Eculizumab in patients with severe coronavirus disease 2019 (COVID-19) requiring continuous positive airway pressure ventilator support: Retrospective cohort study.

BACKGROUND: Complement activation contributes to lung dysfunction in coronavirus disease 2019 (COVID-19). We assessed whether C5 blockade with eculizumab could improve disease outcome.
METHODS: In this single-centre, academic, unblinded study two 900 mg eculizumab doses were added-on standard therapy in ten COVID-19 patients admitted from February 2020 to April 2020 and receiving Continuous-Positive-Airway-Pressure (CPAP) ventilator support from ≤24 hours. We compared their outcomes with those of 65 contemporary similar controls. Primary outcome was respiratory rate at one week of ventilator support. Secondary outcomes included the combined endpoint of mortality and discharge with chronic complications.
RESULTS: Baseline characteristics of eculizumab-treated patients and controls were similar. At baseline, sC5b-9 levels, ex vivo C5b-9 and thrombi deposition were increased. Ex vivo tests normalised in eculizumab-treated patients, but not in controls. In eculizumab-treated patients respiratory rate decreased from 26.8±7.3 breaths/min at baseline to 20.3±3.8 and 18.0±4.8 breaths/min at one and two weeks, respectively (p<0.05 for both), but did not change in controls. Between-group changes differed significantly at both time-points (p<0.01). Changes in respiratory rate correlated with concomitant changes in ex vivo C5b-9 deposits at one (rs = 0.706, p = 0.010) and two (rs = 0.751, p = 0.032) weeks. Over a median (IQR) period of 47.0 (14.0-121.0) days, four eculizumab-treated patients died or had chronic complications versus 52 controls [HRCrude (95% CI): 0.26 (0.09-0.72), p = 0.010]. Between-group difference was significant even after adjustment for age, sex and baseline serum creatinine [HRAdjusted (95% CI): 0.30 (0.10-0.84), p = 0.023]. Six patients and 13 controls were discharged without complications [HRCrude (95% CI): 2.88 (1.08-7.70), p = 0.035]. Eculizumab was tolerated well. The main study limitations were the relatively small sample size and the non-randomised design.
CONCLUSIONS: In patients with severe COVID-19, eculizumab safely improved respiratory dysfunction and decreased the combined endpoint of mortality and discharge with chronic complications. Findings need confirmation in randomised controlled trials.

Introduction

As observed in other experimental and human forms of coronavirus lung infection [1,2], dramatic respiratory dysfunction [3] and dismal outcomes [4] of Severe-Acute-Respiratory-Syndrome Coronavirus-2 (SARS-CoV-2) 2019, or COVID-19 [3,5], are largely mediated by overwhelming release of pro-inflammatory cytokines (cytokine storm) [6] and uncontrolled complement activation [7,8]. Endothelial injury and microangiopathic lesions similar to those observed in the hemolytic uremic syndrome (HUS) [9,10] and deposits of C5b-9 in lung and skin vessels [10], as well as in glomeruli and tubuli [11] of patients dying of COVID-19 confirm that complement activation, in particular of the terminal pathway, may have a key pathogenic role in COVID-19 [12].

Eculizumab is a humanised anti-C5 monoclonal antibody approved for the treatment of paroxysmal nocturnal hemoglobinuria and atypical HUS [13,14]. FDA approved a program of eculizumab off-label compassionate use for the treatment of non-intubated patients with COVID-19. Initial case series and explorative studies showed encouraging effects of C5 blockade in patients with COVID-19 [15-17], even in combination with the JAK1/2 inhibitor ruxolitinib [16]. Based on this background we planned to add two 900 mg eculizumab doses on best supportive therapy in ten patients with COVID-19 who required Continuous-Positive-Airway-Pressure (CPAP) ventilator support and compared their outcomes with outcomes of similar contemporary controls who received the same supportive therapy, but no eculizumab.

Materials and methods

Study population

We included adults admitted at the Azienda-Socio-Sanitaria-Territoriale (ASST) Papa Giovanni XXIII in Bergamo (Italy) because of severe COVID-19 who were receiving CPAP ventilator support from 24 hours or less. Diagnosis was based on WHO Interim guidance criteria [18], and confirmed by detection at admission of SARS-CoV-2 genome from nasal swabs and respiratory samples by using two different molecular methods (GeneFinder COVID-19-Elitech Group, Allplex™ 2019-nCoV Assay—Seegene Inc) according to the manufacturer’s instructions and WHO protocol (Supplementary Methods in S1 Appendix) [18].

Ten consenting participants received compassionate eculizumab treatment along with anti-Neisseria Meningitis and Pneumococcus antibiotic coverage. The drug was freely supplied by the manufacturer (Alexion Pharma Italy S.R.L., Milan). No participant received compensation. Then, when all patients had completed eculizumab therapy, outcomes of eculizumab-treated patients and contemporary controls were compared retrospectively. The prospective compassionate treatment protocol and the retrospective controlled study were both approved by the Ethical Committee of Bergamo. The Ethical Committee that approved our retrospective observational study stated that the informed consent to participate in the study and to use medical records for research purposes had to be collected whenever the patient could be contacted. Thus, we collected the written informed consent from those eculizumab-treated patients and controls who were still alive at the time the study was conducted.

Eculizumab compassionate treatment

Nine-hundreds mg of eculizumab were intravenously infused within 24 hours of CPAP ventilator support and seven to ten days thereafter (this flexibility was justified by the emergency context). The number of patients given eculizumab therapy depended on drug availability, while the selection of eculizumab recipients was at the discretion of the treating physician and influenced by logistic reasons. Indeed, to avoid overlaps of drug administrations and specific laboratory tests to monitor treatment effects in different patients, and prevent possible interference with clinical patient management, we administered eculizumab to one patient every six to seven consecutive potential candidates (Supplementary Methods in S1 Appendix). In no case patients eligible to receive eculizumab objected to the use of their medical data for research purposes. Nonetheless, patients would still have been eligible to receive eculizumab if they had objected to the use of their data in medical research. No systematic change in supportive treatment was introduced. Patients involved in ongoing clinical trials were excluded.

Follow-up

All participants were followed up to death or hospital discharge. History, vital signs, clinical, laboratory and safety parameters and adverse events were recorded in patients’ medical records.

