lp-pla2 inhibitor Our in vitro data show that CyaA does not
Our in vitro data show that CyaA does not play a critical role in the bacterial adherence to mammalian cells. This observation is in contrast to previous studies that reported a role for CyaA in FHA-mediated adherence of B. pertussis bacteria to the respiratory epithelial lp-pla2 inhibitor [37,38]. This discrepancy might be attributed to the difference in the B. pertussis strains used in the present and previous studies, whereby inactivation and/or removal of three toxins in BPZE1 may have altered the overall adherence properties of the bacteria. Furthermore, absence of CyaA overall did not affect the survival's ability of BPZE1 bacteria within macrophages, except at a late stage during infection (48 h pi.). Although B. pertussis has been widely recognized as an extracellular pathogen, a number of studies have reported the ability of B. pertussis to survive intracellularly, and that this intracellular lifestyle is attributed to the immunomodulatory properties of several virulence factors that enable the bacterium to persist within epithelial cells and leukocytes [44,45]. In particular, and consistent with our findings, CyaA-deficient B. pertussis mutants were found to display reduced survival ability within human macrophages . In an infant mouse model of pertussis, we showed that absence of CyaA results in a decreased ability of BPZE1 bacteria to colonize murine lungs in a dose-dependent manner. This observation is consistent with previous studies carried out with non-attenuated B. pertussis strains where the adenylate cyclase and hemolytic activities harbored by the CyaA protein were found to be involved in delayed bacterial clearance in a dose-dependent manner [28,47]. The sub-optimal lung colonization profile observed with the CyaA-deficient strain may be attributed to its impaired ability to resist killing inside phagocytes and persist intracellularly. The ability to colonize the respiratory tract is crucial for the live pertussis vaccine candidate BPZE1 to trigger strong immune priming and protection against B. pertussis. Consequently the faster bacterial clearance observed with the CyaA-deficient BPAL10 strain may result in a less efficient priming of host immunity which would lead to sub-optimal protection against pertussis. Consistently, we observed a decreased ability of BPAL10 to confer protection upon pertussis challenge in a dose-dependent manner. At a vaccine dose of 5 × 105 CFU, BPAL10 conferred a protection level comparable to the parental BPZE1 strain. However, the protective efficacy of BPAL10 was greatly impaired as evidenced by reduced bacterial clearance upon BPSM challenge when the vaccine dose was lower (5 × 103 CFU). Therefore, the data support that CyaA contributes to the protective efficacy of BPZE1 against pertussis. Protection against B. pertussis depends on both humoral and cell-mediated immunity . In addition, a strong correlation between the BPZE1 vaccine dose and the antibody and IFN-γ responses was established in an earlier study with an observed vaccine dose-dependent anamnestic response upon challenge with virulent B. pertussis. BPAL10 was able to induce an antibody response against pertussis comparable to that observed in the BPZE1-immunized animals, thus suggesting that the sub-optimal colonization of BPAL10 was nevertheless sufficient to ensure efficient priming of the host humoral immune system. In addition, removal of CyaA in BPAL10 did not alter the predominant IgG2a/2b isotype profile of the immunized mice. The bias towards Th1 immune responses has been previously reported in mice primed with Pw and is important for the complete clearance upon secondary infections . Interestingly, we observed a significant impairment in the production of IFNγ from BPAL10-primed splenocytes with no significant differences in IL-4 and IL-17 production compared to BPZE1-primed splenocytes. Previous reports have proposed a role for the Hly activity of CyaA in harnessing Th17 response upon co-administration of by-stander antigen and CyaA . However, our study shows that absence of CyaA and hence Hly activity, does not alter the ability of BPZE1 to induce a Th17 response, based on Th17 production. This suggests that Hly activity from CyaA may not be a major player in the induction of BPZE1-mediated Th17 response.