Ornithodoros moubata complement inhibitor is an equally effective C5 inhibitor in pigs and humans

Abstract

Experimental evidence suggests that C inhibition and more particularly combined inhibition of C and the TLR coreceptor CD14 may be of therapeutic benefit in sepsis and other inflammatory conditions. A barrier to the testing and further development of many inhibitors is that their activity is species specific. Pig is a relevant species for experimental models of human disease, and this study undertakes a comprehensive comparison of the inhibitory efficacy of the C5 inhibitor Ornithodoros moubata C inhibitor (OmCI) in human and porcine whole blood ex vivo models of Escherichia coli-induced sepsis. The effect of OmCI on complement activity in pigs undergoing E. coli sepsis was also examined. Porcine and human serum, and whole blood anticoagulated with lepirudin, was incubated with E. coli and the effect of OmCI investigated. The ex vivo results were virtually identical in pig and human. OmCI completely ablated the activity of all three C pathways at 0.64 μM. E. coli-induced C activation and expression of CD11b (wCD11R3 in the pig), was abolished ex vivo at 0.32 μM OmCI. Combining anti-CD14 and OmCI reduced the formation of IL-8 and TNF-α more potently than the single inhibitors. OmCI also efficiently bound E. coli-induced leukotriene B(4) in pig and human plasma. In support of our ex vivo findings, in vivo the activity of all C pathways was inhibited at 0.6 mg OmCI/kg pig. In conclusion, OmCI efficiently inhibited pig and human C activation, has accompanying anti-inflammatory effects and is a promising candidate inhibitor for further in vivo studies of sepsis.

Figure 1. Effect of OmCI on complement functional activity

The effect of OmCI on the functional activity of the classical, lectin and alternative pathways of C were examined in serum from humans and pigs using the Wielisa assay. The data are expressed as % of a standard defined as 100% activity and are presented as mean ± SEM (n = 3).

Figure 2. Effect of OmCI on complement induced formation of C5a and TCC in vitro

Left panel: Human whole blood preincubated with OmCI or equimolar amounts of albumin was incubated with 10⁸ E. coli/ml for 30 minutes. Data are expressed as ng/ml and presented as mean ± SEM (n = 3). Middle panels: Human and pig whole blood preincubated with OmCI or equimolar amounts of albumin was incubated with 10⁸ E. coli/ml for 30 minutes at 37 °C. Data are expressed as AU/ml and presented as mean ± SEM (n = 3 for human and n = 4 for pig). Right panels: Human and pig serum preincubated with 0.64 μM OmCI or albumin was activated with heat aggregated IgG (HAIGG) or zymosan, both at a final concentration of 1 mg/ml. In all experiments, zymosan induced TCC formation in pig whole blood was higher than the upper standard so these are set to 1.0. Data are expressed as TCC ratio, where 1.0 is defined as the amount of TCC formation induced by HAIGG and zymosan, respectively. The data are presented as mean ± SD (n = 5 for human and n = 3 for pig).

Figure 3. Effect of OmCI on E. coli-induced expression of CD11b and wCD11R3

Human and pig whole blood was preincubated with OmCI or equimolar amounts of albumin and then incubated with 10⁸ E. coli/ml for 10 minutes at 37 °C. Expression of human CD11b and pig wCD11R3 were measured using flow cytometry. Median fluorescence intensity (MFI) is expressed as mean ± SD (n = 3 for humans and n = 4 for pigs). To the right, histograms and contour plots of the effect of 1.28 μM OmCI for pig and human.

Figure 4. Effect of inhibiting complement, CD14, and a combination thereof, on E. coli-induced cytokine release

Upper panels: Pig whole blood was preincubated with 0.64 μM OmCI (O), 25 μg/ml anti-CD14 (whole IgG) (α), and the combination of both and then incubated with 10⁶ E. coli/ml for 2 h (TNF-α and IL-8) and/or 10⁵ bacteria/ml for 4 h (IL-1β and IL-8). The effect of anti-CD14 on pig IL-8 could not be measured because of assay intereference. Albumin and an isotype-matched control Ab (IgG2b) were used as controls (Ctr) when analyzing TNF-α and IL-1β, whereas albumin only was used as control when analyzing IL-8. For IL-8, 2 and 4 hour sample data were pooled. Data are normalized to the E. coli group which is defined as 100%. Data are presented as mean ± SEM (n = 3, 4 and 5 for TNF-α, IL-1β and IL-8, respectively). Lower panels: Human whole blood was preincubated with 0.64 μM OmCI, 10 μg/mL anti-CD14 (F(ab′)₂), combinations thereof, and controls for 5 minutes and then incubated for 2 h with 10⁶ E. coli/ml at 37 °C. As control (Ctr) albumin was used in combination with a control F(ab′)₂, both in equimolar amounts to OmCI and anti-CD14, respectively. Data are normalized to the E. coli group which is defined as 100% and presented as mean ± SEM (n=3). Statistical comparisons were performed between the effect of OmCI, anti-CD14 and the combinations of both versus no inhibition, *p < 0.05.

Figure 5. Complement-dependent LTB4 release and binding of LTB4 to OmCI

Left panels: Pig and human whole blood was preincubated with 0.64 μM OmCI or albumin and human whole blood was also preincubated with the human specific C inhibitors compstatin (25 μM) and eculizumab (0.67 μM), thereafter incubated with 10⁶ E. coli/ml for 120 minutes at 37 °C. Data are presented as mean ± SEM (n = 3 for pig and n = 7 for human). Statistics were performed on the effect of the C inhibitors versus the effect of E. coli only, *p < 0.05. Right panels: LTB4-enriched plasma was generated by incubating pig and human whole blood with 10⁶ E. coli/ml for 120 minutes at 37 °C. Thereafter OmCI was added to the activated plasma and incubated for 15 minutes at 37 °C. A control peptide or eculizumab were used as controls for pig and human respectively. Samples were then analysed for LTB4. Data are normalized to LTB4 in E. coli activated plasma (defined as 100%). Data are presented as mean ± SEM for human (n = 4), and as mean for pig (n = 2).

Figure 6. In vivo effects of OmCI in pigs

Different amounts of OmCI were tested in a pig model of E. coli-induced sepsis. Five 15 kg pigs received the following boluses of OmCI: 22.5 mg (n = 1), 15 mg (n = 1), 7.5 mg (n = 2) or 3.75 mg (n = 1) prior to the induction of sepsis, and thereafter a continuous infusion of 0.5 mg OmCI/h. Two animals served as positive controls, receiving E. coli and saline (9 mg/ml). Blood samples were drawn at baseline (Tbasis), and then at 0 (T0), 30, 60, 120, 180 and 240 minutes. The effect of OmCI on the functional activity of the three C pathways is shown using the Wielisa assay. The data are expressed in % of a standard defined as 100% activity. Data points are given as single values or as mean with range for 7.5 mg dose and the E. coli positive control.