Antibody dependent complement activation is critical for boosting opsonophagocytosis of Staphylococcus epidermidis in an extremely preterm human whole blood model

Abstract

Staphylococcus epidermidis is a major cause of late onset sepsis in extremely preterm neonates. Antibody therapies are considered as an interesting strategy to prevent sepsis. However, previous clinical trials with intravenous immunoglobulin (IVIG) and a monoclonal antibody (mAb) targeting staphylococcal lipoteichoic acid (LTA) (Pagibaximab) failed to show significant protection from invasive infections in extremely preterm neonates. Here we use an age-specific in vitro platform to compare immune protection by Pagibaximab with two other mAbs recognizing invasive S. epidermidis in the context of the neonatal immune system. We demonstrate poor activity of Pagibaximab in inducing complement C3b opsonization and neutrophil opsonophagocytosis in neonatal plasma. MAbs CR5133 and CR6453 [recognizing wall teichoic acid (WTA)] potently induced S. epidermidis opsonophagocytosis in an (extreme) preterm reconstituted whole blood model, especially after introduction of hexamer-enhancing mutations in the IgG-Fc tail. In conclusion, using age-specific in vitro assays, we show that mAbs with strong complement-inducing potential may be effective in preventing neonatal bacterial sepsis in extreme preterm neonates.

Fig. 1

Binding of mAbs CR6453, CR5133 and Pagibaximab to S. epidermidis clinical isolate N2279. (A) Binding curve of mAbs to S. epidermidis N2279. Bacteria were incubated with a concentration curve of mAbs or a fixed concentration of the negative control mAb. (B) Antibody binding at a fixed concentration of 3 µg/mL. (A) and (B) Binding of the bacteria by mAbs was detected using a secondary antibody, goat-anti-human Fc-A647 and analyzed using flow cytometry. B12 was used as the negative control mAb. One-way ANOVA was performed to analyze binding and displayed as ***P ≤ 0.001; NS not significant.

Fig. 2

Binding of mAbs CR5133, CR6453 and Pagibaximab to staphylococcal antigens. ELISA plates were coated with (A) purified LTA (B) purified PNAG, (C) purified PG and incubated with mAbs. Anti-LTA antibody A120 (a-LTA A120), anti-PNAG antibody F598 (a-PNAG F598) and anti-PG antibody M130 (a-PG 130) were used as a positive control, B12 was used as the negative control mAb. One-way ANOVA was performed to analyze binding, with Bonferroni correction for multiple testing. Only significant results are displayed as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001. (D) Binding of mAbs to synthetic beads coated with S. aureus WTA with either RboP backbone only or with modifications TarS (β-GlcNAc) or TarM (α-GlcNAc). Binding was detected with goat-anti-human-IgG/IgM AF647 and measured by flowcytometry. Anti-α-GlcNac IgG1 4461 and anti-β-GlcNac IgG1 4497 were used as positive controls, B12 was used as the negative control mAb. Two-way ANOVA was performed to analyze binding, with Bonferroni correction for multiple testing. Significant results are displayed with brackets to indicate the comparison and asterixes to indicate respective P values as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001. Binding of (E) CR5133 and (F) CR6453 to S. epidermidis E73 WT, ΔtarM2, ΔtarM2 pRB474-tarM2 and ΔtarM2 pRB474-EV. Binding was detected with Protein G-AF488 and measured by flow cytometry.

Fig. 3

Immature complement activity in neonatal plasma of different gestational ages compared to healthy adult plasma. (A) classical pathway activity (B) alternative pathway activity. < 32 = before 32 + 0 weeks GA, 32–36 = between 32 + 0 and 36 + 6 weeks GA, > 37 = after 37 + 0 weeks GA. Data represent the mean ± SD of six different neonatal plasma samples per age group, performed in two separate experiments. Adult plasma consists of a serum pool of 27 healthy adults. GA = gestational age in weeks; OD405 = optical density at 405 nm.

Fig. 4

C3b deposition on S. epidermidis is improved by mAbs in extreme preterm neonatal plasma. The effect of (A) CR6453 and (B) CR5133, and Pagibaximab on C3b deposition on S. epidermidis strain N2279 in extreme preterm plasma. Staphylococcus epidermidis N2279 bacteria were FITC labeled and incubated with 1% neonatal plasma supplemented with mAbs as indicated. The dotted line represents bacteria incubated with 1% pooled healthy adult plasma without any supplemented mAbs. To the neonatal plasma, a concentration range of mAbs or a fixed concentration (8 µg/mL) of the negative control mAb (B12) was added. Flow cytometry was used to detect C3b deposition using an anti-C3b-AF647 antibody conjugate. Data were plotted as AF647 GeoMFI of the FITC-positive (FITC + ve) bacterial population. Data represent mean ± SD of 5 different plasma samples. Gestational age ranges from 27 + 0 to 31 + 5 weeks GA. GA = weeks gestational age.

Fig. 5

Hexamer-enhancing mutation E345K improves C3b deposition by CR5133 and CR6453 on S. epidermidis in extreme preterm neonatal plasma. The effect of (A) CR6453 and CR5133 WT versus E345K variant, and (B) E345K variants of CR5133 and CR6453 vs Pagibaximab on C3b deposition on S. epidermidis strain N2279 in extreme preterm plasma. Staphylococcus epidermidis N2279 bacteria were FITC labeled and incubated with 1% neonatal or 1% pooled healthy adult plasma (dotted line). To the pooled adult plasma, no mAbs were added. To the neonatal plasma, a concentration range of mAbs or a fixed concentration of the negative control mAb (B12) (dotted line) was added. Flow cytometry was used to detect C3b deposition using an anti-C3b-AF647 antibody conjugate. Data were plotted as AF647 GeoMFI of the FITC + ve bacterial population. Data represent mean ± SD of 5 different plasma samples. Gestational age ranges from 27 + 0 to 31 + 5 weeks GA. GA = weeks gestational age.

Fig. 6

mAbs CR5133(-E345K) and CR6453(-E345K) improve phagocytosis of S. epidermidis in preterm neonatal reconstituted whole blood. Phagocytosis of FITC labelled S. epidermidis strain N2279 in neonatal reconstituted whole blood in the presence of a concentration range of mAbs CR5133, CR6453 (both as WT-IgG1 or E345K-IgG1), Pagibaximab or a fixed concentration of the negative control mAb (B12) of (A) 31 + 2 (B) 32 + 6 weeks GA. Whole blood was reconstituted with 2.5% neonatal plasma for the antibody titration or 2.5% pooled healthy adult plasma as a control (dotted line). To the pooled healthy adult plasma, no mAbs were added. Data were plotted as FITC GeoMFI of the PMN population. (C) Representative images of internalization of FITC-labeled bacteria (green) by PMNs in reconstituted whole blood after addition of CR6453-E345K (1 µg/mL) visualized by confocal microscopy. The membrane of PMNs was stained with WGA-AF647 (red). GA = gestational age.