Anti-FIM and Anti-FHA Antibodies Inhibit Bordetella pertussis Growth and Reduce Epithelial Cell Inflammation Through Bacterial Aggregation

Abstract

The pertussis vaccination is highly recommended for infants, children, and pregnant women. Despite a high coverage of vaccination, pertussis continues to be of public health concern as a re-emerging infectious disease. The mechanism by which vaccine-elicited anti-pertussis antibodies mediate direct bactericidal effects is poorly understood. In this study, we showed that the interaction of B. pertussis with A549 epithelial cells induce release of biological factors which enhance bacteria growth. Complement-depleted antisera from vaccine-immunized guinea pigs or monoclonal antibodies targeting FHA and FIM mediate bacteria aggregation and elicit bactericidal effects. Our in vitro results indicated that aggregation of bacteria through anti-FIM and anti-FHA specific antibodies is one of the major biological mechanisms to clear bacterial infections and restore epithelial cell survival in vitro. Our data also indicates that the anti-pertussis antibodies reduce secretion of proinflammatory chemokines and cytokines by preventing interaction of B. pertussis with host cells. The results of this study not only demonstrate mechanism of action of anti-FIM and anti-FHA antibodies, but also opens translational applications for potential therapeutic approaches or development of analytical assays such as in vitro potency assays.

Keywords: aggregation; anti-pertussis antibodies; bacterial growth inhibition; cytokines; epithelial inflammation.

Figure 1

Bordetella pertussis interactions with A549 epithelial cells are essential for bacterial growth which is inhibited by specific mAbs. (A) B. pertussis growth significantly increased in a dose dependent manner in presence of 1 and 0.5 million of A549 cells. After 72 h co-culture of B. pertussis with A549 cells, extracellular bacterial concentration in the medium was determined. (B, C) Enumeration of live bacteria on Bordet-Gengou blood agar showed significant inhibition of B. pertussis growth by anti-sera (B) compared to naïve control sera. Mouse mAb targeting FIM (G10, 1-7, and 1-10) and FHA (28-1, 1-11, and 1-9) significantly inhibited bacterial growth compared to anti-RSV control mAb (C). Unpaired Student’s t-test. Mean ± SEM. *p < 0.05, and **p < 0.01, n ≥ 4 experiments. ns, non significant.

Figure 2

Antibodies against B. pertussis key adhesins induced bacterial aggregation and inhibit adhesion. (A) Naïve control sera did not protect infected A549 cells which exhibited cluster formation and cell death (blue arrows) compared to confluent resting cells. Naïve sera did not induce bacteria aggregation (A1). Antisera containing antibodies against aP vaccine or single immunogens, FHA, and FIM, induced bacterial aggregation (red arrows) in 100% of tested sera as certain anti-FHA and anti-FIM mAbs induced bacterial aggregation (A2). (B) Specific anti-pertussis antibodies inhibit bacterial adhesion. After 48 h of co-culture, anti-FIM and anti-FHA mAb significantly inhibited bacteria adhesion compared to control anti-PRN mAb (white arrow) to A549 cells which was confirmed using bacteria enumeration on Bordet-Gengou plate (C). Mean ± SEM. ***p < 0.001 and **p < 0.01, n ≥ 3 experiments.

Figure 3

B. pertussis sequestration by aggregation inducing antibodies mediated direct bactericidal effects. B. pertussis was incubated for 48 h with anti-pertussis antibodies without A549 cells to test the direct bactericidal effect of anti-FIM and anti-FHA antibodies. Flow cytometry-based analysis of bacteria death shows that anti-pertussis antibodies against FIM and FHA exhibit direct bactericidal effect (A). Furthermore, analysis by imaging flow cytometry showed that majority of dead bacteria are within the aggregates (B). Unpaired Student’s t-test. Mean ± SEM. **p < 0.01 and ***p < 0.001, n ≥ 3 experiments.

Figure 4

Bacteria aggregation and inhibition of adhesion improve A549 cell survival. After 72 h of infection, remaining adherent A549 cells were stained with Neutral red for cell viability/proliferation. Anti-FIM and anti-FHA anti-sera, which induced B. pertussis aggregation have a beneficial impact on A549 cell survival (A). This observation was reproducible by certain specific anti-FIM and anti-FHA mAbs (B). Unpaired Student’s t-test. Mean ± SEM. *p < 0.05 and **p < 0.01. n ≥ 3 experiments.

Figure 5

Antibodies mediating B. pertussis aggregation reduced A549 pro-inflammatory signaling. (A) Anti-FHA and anti-FIM polyclonal sera reduced NF-κB activation and A549 cell inflammatory response. Cytoplasmic NF-κB levels remains higher in presence of anti-FIM and anti-FHA sera compared to control sera. (B) Complementary analysis of pro-inflammatory cytokines and chemokines released in the culture supernatant in response to B. pertussis infection were significantly reduced by anti-FIM and anti-FHA compared to naive sera. Unpaired Student’s t-test. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 n ≥ 3 experiments.