Fig. 6: Combining capsule targeting antibodies improves complement activation on KpnO2.

a, b Antibody-dependent C3b deposition. KpnO2 was pre-incubated with antibodies for 30 min at 4 °C. After antibody binding, bacteria were incubated in 3% NHS as a complement source for 30 min at 37 °C. C3b-deposition was detected using anti-hu-C3b-AF647 by flow cytometry. c Phagocytosis of KpnO2-GFP by human neutrophils. KpnO2-GFP was incubated with antibodies in the presence of 3% KpnO2∆NHS. Percentage of neutrophils that phagocytosed was determined by flow cytometry. d Antibody-mediated neutrophil killing was analyzed by incubating neutrophils with KpnO2 at MOI of 0.1 together with 8% KpnO2∆NHS and 0.11 µg/ml antibodies for 2 h at 37 °C. CFU/ml was calculated the next day and expressed as relative survival by dividing with the no antibody control. KpnO2 was pre-incubated with (e) UKpn2 or (f) the combination of UKpn1 and UKpn3 in the presence or absence of 10 µg/ml SpA-B for 30 min at 4 °C. After antibody binding, 3% NHS as complement source with or without SpA-B was added for 30 min at 37 °C. C3b deposition was measured using anti-hu-C3b-A647 by flow cytometry. a–d, f In the conditions where antibodies were combined, both antibodies were added in the same concentration. The total antibody concentration in the assay is the same for single or combination of antibodies. a, b, e, f Flow cytometry data are represented as gMFI values of bacterial populations. a–c, e, f Data represent mean ± SD of three independent experiments. d Data represents mean ± SD of four independent experiments and were analyzed by one-way ANOVA with Dunnett multiple comparison analysis compared to without mAb control; significant differences are indicated, * p < 0.05.