Blood samples to evaluate a marker of complement activation in plasma (sC5b-9) and ex vivo serum-induced complement deposition and thrombus formation on ADP-activated human microvascular endothelial cells (HMEC-1) of dermal origin [19-21] were collected from all eculizumab-treated patients (before eculizumab administration) and four controls (“Biochemical Controls”) within 24 hours from CPAP initiation (baseline) along with samples for genetic analyses. In vivo and ex vivo tests were repeated 1–4 days and 7–16 days after baseline, respectively, and 30–60 days after discharge (recovery visit). Because of resource restrictions, ex vivo thrombi deposition at 7–16 days was not evaluated. Complement and genetic analyses were freely performed at the Laboratories of the Istituto di Ricerche Farmacologiche Mario Negri IRCCS in Bergamo (Italy). Other laboratory parameters were evaluated at the hospital clinical laboratories. All data were recorded in the same dedicated database (further details in Study Protocol in S2 Appendix).

Complement evaluation and genotyping

SC5b-9 levels were evaluated in plasma EDTA by MicroVue SC5b-9 Plus EIA (SC5b-9 Plus; Quidel). Ex-vivo serum-induced C5b-9 deposits and thrombus formation on HMEC-1 lines were evaluated as described previously, with minor modifications (Supplementary Methods in S1 Appendix and S1 Fig) [19-21]. CFH, MCP, CFI, CFB, C3, THBD coding sequences were screened by amplicon-based next generation sequencing and H3 CFH haplotype and CFH/CFHR genomic abnormalities were evaluated as previously described (Supplementary Methods in S1 Appendix) [22].

Sample size and statistical analyses

We calculated that with the administration of two 900 mg doses per patient, drug supply would have been sufficient to treat ten patients. Considering that eculizumab would have been administered to one patient every six to seven potential candidates we predicted that at inclusion of ten eculizumab-treated patients we would have identified a total of 60 to 70 potential controls. Among potential controls we selected those with the same age-range of eculizumab-treated patients.

Due to the retrospective and observational nature of the study the sample size was not calculated. Primary outcome was the absolute change in respiratory rate at week one versus baseline. Secondary short-term outcomes included changes in respiratory rate at two weeks and changes in arterial partial pressure of oxygen (PaO2) and carbon dioxide (PaCO2), PaO2 to fractional inspired oxygen (FiO2) ratio (PaO2/FiO2); heart rate, systolic, diastolic and mean blood pressure; C-Reactive Protein (CRP), D-dimer test, serum creatinine, blood cell count at one and two weeks after baseline and concomitant changes in complement parameters in a subgroup. Complement parameters were evaluated also at recovery visits.

The combined endpoint of in-hospital death or discharge with chronic severe complications was considered as the most clinically relevant long-term outcome. Secondary long-term outcomes included death during hospitalisation as single endpoint and discharge without chronic complications. Any serious adverse event was recorded and monitored up to patient discharge or death.

We analysed continuous variables through descriptive statistics. We reported data as mean (SD) or median [IQR], within-group changes vs. baseline with paired t-test or Wilcoxon signed-rank test, and between-group differences by analysis of covariance (ANCOVA) or chi-square or Fisher’s exact test, as appropriate. Survival analysis was performed by means of Cox proportional hazard regression models and results were expressed as hazard ratio (HR) and 95% confidence interval (CI). Analyses were adjusted by pre-defined covariates age and sex, and serum creatinine, the only covariate that, among baseline covariates listed in Table 1, at explorative univariable analysis significantly associated with all considered long-term outcomes. Cumulative events were constructed using the Kaplan-Meier method. Correlation analysis was carried out using Pearson’s r or Spearman’s rho correlation coefficient [23]. A further exploratory repeated measures correlation analysis was considered using package ‘rmcorr’. No imputation method was used for missing values. Data were analysed by SAS (version 9.4), STATA (version 15) and package ‘rmcorr’ (version 0.4.1). Statistically significant differences were assumed at 5% level of probability.

Table 1

Patient characteristics at baseline according to treatment group.

	Overall (n = 75)	Eculizumab (n = 10)	Controls (n = 65)
Demographic and clinical characteristics
Age, years	65.5 ± 12.9	60.0 ± 15.1	66.1 ± 12.5
Males, n (%)	56 (75)	7 (70)	49 (75)
Smokers, n (%)	12 (16)	2 (20)	10 (15)
Comorbidities per patient	2 [1–3]	1 [0–2]	2 [1–3]
Patients without comorbidities, n (%)	24 (32)	3 (30)	21 (32)
Hypertension alone, n (%)	10 (13)	1 (10)	9 (14)
Other comorbidities and hypertension, n (%)	27 (36)	1 (10)	26 (40)
Other comorbidities without hypertension, n (%)	14 (19)	5 (50)	9 (14)
Systolic blood pressure, mmHg	131.7 ± 23.3	139.7 ± 37.6	130.4 ± 20.2
Diastolic blood pressure, mmHg	73.7 ± 12.0	77.6 ± 7.0	73.1 ± 12.6
MAP, mmHg	93.1 ± 13.7	98.3 ± 13.5	92.2 ± 13.7
Heart rate, bpm	82.9 ± 14.0	85.4 ± 12.5	82.5 ±14.3
Respiratory functional parameters
Respiratory rate, breaths/min	26.6 ± 7.4	26.8 ± 7.3	26.6 ± 7.5
PaO2, mmHg	70.5 [59.6–92.0]	80.5 [66.0–90.0]	69.0 [58.0–92.0]
PaCO2, mmHg	32.0 [30.0–37.0]	31.5 [30.0–37.0]	32.0 [29.8–36.5]
PaO2/FiO2, mmHg	125.7 [100.0–183.8]	138.1 [110.0–159.3]	124.3 [97.1–184.0]
FiO2	0.6 [0.5–0.7]	0.6 [0.6–0.6]	0.6 [0.5–0.7]
Arterial pH	7.46 [7.44–7.49]	7.47 [7.44–7.48]	7.46 [7.44–7.50]
Laboratory parameters
White blood cell count, x109/L	8.12 [5.89–12.41]	10.72 [7.54–12.41]	7.94 [5.75–12.26]
Neutrophil count, x109/L	6.10 [4.39–9.58]	8.79 [6.02–11.31]	5.85 [4.08–8.95]
Lymphocyte count, x109/L	0.75 [0.52–1.12]	0.63 [0.44–0.76]	0.79 [0.57–1.14]
Monocyte count, x109/L	0.38 [0.28–0.51]	0.42 [0.32–0.49]	0.37 [0.28–0.55]
Platelet count, x109/L	237.0 [150.0–308.0]	303.5 [230.0–447.0]§	229.5 [137.0–286.5]
NLR	7.44 [4.83–19.37]	16.88 [7.67–20.08]	6.91 [4.68–12.63]
PLR	264.3 [187.7–514.4]	402.3 [280.3–879.3] §	244.4 [176.2–500.0]
Hemoglobin, g/dL	13.4 [12.1–14.7]	13.7 [12.2–14.7]	13.4 [12.0–14.6]
C-reactive protein, mg/dL	14.4 [9.1–21.9]	15.4 [11.2–22.1]	14.2 [9.1–21.5]
Aspartate aminotransferase, U/L	53.0 [36.0–81.5]	42.0 [35.5–50.5]	56.0 [36.5–84.5]
Alanine aminotransferase, U/L	44.0 [31.0–76.0]	43.5 [29.0–69.0]	44.0 [31.0–76.0]
LDH, U/L	610 ± 301	499 ± 144	626 ± 315
Serum creatinine, mg/dL	0.89 [0.70–1.09]	0.68 [0.60–0.95]	0.90 [0.73–1.09]
Estimated GFR, mL/min#	67.0 ± 23.2	79.7 ± 25.0	65.1 ±22.5
D-dimer, ng/mL*	942 [624–2132]	871 [568–1044]	1002 [624–2360]
IL-6, pg/mL°	86.5 [53.2–132.0]	83.6 [25.3–121.9]	87.3 [53.2–132.0]
sC5b-9, ng/mL‡	1022.0 ± 461.2	1145.1 ± 458.0	612.5 ± 72.5
C5b-9, % increase versus control¥	276.6 ± 71.7	270.3 ± 69.4	292.3 ± 85.7
Thrombus formation, pixel†	2821.8 ± 1326.7	2881.9 ± 1434.7	2661.7 ± 1242.5
Patients with medications, n (%)
ACE inhibitors or ARB&	26 (35)	0 (0) §	26 (40)
Darunavir/cobicistat	39 (52)	7 (70)	32 (49)
Hydroxychloroquine	51 (68)	9 (90)	42 (65)

