against the Three Classical Bordetella spp.

Acute pathogens such as Bordetella pertussis can cause severe disease but are ultimately cleared by the immune response. This has led to the accepted paradigm that convalescent immunity is optimal and therefore broadly accepted as the "gold standard" against which vaccine candidates should be compared. However, successful pathogens like B. pertussis have evolved multiple mechanisms for suppressing and evading host immunity, raising the possibility that disruption of these mechanisms could result in substantially stronger or better immunity. Current acellular B. pertussis vaccines, delivered in a 5-dose regimen, induce only short-term immunity against disease and even less against colonization and transmission. Importantly, they provide modest protection against other Bordetella species that cause substantial human disease. A universal vaccine that protects against the three classical Bordetella spp. could decrease the burden of whooping cough-like disease in humans and other animals. Our recent work demonstrated that Bordetella spp. suppress host inflammatory responses and that disrupting the regulation of immunosuppressive mechanisms can allow the host to generate substantially stronger sterilizing immunity against the three classical Bordetella spp. Here, we identify immune parameters impacted by Bordetella species immunomodulation, including the generation of robust Th17 and B cell memory responses. Disrupting immunomodulation augmented the immune response, providing strong protection against the prototypes of all three classical Bordetella spp. as well as recent clinical isolates. Importantly, the protection in mice lasted for at least 15 months and was associated with recruitment of high numbers of B and T cells in the lungs as well as enhanced Th17 mucosal responses and persistently high titers of antibodies. These findings demonstrate that disrupting bacterial immunomodulatory pathways can generate much stronger and more protective immune responses to infection, with important implications for the development of better vaccines. IMPORTANCE Infectious diseases are a major cause of morbidity and mortality in the United States, accounting for over 40 million hospitalizations since 1998. Therefore, novel vaccine strategies are imperative, which can be improved with a better understanding of the mechanisms that bacteria utilize to suppress host immunity, a key mechanism for establishing colonization. Bordetella spp., the causative agents of whooping cough, suppress host immunity, which allows for persistent colonization. We discovered a regulator of a bacterial immunosuppressive pathway, which, when mutated in Bordetella spp., allows for rapid clearance of infection and subsequent generation of protective immunity for at least 15 months. After infection with the mutant strain, mice exhibited sterilizing immunity against the three classical Bordetella spp., suggesting that the immune response can be both stronger and cross-protective. This work presents a strategy for vaccine development that can be applied to other immunomodulatory pathogens.

Modeling Immune Evasion and Vaccine Limitations by Targeted Nasopharyngeal Bordetella pertussis Inoculation in Mice.

Conventional pertussis animal models deliver hundreds of thousands of Bordetella pertussis bacteria deep into the lungs, rapidly inducing severe pneumonic pathology and a robust immune response. However, human infections usually begin with colonization and growth in the upper respiratory tract. We inoculated only the nasopharynx of mice to explore the course of infection in a more natural exposure model. Nasopharyngeal colonization resulted in robust growth in the upper respiratory tract but elicited little immune response, enabling prolonged and persistent infection. Immunization with human acellular pertussis vaccine, which prevents severe lung infections in the conventional pneumonic infection model, had little effect on nasopharyngeal colonization. Our infection model revealed that B. pertussis can efficiently colonize the mouse nasopharynx, grow and spread within and between respiratory organs, evade robust host immunity, and persist for months. This experimental approach can measure aspects of the infection processes not observed in the conventional pneumonic infection model.

Enhancement of immune response against Bordetella spp. by disrupting immunomodulation.

Well-adapted pathogens must evade clearance by the host immune system and the study of how they do this has revealed myriad complex strategies and mechanisms. Classical bordetellae are very closely related subspecies that are known to modulate adaptive immunity in a variety of ways, permitting them to either persist for life or repeatedly infect the same host. Exploring the hypothesis that exposure to immune cells would cause bordetellae to induce expression of important immunomodulatory mechanisms, we identified a putative regulator of an immunomodulatory pathway. The deletion of btrS in B. bronchiseptica did not affect colonization or initial growth in the respiratory tract of mice, its natural host, but did increase activation of the inflammasome pathway, and recruitment of inflammatory cells. The mutant lacking btrS recruited many more B and T cells into the lungs, where they rapidly formed highly organized and distinctive Bronchial Associated Lymphoid Tissue (BALT) not induced by any wild type Bordetella species, and a much more rapid and strong antibody response than observed with any of these species. Immunity induced by the mutant was measurably more robust in all respiratory organs, providing completely sterilizing immunity that protected against challenge infections for many months. Moreover, the mutant induced sterilizing immunity against infection with other classical bordetellae, including B. pertussis and B. parapertussis, something the current vaccines do not provide. These findings reveal profound immunomodulation by bordetellae and demonstrate that by disrupting it much more robust protective immunity can be generated, providing a pathway to greatly improve vaccines and preventive treatments against these important pathogens.

Blood or Serum Exposure Induce Global Transcriptional Changes, Altered Antigenic Profile, and Increased Cytotoxicity by Classical Bordetellae.

The classical bordetellae sense and respond to a variety of environments outside and within their mammalian hosts. By causing inflammation and tissue damage, we reasoned that bordetellae are likely to encounter components of blood and/or serum during the course of a respiratory infection, and that detecting and responding to these would be advantageous. Therefore, we hypothesized that classical bordetellae have the ability to sense and respond to blood or serum. Blood or serum exposure resulted in substantial transcriptional changes in Bordetella bronchiseptica, including enhanced expression of many virulence-associated genes. Exposure to blood or serum additionally elicited production of multiple antigens not otherwise detectable, and led to increased bacterial cytotoxicity against macrophages. Transcriptional responses to blood/serum were observed in a Bvg- phase-locked mutant, indicating that the response is not solely dependent on a functional BvgAS system. Similar transcriptional responses to blood/serum were observed for the other classical bordetellae, Bordetella pertussis and Bordetella parapertussis. These data suggest the classical bordetellae respond to signals present in blood and serum by changing their behavior in ways that likely contribute to their remarkable success, via effects on pathogenesis, persistence and/or transmission between hosts.