A highly attenuated B pertussis strain named BPZE

A highly attenuated B. pertussis strain, named BPZE1, has recently been described [10]. It produces enzymatically inactive pertussis toxin (PT), no dermonecrotic toxin and only trace amounts of tracheal cytotoxin. Markedly reduced lung pathology was observed in mice intranasally (i.n.) infected with BPZE1, compared to the virulent parent strain, while maintaining the ability to colonize and induce a strong protective immunity. In infant mice a single i.n. administration of BPZE1 induced stronger and longer-lasting protection than two administrations of current acellular pertussis vaccines [10,11]. In addition, the high genetic stability in vitro and in vivo[12], and a good safety profile in immunocompromised animals [13] have allowed BPZE1 to undergo the first-in-man clinical trial in adult healthy volunteers (http://www.child-innovac.org). BPZE1-induced protective immune responses were found to involve both BIIE 0246 and CD4+ T cells. Further analyses into the protective CD4+ T cells revealed IFN-γ-producing and IL-17-producing subsets, indicating that BPZE1 induces both Th1 and Th17 CD4+ T cells in mice [14]. To further study the mechanisms involved in BPZE1-mediated protection and in particular the contribution of some virulence factors in the protective efficacy, we investigated the role of adenylate cyclase (CyaA) which is produced at wild type levels in BPZE1. CyaA is a dual activity toxin composed of two functional domains; the N-terminal cell invasive adenylyl cyclase enzyme, which elevates cAMP levels in phagocytes, and the C-terminal pore-forming ‘hemolysin-toxin’ activity (Hly) [15]. In vivo studies have suggested that CyaA exerts immunosuppressive effects on both the innate and adaptive immune system, specifically targeting CD11b/CD18 expressing-myeloid cells such as macrophages, neutrophils and dendritic cells [16–18]. The CyaA Hly domain mediates the binding to CD11b/CD18 host receptor and internalization of the enzymatic moiety into the host cells [19]. Upon entry into the cell, the AC enzymatic moiety is activated by host calmodulin and subverts cellular signaling by unregulated conversion of ATP to cAMP. Intracellular cAMP accumulation yields to suppression of bactericidal activities of phagocytes. In particular, it was shown that CyaA primarily and very sensitively blocks complement-mediated opsonophagocytic killing of bacteria by neutrophils [20]. Other functions such as chemotaxis, FcR-mediated phagocytosis and superoxide production were also found impaired by CyaA [20–23] which eventually leads to apoptosis [24]. The role of the Hly activity of CyaA in Bordetella virulence remains unclear, but its pore-forming activity may potentially be important in harnessing the pro-inflammatory response during bacterial colonization, yielding IL-1 induction and promoting Th17 response that appears required for bacterial clearance [25]. CyaA was previously shown to be involved in bacterial colonization of the airways and displays important immunomodulatory properties [26,27]. Infection with CyaA deficient B. pertussis strains results in reduced pathology and inability to cause lethality in infant mice [16,28–30]. On the other hand, CyaA has been reported to play a protective role against pertussis [25,31] and also displays adjuvant properties [32]. In this study, we constructed a CyaA-deficient BPZE1 mutant and analyzed its lung colonization profile, immunogenicity, and protective efficacy in an infant mouse model of B. pertussis infection.
Materials and methods
Results
Discussion Despite broad vaccination coverage, pertussis remains an important respiratory infectious disease and the least-controlled vaccine-preventable infectious disease in children. The resurgence has been attributed at least partly to the waning of vaccine-induced protective immunity over the time [43]. Therefore, development of a new generation of pertussis vaccines conferring strong immune responses and long-lasting protection are highly desirable and might exert a profound impact on public health. Recently, a live, highly attenuated pertussis vaccine candidate, named BPZE1, developed to be administered early in life in order to protect young children against pertussis, has shown great promise in pre-clinical models [10,12,13] and has recently completed phase I clinical trial in healthy human adult volunteers. While the BPZE1-induced protective immune responses have been reported [14], contribution of the virulence factors in the BPZE1-mediated protection remains to be investigated. Hence, we studied the contribution of adenylate cyclase (CyaA) in the protection afforded by BPZE1 vaccination. To do so, we constructed BPAL10, a CyaA-deficient BPZE1 derivative, and compared its protective efficacy to its parental counterpart BPZE1 in a 3 week-old mouse model.