Data are numbers (percentages), mean ± SD or median [IQR], as appropriate. Abbreviations: ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blockers; FiO2, fraction of inspired oxygen; GFR, glomerular filtration rate; IL-6, interleukin-6; LDH, lactate dehydrogenase; MAP, mean arterial pressure; NLR, neutrophil-to-lymphocyte ratio; PaCO2, partial pressure of arterial carbon dioxide; PaO2/FiO2, ratio of partial pressure of arterial oxygen to fractional inspired oxygen; PLR, platelet-to-lymphocyte ratio. Comorbidities: Hypertension, cancer, cardiovascular disease, cerebrovascular disease, chronic kidney disease, chronic liver disease, chronic obstructive pulmonary disease, diabetes mellitus and obesity.

#Estimated through the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

*D-dimer measurement was available in 8 patients of the eculizumab group and in 48 patients of the control group.

°IL-6 measurement was available in 8 patients of the eculizumab group and in 42 patients of the control group.

sC5b-9 levels were available in 10 patients of the eculizumab group and in 3 patients in the control group.

C5b-9 was available in 10 patients of the eculizumab group and in 4 patients in the control group.

†Thrombus formation was available in 8 patients of the eculizumab group and in 3 patients in the control group.

&In the control group 17 patients were given an ACE inhibitor and nine an ARB.

§P<0.05 versus controls.

Results

From February 2020 to April 2020 we included 75 participants: ten eculizumab-treated patients and 65 controls. All of them received CPAP ventilator support from 24 hours or less and standard treatment including low-dose steroid, low-molecular-weight heparin and antimicrobial prophylaxis with cephtriaxone and azithromycin (which served also as prophylaxis for Neisseria Meningitis and Pneumococcus infection in eculizumab-treated patients). Most patients received also hydroxycholoroquine and/or antiviral therapy (darunavir and cobicistat combination). Tachypnea, hypoxia, hypocapnia, markedly decreased PaO2/FiO2 ratio and increased FiO2 were consistent with severe respiratory distress at inclusion (Table 1). Other detailed demographic, clinical and laboratory parameters are reported in Table 1 and Supplementary Methods (S1 Appendix).

Complement activity and ex vivo complement deposition and thrombogenesis

At baseline, sC5b-9 plasma levels were significantly higher in COVID-19 patients than in 10 contemporary healthy controls and even as compared to levels observed in historical patients with atypical HUS (Fig 1A). Sera from all the 14 analysed patients induced strong C5b-9 deposition (>149% of a control serum pool) on ADP-activated HMEC-1 (Fig 1B). Pre-exposure to sera from 11 patients induced massive thrombus formation on ADP-activated HMEC-1 flowed with normal heparinised whole blood added with mepacrine (S1 Fig). C5b-9 and thrombus deposition were significantly stronger than those induced by sera from healthy controls and similar to those induced by sera from historical patients with atypical HUS (Fig 1B and 1C).

Fig 1

Circulating complement profile and ex-vivo effect of serum on C5b-9 deposition and thrombus formation on HMEC-1 in COVID-19 patients.

