Long Lasting Antibodies From Convalescent Pertussis Patients Induce ROS Production and Bacterial Killing by Human Neutrophils

Abstract

Pertussis is a respiratory infection caused by the Gram-negative bacterium Bordetella pertussis. Despite high vaccination coverage this disease remains a public health concern worldwide. A better understanding of the protective immune responses to B. pertussis is required for the development of improved vaccines. The aim of this study was to determine the production of reactive oxygen species (ROS) by human neutrophils in response to B. pertussis and to determine the contribution of opsonizing antibodies from convalescent pertussis patients in this response. The serum samples from convalescent patients were taken at <3, 9, 18 and 36 months after diagnosis of pertussis. Also included were sera from healthy age-matched controls. We show that neutrophils produced high levels of ROS in response to opsonized, compared to non-opsonized, B. pertussis and that this effect was independent of the time the convalescent serum samples were taken. This indicates the presence of functional opsonizing antibodies up to 3 years after B. pertussis infection. While opsonization of B. pertussis with serum samples from uninfected controls also induced ROS production, sera from infected individuals induced significantly higher ROS levels. Spearman correlations analysis showed that IgG antibodies targeting fimbriae3 followed by pertactin, and BrkA correlate with ROS production. Additionally, we observed that neutrophils killed opsonized B. pertussis in a ROS-dependent manner. Searching for other antigen-specific antibodies from convalescent pertussis patients involved in ROS production by neutrophils may assist in the identification of novel antigens to improve the current pertussis vaccines.

Keywords: Bordetella pertussis; Reactive Oxygen Species (ROS); antibody; neutrophil; opsonization.

Figure 1

Schematic representation of the study population. Shown are the number (n) of serum samples in the convalescent pertussis ex-patients group (green) which were sampled in a longitudinal fashion at <3, 9, 18 and 36 months after diagnosis as well as that of the age matched control group (purple). Age median and range in years as well as male/female ratio are shown per sample group.

Figure 2

Neutrophil ROS production induced by opsonized B. pertussis. (A) ROS production was measured in real-time for 30 minutes after incubation of neutrophils in the presence (squares) or absence (circles) of (opsonized) B. pertussis at MOI 10. Data is represented as individual AUC values with geometric mean. # indicate significance to “No serum, No B. pertussis” condition (blue circle). Ctrl (purple) indicates serum derived from healthy control individuals (n=21) and BP (green) indicate serum derived from convalescent pertussis patients (n=18 for timepoint <3 months, n=20 for remaining timepoints). *p <0.05, ^##p < 0.01, ^###p < 0.001, ****p < 0.0001. (B) Spearman correlations plots were performed between neutrophil ROS production at all time points and the levels of nine different antigen-specific IgG antibody levels in plasma of the same convalescent pertussis patients. The ROS production was measured after incubating neutrophils with Bp-serum only (ROS) or with B. pertussis opsonized with BP-serum (ROS + Bp). Circles indicate a correlation with adjusted p < 0.05 and circle colors and size indicate the correlation r_s value.

Figure 3

Neutrophil killing of opsonized B. pertussis and the role of ROS in this process. Live B. pertussis (MOI 10) were opsonized with serum samples from healthy controls (Ctrl-serum) shown in purple or from convalescent pertussis ex-patients (BP-serum) (<3 month after diagnosis) in green. The opsonized bacteria were incubated in the presence or absence of neutrophils and/or NOX inhibitor for 4 hours after which neutrophils were lysed and total CFU were determined by plating on BG plates. Bacterial growth is represented as % CFU which was calculated relative to CFU from non-opsonized bacteria incubated with neutrophils (dotted line). § p = 0.075 + ES = 0.605 indicating significant differences. The data represented are of 2 independent experiments (each represented by symbol shapes).