Fig 1.
Schematic representation of the time periods in which the strains used in this study were grouped, based on their year of isolation.
The periods included strains isolated before or around the time the WCVs were introduced (Introd-Vac-strains, 1949–1960) and after the introduction of the WCVs (WCV-strains, 1970–2000) or ACVs (ACV-strains, 2005–2014). The three groups are indicated in green, blue and pink respectively. The WCV was introduced in the Dutch National Immunisation programme (NIP) in 1957. In 2001, the ACVs were included in the NIP as a four year old (4yo) booster. The WCVs were fully replaced by the ACVs in 2005 (black arrows).
Table 1.
B. pertussis strains used in this study isolated from Dutch patients between 1949 and 2014.
Fig 2.
Activation of human TLR2, TLR4, NOD2 and NOD1 by B. pertussis strains isolated from patients from 1949 to 2014.
HEK-Blue cell lines either expressing TLR2, TLR4, NOD2 or NOD1 were stimulated with 92 different B. pertussis strains (MOI 40) isolated from pertussis patients either around the time the WCVs were introduced (Introd-Vac-strains) or when the WCVs (WCV-strains) or ACVs (ACV-strains) were used. PRR activation measured by SEAP activity of (A) HEK-Blue-TLR2 cells, (B) HEK-Blue-TLR4 cells, (C) HEK-Blue-NOD2 cells or (D) HEK-Blue-NOD1 cells by Introd-Vac-strains (1949–1960), WCV-strains (1970–2000) and ACV-strains (2005–2014). SEAP activity induced by these same strains grouped according to their ptxP alleles, ptxP1 (P1), ptxP2 (P2) or ptxP3 (P3), upon stimulation of (E) HEK-Blue-TLR2, (F) HEK-Blue-TLR4 cells or (G) HEK-Blue-NOD2 cells. SEAP activity of cells in medium only or with the respective ligands, Pam3CSK4 (100ng/ml), LPS-EK (10ng/ml), MDP (200ng/ml) or iE-DAP (100ng/ml) are included for each HEK-Blue cell line. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
Fig 3.
Cytokine production by moDCs stimulated with B. pertussis strains isolated from patients from 1949 to 2014.
MoDCs were stimulated with 29 different strains either isolated around the time the WCVs were introduced (Introd-Vac-strains) or when the WCVs (WCV-strains) or ACVs (ACV-strains) were used at an MOI of 10. MoDC production of (A) IL-10, (B) G-CSF, (C) TNF-α and (D) IL-8 production by Introd-Vac-strains (1949–1960), WCV-strains (1970–2000) and ACV-strains (2005–2014). Medium stimulation values for IL-8, TNF-α, G-CSF and IL-10 are depicted in the appropriate graphs * p ≤ 0. 05, ** p ≤ 0.01, **** p ≤ 0.0001.
Fig 4.
Surface marker expression of moDCs stimulated with Introd-Vac and ACV- B. pertussis strains isolated from pertussis patients.
MoDCs were stimulated with 12 different B. pertussis strains (MOI 10) isolated from patients at around the time the WCVs were introduced (Introd-Vac-strains) or when the ACVs (ACV-strains) were used, either in the presence or absence of 1 μg/ml anti-TLR2 blocking antibody. MoDC surface expression of (A) CD80, (B) CD86, (C) PD-L1/CD274 and (D) CD101 by Introd-Vac-strains (1949–1960) and ACV-strains (2005–2014) alone. (E) MoDC surface expression of PD-L1/CD274 by Introd-Vac-strains (1949–1960) and ACV-strains (2005–2014) in the presence or absence of 1 μg/ml anti-TLR2 blocking antibody. Medium stimulation values for CD80, CD86, PD-L1/CD274 and CD101 are depicted in the corresponding graphs * p ≤ 0. 05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Fig 5.
Cytokine production by moDCs stimulated with B. pertussis strains isolated from patients from 1949 to 2014 grouped according to ptxP allele.
moDCs were stimulated with 29 different strains either expressing the ptxP2 (P2), ptxP1 (P1) or ptxP3 (P3) allele, isolated around the time the WCVs were introduced (Introd-Vac-strains) or when the WCVs (WCV-strains) or ACVs (ACV-strains) were used at an MOI of 10. MoDC production of (A) IL-10, (B) G-CSF, (C) TNF-α and (D) IL-8 production by P2, P1 and P3 strains. The production of (E) IL-10, (F) G-CSF, (G) TNF-α and (H) IL-8 upon stimulation of MoDC by P3 strains isolated from 1995–2000 or P3 strains isolated from 2005–2014 was also investigated. Medium stimulation values for IL-8, TNF-α, G-CSF and IL-10 are depicted in the appropriate graphs * p ≤0. 05, ns = non-significant.
Fig 6.
Cytokine production by moDCs stimulated with recently circulating Prn deficient or Prn expressing B. pertussis strains isolated from patients.
MoDC production of (A) IL-10, (B) G-CSF, (C) TNF-α and (D) IL-8 production upon stimulation with P3 strains either expressing (Prn+) or not expressing Prn (Prn-). Medium stimulation values for IL-8, TNF-α, G-CSF and IL-10 are depicted in the corresponding graphs. ns = non-significant.