(A) SC5b-9 plasma levels in ten healthy subjects (healthy CTRs, negative controls), in 13 COVID-19 patients evaluated within 24 hour initiation of CPAP ventilator support and in patients with atypical hemolytic uremic syndrome studied in remission (aHUS, positive controls, n = 30). (B) Effect of serum from COVID-19 patients on C5b-9 formation on microvascular endothelial cells (HMEC-1). ADP-activated HMEC-1 were incubated for 2 hr with 50% serum, diluted with test medium (HBSS with 0.5% BSA), from healthy subjects (healthy CTRs, n = 17), or COVID-19 patients evaluated within 24 hour initiation of CPAP ventilator support (n = 14), or aHUS patients studied in remission (aHUS positive controls, n = 14) or with a pool of sera from healthy controls. At the end of incubation, cells were washed, fixed, and stained with rabbit anti-human complement C5b-9 complex antibody followed by FITC-conjugated secondary antibody. An AXIO Image.Z2 laser microscope was used to view the fluorescent staining on the endothelial cell surface, the HMEC-1 area covered by C5b-9 staining was calculated by automatic edge detection (Image J software), and values were expressed as the percentage of C5b-9 deposits induced by a pool of sera run in parallel (control, reference 100%). (C) Effect of serum from COVID-19 patients on thrombus formation on microvascular endothelial cells (HMEC-1). HMEC-1 were treated with ADP and exposed for 2 h in static conditions to 50% serum, diluted with test medium, from COVID-19 patients evaluated within 24 hour initiation of CPAP ventilator support (n = 11), or aHUS patients studied in remission (aHUS positive controls, n = 4) or with a pool of sera from healthy controls (healthy CTR pool). Perfusion of heparinised whole blood from healthy subjects (added with mepacrine) was then performed in a thermostatic flow chamber in which one surface of the perfusion channel was a glass slide seeded with a monolayer of endothelial cells, at a constant flow rate of 1500 sec-1 (60 dynes/cm2). After 3 min, perfusion was stopped, and the slide with the endothelial cell monolayer was dehydrated and fixed. A confocal inverted laser microscope was used to view the fluorescent staining on the endothelial cell surface, and the HMEC-1 area covered by thrombi was calculated by automatic edge detection (Image J software), and values expressed in pixels2. Data are mean ± SD. One-way Anova test. Abbreviations: CTRs, controls °°°P<0.0001, °°P<0.01, °P<0.05 versus healthy CTRs. ###P<0.0001 versus aHUS.

Genotyping

Genetic analysis was done in all eculizumab-treated COVID-19 patients and four COVID-19 controls (“Biochemical Controls”). Next generation sequencing screening revealed only a new missense heterozygous variant in C3 (c.C1426A, p.L476I) in one of the four COVID-19 controls. This variant has not been reported in patients with atypical HUS or other complement-related genetic diseases. No functional studies are available and the variant is predicted damaging only by two of 12 in silico tools (CADD 9.844). We classified the p.L476I as variant of unknown significance. The H3 CFH haplotype, which had been previously associated with the risk of atypical HUS [24] and with reduced plasma levels of factor H, was identified only in 2 patients (both are heterozygous) with an allele frequency of 0.11, which is not different from the allele frequency (0.17) that we previously reported in healthy subjects [25].

Copy number variation analysis in the genomic region including CFH and the 5 CFHR genes revealed the common CFHR3-CFHR1 heterozygous deletion in 6 out of 14 patients (frequency of the deleted allele 0.21 compared to 0.19 in 100 healthy controls, p = 0.6 Fisher’s exact test). We did not identify any CFH/CFHR hybrid gene or other rare genomic rearrangements.

One patient received only the first eculizumab dose because he was transferred to another hospital before the second dose could be administered. His data were recorded and analysed as for the other patients. Baseline characteristics of eculizumab-treated patients were similar to those of the whole patient population (Table 1).

Eculizumab was well tolerated and no treatment-related adverse event was observed. Eculizumab was associated with a significant reduction in respiratory rate at one and two weeks that was paralleled by a concomitant increase in arterial PaCO2 and a decrease in mean and diastolic blood pressure (Table 2). CRP and lactate dehydrogenase serum levels decreased as well, whereas estimated glomerular filtration rate significantly increased. Other changes are shown in Table 2.

Table 2

Clinical, respiratory and laboratory parameters at baseline and during follow-up according to study group.

	Eculizumab (n = 10)			Controls (n = 65)
	Baseline	Week 1	Week 2	Baseline	Week 1	Week 2
Clinical parameters
Systolic BP, mmHg	139.7±37.6	129.2±13.1	117.0±11.1#	130.4±20.2	131.6±19.8	128.6±21.5
Diastolic BP, mmHg	77.6±7.0	68.9±9.5*##	65.4±12.2*###	73.1±12.6	72.4±13.1	71.3±11.9
MAP, mmHg	98.3±13.5	89.0±8.5#	82.6±7.7*###	92.2±13.7	92.1±13.6	90.4±13.3
Heart rate, bpm	85.4±12.5	79.1±8.2#	81.2±9.8	82.5±14.3	82.9±14.8	80.9±12.7
Respiratory parameters
Respiratory rate, breaths/min	26.8±7.3	20.3±3.8*##	18.0±4.8*##	26.6±7.5	26.0±7.4	24.3±6.5
PaO2, mmHg	80.5 [66.0–90.0]	78.0 [70.0–89.0]	75.1 [63.0–88.0]	69.0 [58.0–92.0]	69.0 [58.0–90.4]	69.5 [57.0–89.0]
PaCO2, mmHg	31.5 [30.0–37.0]	55.0 [36.5–58.9]** #	54.7 [38.6–68.4]	32.0 [29.8–36.5]	41.0 [34.0–47.0]***	40.3 [32.7–48.0]**
PaO2/FiO2, mmHg	138.1 [110.0–159.3]	162.6 [100.0–180.0]	97.6 [75.0–179.3]	124.3 [97.1–184.0]	95.7 [71.4–142.9]	111.1 [90.0–162.9]
FiO2	0.6 [0.6–0.6]	0.6 [0.3–0.8]	0.3 [0.2–0.8]	0.6 [0.5–0.7]	0.7 [0.5–0.8]*	0.6 [0.4–0.7]
Arterial pH	7.47 [7.44–7.48]	7.38 [7.38–7.39]	7.41 [7.36–7.44]	7.46 [7.44–7.50]	7.44 [7.40–7.48]	7.45 [7.41–7.48]
Laboratory parameters
WBC count, x109/L	10.72 [7.54–12.41]	10.65 [8.84–12.80]	8.23 [6.59–12.69]	7.94 [5.75–12.26]	12.15 [7.64–17.16]***	12.24 [9.96–15.46]***
Neutrophil count, x109/L	8.79 [6.02–11.31]	8.91 [7.50–11.67]	5.50 [4.19–10.12]	5.85 [4.08–8.95]	11.09 [6.84–14.89]***	10.01 [7.45–12.54]***
Lymphocyte count, x109/L	0.63 [0.44–0.76]	0.76 [0.34–1.02]	1.04 [0.89–1.99]*	0.79 [0.57–1.14]	0.66 [0.45–1.08]	1.32 [0.70–1.95]**
Monocyte count, x109/L	0.42 [0.32–0.49]	0.62 [0.52–0.86]*	0.67 [0.53–0.73]**	0.37 [0.28–0.55]	0.55 [0.32–0.76]***	0.80 [0.58–0.93]***
Platelet count, x109/L	303.5 [230.0–447.0]#	306.5 [251.0–378.0]	192.0 [177.0–317.0]	229.5 [137.0–286.5]	244.0 [165.0–333.0]	243.5 [184.0–335·5]
NLR	16.88 [7.67–20.08]	9.51 [8.44–15.46]	5.09 [2.08–16.85]	6.91 [4.68–12.63]	15.74 [9.27–34.50]	6.91 [3.36–14.64]
PLR	402.3 [280.3–879.3] #	322.3 [255.4–561.3]	157.4 [98.9–388.3]	244.4 [176.2–500.0]	367.7 [178.6–545.8]	244.3 [86.3–383.5]
Hemoglobin, g/dL	13.7 [12.2–14.7]	13.3 [11.0–13.7]	11.4 [10.0–13.0]*	13.4 [12.0–14.6]	11.8 [10.9–13.1] ***	11.3 [9.7–12.4] ***
C-reactive protein, mg/dL	15.4 [11.2–22.1]	1.0 [0.5–1.4]**##	0.8 [0.2–9.5]*	14.2 [9.1–21.5]	5.9 [2.0–15.9]***	4.2 [0.7–8.4]***
AST, U/L	42..0 [35.5–50.5]	32.5 [21.0–46.0]	27.0 [20.5–46.0]	56.0 [36.5–84.5]	36.0 [26.0–48.0]***	27.0 [20.0–40.5]***
ALT, U/L	43.5 [29.0–69.0]	86.5 [61.0–111.0] **#	68.0 [52.0–96.5]*	44.0 [31.0–76.0]	53.0 [34.0–77.0]**	59.0 [34.0–94.0]**
LDH, U/L	499±144	301±112***	267±73***	626±315	446±192***	388±166**
Serum creatinine, mg/dL	0.68 [0.60–0.95]	0.56 [0.52–0.73]	0.56 [0.49–0.74]	0.90 [0.73–1.09]	0.79 [0.61–1.00]*	0.70 [0.52–0.88]**
Estimated GFR, mL/min	79.7±25.0	90.3±22.1**	89.9±28.9*	65.1±22.5	71.8±22.8***	76.8±22.8***
D-dimer, ng/mL	871 [568–1044]	916 [378–2551]	1336 [278–3370]	1002 [624–2360]	3139 [1045–6306]***	2743 [1072–4008]***
IL-6, pg/mL	83.6 [25.3–121.9]	8.0 [7.6–11.5]	49.2 [38.9–84.3]	87.3 [53.2–132.0]	35.8 [2.0–58.5]	19.9 [2.0–37.8]
sC5b-9, ng/mL‡	1145.1±458.0	761.5±247.3*	831.5±211.2	612.5±72.5	578.6±144.5	704.8±192.7
C5b-9, % increase vs control¥	270.3±69.4	78.5±19.0***###	128.8±54.3**###	292.3±85.7	316.5±37.6	322·4±44·1
Thrombus formation, pixel2†	2881.9±1434.7	638.1±251.4**###		2661.7±1242.5	3593.7±1018.3

Data are mean (SD) or median [IQR], as appropriate. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BP, blood pressure; CRP, C-reactive protein; GFR, glomerular filtration rate; IL-6, interleukin-6; LDH, lactate dehydrogenase; MAP, mean arterial pressure; NLR, neutrophil-to-lymphocyte ratio; PaCO2, partial pressure of arterial carbon dioxide; PaO2/FiO2, ratio of partial pressure of arterial oxygen to fractional inspired oxygen; PLR, platelet-to-lymphocyte ratio.

sC5b-9 levels were available in 10 patients of the eculizumab group and in 3 patients in the control group.

C5b-9 was available in 10 patients of the eculizumab group and in 4 patients in the control group.

†Thrombus formation was available in 8 patients of the eculizumab group and in 3 patients in the control group. For complement activity, ex vivo complement deposition and thrombus formation, Week 1 corresponds to 1–4 days post-CPAP, and Week 2 corresponds to 7–16 days post-CPAP. T-test or Wilcoxon Signed Rank *p<0.05, **p<0.01, ***p<0.001 vs baseline, ANCOVA test #p<0.05, ##p<0.01, ###p<0.001 vs controls.

Plasma sC5b-9 levels significantly decreased after the first eculizumab administration versus baseline, but mean values persistently exceeded normal range and fully normalised only at post-discharge recovery visits (Fig 2A). Serum-induced ex-vivo C5b-9 deposition and thrombus formation on HMEC-1 significantly and persistently decreased to normal range after eculizumab administration up to post-discharge recovery visits (Fig 2B and 2C).

Fig 2

Changes in sC5b-9 plasma levels and in ex vivo serum induced C5b-9 and thrombus formation on HMEC-1 lines in eculizumab-treated patients and “biochemical” controls.

SC5b-9 plasma levels (A), ex vivo serum induced C5b-9 (B) and thrombus formation on HMEC-1 lines (C) at baseline, at 1–4 days and 7–16 days of CPAP ventilator support and at post discharge recovery visit in eculizumab-treated patients (Black columns) and “biochemical” controls (diagonal stripes), respectively. Numbers under the columns describe the number of eculizumab—treated patients or controls evaluated at each time-point. Horizontal dashed lines are the upper and lower limit of normal range (mean±2SD) of each considered parameter. Abbreviations: CPAP, Continuous Positive Airway Pressure; Ecul, Eculizumab. Data are mean ± SD. One-way Anova test., *P<0.05, **P<0.01, ***P<0.001 versus baseline; ^^P<0.01 versus recovery; ###P<0.0001 versus “biochemical controls” at the same time-point.

Controlled study

Baseline characteristics were similar between eculizumab-treated patients and controls (Table 1), with the exception of lower platelet count and platelet-to-lymphocyte ratio, and more frequent use of renin-angiotensin system (RAS) blockers in controls. In the four “biochemical” controls baseline sC5b-9 plasma levels, ex-vivo serum-induced C5b-9 deposition and thrombus formation were similarly elevated as in eculizumab-treated patients (Fig 2A–2C).

Respiratory rate did not change appreciably at one and two weeks of observation vs. baseline in controls, whereas arterial PaCO2 significantly increased at both time-points. No significant changes were observed in heart rate and arterial blood pressure. Changes in other considered parameters are shown in Table 2. Changes in respiratory rate, PaCO2, mean and diastolic blood pressure, and median CRP values at one and two weeks (Fig 3A–3D) versus baseline significantly differed between treatment groups (Table 2). Other changes are shown in Table 2.

Fig 3

Changes in clinical, respiratory and laboratory parameters in eculizumab-treated patients and all controls, and correlations between changes in respiratory rate and ex vivo C5b-9 deposition at different time points in eculizumab-treated patients and “biochemical” controls considered as a whole.

Respiratory rate (A), PaCO2 (B), mean arterial pressure (C) and C-reactive protein plasma levels (D) at baseline and at 1–4 days and 7–16 days of CPAP ventilator support in eculizumab-treated patients (black circles and black continuous line) and in controls (white circles and grey dashed line). Data are mean ± SEM or median. Correlations between changes in serum-induced C5b-9 deposits at 1 week (E) and 2 weeks (F) of CPAP ventilator support and concomitant changes in respiratory rate in eculizumab-treated patients (black circles) and controls (white circles) considered as a whole. Number of patients, rs and p values for each correlation are shown in the two panels. Abbreviations: CRP, C-reactive protein; MAP, mean arterial pressure; RR, respiratory rate. *P<0.05, **P<0.01, ***P<0.001 versus baseline. #P<0.05, ##P<0.01 versus controls.

Ex-vivo C5b-9 and thrombus deposition in “biochemical” controls did not change at one and two weeks vs. baseline and normalised only at recovery visits (Fig 2B and 2C). Thus, follow-up changes in both parameters significantly differed between groups during the first two weeks of CPAP ventilator support. At recovery visits all considered parameters were in normal range and similar between groups.

Long-term endpoints

Over a median (IQR) observation period of 47.0 (14.0–121.0) days, four of the ten eculizumab-treated patients died or were discharged with chronic complications as compared to 52 of the 65 controls (80.0%) (Fig 4A). Event rate was significantly lower in eculizumab-treated patients than in controls [HRCrude (95% CI): 0.26 (0.09–0.72), p = 0.010]. The difference between groups was statistically significant even after adjustment for age and sex [HRAdjusted (95% CI): 0.29 (0.10–0.84), p = 0.022], and further adjustment for baseline serum creatinine [HRAdjusted (95% CI): 0.30 (0.10–0.84), p = 0.023]. Causes of death and chronic complications at discharge are shown in S1 and S2 Tables, respectively.

Fig 4

Kaplan-Meier curves for the combined endpoint of mortality or discharge with chronic complications, and for mortality as a single endpoint and discharge without chronic complications in eculizumab-treated patients and controls.

Kaplan-Meier curves show the proportion of eculizumab-treated patients and controls who reached the combined endpoint of mortality or discharge with disabling chronic complications (A), mortality as a single endpoint (B) or discharge without chronic complications (C) during the observation period. Hazard ratios (HRs) and 95% confidence intervals are crude and adjusted for sex and age classes (model 1), or for sex, age classes and baseline serum creatinine (model 2). The number of patients at risk is shown in the bottom table. Black continuous line, eculizumab group; grey dashed line, control group.

Two eculizumab-treated patients versus 31 (47.7%) controls died. Mortality rate, however, did not significantly differ between groups, even after adjustment for age and sex, and further adjustment for baseline serum creatinine (Fig 4B). Six patients and 13 controls (20.0%) were discharged without chronic complications. The event rate was significantly higher in eculizumab-treated patients than in controls [HRCrude (95% CI): 2.88 (1.08–7.70), p = 0.035, (Fig 4C)]. Between-group difference approached the nominal significance after adjustment for age and sex [HRAdjusted (95% CI): 2.92 (0.99–8.67), p = 0.053], but was not significant after adjustment for baseline serum creatinine [HRAdjusted (95% CI): 2.21 (0.71–6.88), p = 0.171].

Other endpoints

During the observation period, 5 (50%) eculizumab-treated patients and 21 (32.3%) controls required mechanical ventilation (p = 0.301). There were six (9.2%) cardiovascular events (two cardiogenic shocks, one myocardial infarction, one atrial fibrillation, one atrioventricular block and one supraventricular tachycardia) and six (9.2%) thromboembolic events in controls versus none in eculizumab-treated patients. None of the patients in either group required renal replacement therapy.

Correlation analyses

At baseline, plasma sC5b-9 levels positively correlated with D-dimer concentrations (rs = 0.849, p = 0.002) and serum-induced C5b-9 deposits with respiratory rate (rs = 0.590, p = 0.026). At one-week follow-up, changes in plasma sC5b-9 levels versus baseline directly correlated with concomitant changes in D-dimer concentrations (rs = 0.925, p = 0.001). Changes in serum-induced C5b-9 deposits at one (rs = 0.706, p = 0.010, Fig 3E) and two (rs = 0.751, p = 0.032, Fig 3F) weeks positively correlated with concomitant changes in respiratory rate. At baseline, neutrophil-to-lymphocyte ratio (r = 0.675, p = 0.046) and platelet-to-lymphocyte ratio (r = 0.807, p = 0.009) directly correlated with thrombus formation. Changes in neutrophil-to-lymphocyte (r = 0.884, p = 0.046) and platelet-to-lymphocyte (r = 0.908, p = 0.033) ratios at one week of follow-up positively correlated with concomitant changes in thrombus formation.

Discussion

In this fully academic, single centre, two-phase study we first found that eculizumab compassionate therapy was safe and well tolerated in ten patients with severe COVID-19 requiring CPAP ventilator support. No treatment-related adverse event was reported. Respiratory distress promptly improved, as documented by significant reduction in respiratory rate and concomitant increase in PaCO2 at one and two weeks of CPAP ventilator support. Inflammation also improved as demonstrated by reduction in CRP levels at the same time points. Conversely, in the second controlled phase we found that in controls respiratory rate, PaCO2 and CRP levels did not change appreciably at one and two weeks of follow-up. Finding that at one and two weeks changes in these parameters significantly differed between groups confirmed that eculizumab remarkably and promptly improved respiratory distress and inflammation during the acute phase of the disease versus standard therapy alone.

Over a median observation period of 47 days, eculizumab-treated patients were also significantly protected against the combined endpoint of in-hospital death or discharge with invalidating chronic sequelae, including residual respiratory insufficiency in most cases. Moreover eculizumab associated with increased probability of discharge without chronic complications and a trend to better survival rate versus controls. Treatment effect on the combined endpoint was significant even after adjustment for age, sex and baseline serum creatinine. On the other hand, treatment effect on mortality rate considered as a single endpoint failed to reach the nominal significance most likely because of the too small number of events. Eculizumab therapy also reduced arterial blood pressure versus controls. Whether this effect was explained by reduced sympathetic tone associated with amelioration of respiratory distress and/or by blunted complement-mediated arteriolar vasoconstriction remains elusive [26].

Study findings were unlikely confounded by differences in the distribution of risk factors or in patient’s care because at inclusion patients’ characteristics were quite similar between groups, and all patients were followed by the same “COVID teams” during the same observation period and were managed according to the same standardised monitoring and treatment protocols. Findings were not confounded by concomitant experimental treatments because patients included in clinical trials were not considered. The more frequent use of RAS blockers in controls should not have confounded the results because these medications do not appear to affect disease progression and case-fatality in COVID-19 [27].

The sub-study in the ten eculizumab-treated patients and four “biochemical” controls showed a marked increase in plasma sC5b-9 levels along with extremely activated ex vivo complement and thrombus deposition on human cultured endothelial cells induced by patients’ sera. SC5b-9 plasma levels decreased and ex-vivo C5b-9 and thrombus deposition fully normalised with eculizumab, and all parameters in eculizumab-treated patients normalised at recovery visits. In controls, plasma sC5b-9 levels and ex vivo C5b-9 and thrombus deposition did not appreciably change at one to four and seven to 16 days of follow-up versus baseline and normalised only at recovery visit. Between-group differences in ex vivo C5b-9 formation at the same time points and in thrombus deposition at one to four days were highly significant. Thus, eculizumab fully blunted ex vivo complement deposition and thrombogenesis induced by patients’ SARS-CoV-2 infected sera. These data suggest that in COVID-19, disease severity could be sustained by extreme activation of the complement terminal pathway, both in the circulation and on the endothelial cell surface [19,20]. Benefits of C5 blockade could be mediated by prompt and effective systemic complement inhibition and protection from complement deposition and thrombus formation on endothelial cell surface during the acute phase of the disease. Consistently, respiratory rate positively correlated with ex vivo C5b-9 formation on endothelial cells at inclusion and its reduction at one to four days and seven to 16 days of follow up correlated with concomitant reductions in ex vivo C5b-9 formation.

In healthy subjects, complement modulates pro- and anti-inflammatory functions and facilitates the clearance of pathogens and apoptotic cells [28]. This modulatory function is disrupted by SARS-CoV-2 that triggers uncontrolled cleavage of the terminal complement protein C5 with consequent excess production of the proinflammatory anaphylatoxin C5a and of the terminal complement complex C5b-9. These changes activate endothelial and phagocytic cells and sustain production of reactive oxygen species [7]. The C5b-9 complex may also directly injure endothelial and alveolar cells [29] with consequent disruption of the lung tissue. C5a and C5b-9 may also have pro-thrombotic effects by acting on endothelium, neutrophils and platelets [12,29,30]. Thus, C5 blockade by eculizumab might also serve to prevent the thromboembolic complications of COVID-19 [9]. These effects are achieved without affecting upstream immune-modulatory and immune-protective functions of the complement cascade [28], which might explain—along with antimicrobial prophylaxis against gram-positive encapsulated bacteria—why in our patients eculizumab treatment was not associated with excess risk of infectious complications. However, the risk of infection with Klebsiella pneumonie despite antimicrobial prophylaxis during C5-blocking therapy [31] should be taken into consideration because this gram-negative encapsulated bacterium often produces large spectrum beta-lactamases that can inactivate antibiotics commonly recommended to prevent meningococcal infection upon exposure to eculizumab [32]. The findings are not affected by doses of heparin or low molecular weight heparin that are used in clinics to prevent or treat pulmonary thromboembolism [21,33].

At variance with previous scanty reports [34], none of our patient disclosed signs of thrombotic microangiopathy. Lack of predisposing genetic abnormalities in the complement system could explain these findings and even suggests that SARS-CoV-2 infection is per se sufficient to induce complement activation and precipitate thrombo-embolic complications independent of host genetic predisposition [35].

Major study limitations were the relatively small number of patients receiving eculizumab therapy and the non-randomised and unblinded design. Major strengths were the controlled design and the integrated evaluation of potential mediators of the disease, markers of respiratory distress and long-term hard endpoints. All considered outcomes were pre-specified and data assessors were blinded to treatment. Treatment assignment was based on predefined guidelines, which limited the risk of investigator bias in the allocation to treatment groups. When we designed our study, few early doses of eculizumab had been reported to inhibit complement activation in severe cases of Shiga-toxin associated HUS [36]. With this background we administered only two 900 mg doses of eculizumab one week apart at acute onset of severe COVID-19. We found that this treatment protocol was safe and effective in our patients. Recent data suggest that intensified treatment compared to that indicated for atypical HUS appeared to improve outcomes of patients with COVID-19, but was associated with excess infectious complications in particular of life-threatening ventilator-associated pneumonia [15]. However, assessing whether higher eculizumab doses, shorter intervals between drug administrations or longer treatment periods would have been more effective, or rather less safe, was beyond our purposes. We did not measure the degree of circulating C5 blockade by eculizumab through CH50 or similar assays. However finding that ex vivo serum-induced C5b-9 deposition on HMEC-1 fully normalised in COVID-19 patients after eculizumab, would support an effective degree of C5 inhibition as we previously documented in patients with aHUS [20].

In conclusion, our findings–that need confirmation in a prospective randomised clinical trial—suggest that adding only two 900 mg doses of eculizumab to standard therapy in patients with severe COVID-19 who were receiving CPAP support for 24 hours or less, can safely improve respiratory dysfunction and decrease the combined endpoint of long-term mortality and chronic complications. These findings may have major implications, since effective complement C5 blockade restricted to the early acute phase of the disease could have better risk/benefit profiles than standard or intensified treatement protocols. Optimised cost/effectiveness could also facilitate patients’ access to compassionate treatment with this expensive medication, particularly in resource-restricted settings.

Experimental design of ex vivo serum-induced thrombus formation experiments on microvascular endothelial cells.

HMEC-1 were treated with ADP and exposed for 2 h in static conditions to 50% serum from COVID-19 patients, from aHUS patients studied in remission (aHUS positive controls) or from control serum pool diluted with test medium. Perfusion of heparinised whole blood from healthy subjects (added with mepacrine) was then performed in a thermostatic flow chamber in which one surface of the perfusion channel was a glass slide seeded with a monolayer of endothelial cells, at a constant flow rate of 1500 sec-1 (60 dynes/cm2). After 3 min, perfusion was stopped, and the slide with the endothelial cell monolayer was dehydrated and fixed. The values were expressed in pixels2.

(TIF)

Click here for additional data file.

Causes of death in the study group as a whole (overall) and in cases and controls considered separately.

(DOCX)

Click here for additional data file.

Age, gender and chronic complications at hospital discharge in cases and controls.

(DOCX)

Click here for additional data file.

Supplementary methods.

(DOCX)

Click here for additional data file.

Study protocol.

(PDF)

Click here for additional data file.

STROBE checklist.

(DOCX)

Click here for additional data file.

7 Oct 2021

PONE-D-21-17700Eculizumab in patients with severe coronavirus disease 2019 (COVID-19) requiring continuous positive airway pressure ventilator support: retrospective cohort studyPLOS ONE

Dear Dr. Remuzzi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers, as well as the academic editor had significant concerns. Specifically, the description of the study was judged to be confusing and the conclusions may not be fully supported by the data considering the limited number of patients studied. There are also a variety of other variables/outcomes that could be included in the description of the patient populations, as outlined by reviewer 2. Further, a bit more context, in terms of relationships to other studies (see Reviewer 1) and to an ongoing clinical trial (see Reviewer 2), is required.

Please submit your revised manuscript by Nov 21 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ruud AW Veldhuizen

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

1) As a non-expert reader of his manuscript, I found the description of the study protocols and patient populations very confusing. It seems this is a retrospective study but written as a prospective study.

2) The authors conclude: "Eculizumab safely improved respiratory dysfunction and long-term outcomes of patients with severe COVID-19" (Abstract and a similar statement in the discussion). Considering this is a non-blinded study, with only 10 patients receiving the in the drug of interest, in an extremely complex disease, the authors should downplay this conclusion. This includes being specific regarding their findings and staying away from statements like "long-term outcomes" which can easily be misinterpreted by readers. After reading this manuscript, my conclusion would be that this data support the safety and rationalizes a more extensive, blinded, multi-center trial for the use of Eculizumab in this patient population, or something of that nature.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for including your ethics statement: "The compassionate treatment protocol and the controlled study were both approved by the local Ethical Committee. Participants provided written informed consent. Local Ethical Committee approved the study. "

a) Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

4. Thank you for stating in your Funding Statement: " The ASST Papa Giovanni XXIII in Bergamo (Italy) sponsored the trial, Brembo SpA (Curno, Bergamo, Italy) partially covered study costs by a liberal grant under the initiative "Progetto TrexUno" and Alexion Pharma Italy S.R.L. (Milan, Italy) freely supplied the study drug. Neither the sponsor nor the companies had any role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. All authors had full access to all the data in the study and accept responsibility to submit for publication. "

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

5. Thank you for stating the following financial disclosure: "The ASST Papa Giovanni XXIII in Bergamo (Italy) sponsored the trial, Brembo SpA (Curno, Bergamo, Italy) partially covered study costs by a liberal grant under the initiative "Progetto TrexUno" and Alexion Pharma Italy S.R.L. (Milan, Italy) freely supplied the study drug. Neither the sponsor nor the companies had any role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. All authors had full access to all the data in the study and accept responsibility to submit for publication."

We note that one or more of the authors is affiliated with the funding organization, indicating the funder may have had some role in the design, data collection, analysis or preparation of your manuscript for publication; in other words, the funder played an indirect role through the participation of the co-authors. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please do the following:

a. Review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. These amendments should be made in the online form.

b. Confirm in your cover letter that you agree with the following statement, and we will change the online submission form on your behalf:

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section."

6. Thank you for stating the following in the Competing Interests section: "M.G. reported grants Omeros Corporation, Alexion Pharmaceutical, F. Hoffman-La Roche Ltd and Novartis Pharma AG (payments were made to her institution); M.N. reported grants from Omeros Corporation, Novartis Pharma AG, Roche and BioCryst Pharmaceutical (payments were made to her institution) as well as personal fees from Inception Sciences and BioCryst Pharmaceutical. A.B. reported personal fees from Akebia Pharmaceuticals, Alexion Pharmaceutical, BioCryst Pharmaceutical, Janssen Research & Development LLC, as well as speaker honorarium/travel reimbursements from Alnylam, Boehringer Ingelheim and Inception Science Canada. G.R. reported personal fees from Akebia Pharmaceuticals, Alexion Pharmaceutical, BioCryst Pharmaceutical and Janssen Research & Development LLC, as well as speaker honorarium/travel reimbursements from Alnylam, Boehringer Ingelheim and Inception Science Canada. All the other authors have nothing to disclose."

We note that you received funding from a commercial source: Omeros Corporation, Alexion Pharmaceutical, F. Hoffman-La Roche Ltd, Novartis Pharma AG, Novartis Pharma AG, Roche and BioCryst Pharmaceutical, Akebia Pharmaceuticals, and Janssen Research & Development LLC.

Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.

Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf.

7. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a good, comprehensive retrospective cohort study using Eculizumab in patients with severe coronavirus disease 2019 (COVID-19). However, some omissions should be addressed.

1. Authors should explain why the standard dose of LMWH may be sufficient or insufficient, as in some reported cases (Stattin K, et al. Inadequate prophylactic effect of low-molecular weight heparin in critically ill COVID-19 patients. J Crit Care. 2020 Dec; 60:249-252). Taking into account that LMWH significantly suppresses the cleavage of C3 (Amara U, et al., Molecular intercommunication between the complement and coagulation systems. J Immunol. 2010 Nov 1;185(9):5628-36), and therefore the levels of sC5b-9.

2. The authors need to explain in Table 2, during the follow-up of the study group (Baseline, Week 1 and Week 2) with Eculizumab; why the levels of D-Dimer increase slightly, however, the thrombus formation decreases, at least during the week1?

3. In addition, Table 2 could include the neutrophils to lymphocytes ratio and the platelets to lymphocytes ratio in order to discuss and correlate them with laboratory parameters.

4. The authors mention in lines 322-324 “Mortality rate, however, did not significantly differ between groups, even after adjustment for age and sex, and further adjustment for baseline serum creatinine (Fig 4B)”; but they should explain this.

5. The authors should include the pH of the patients in Table 2. Taking into consideration that there could be some correlation with complement activation in patients with severe COVI-19 since a relationship has been shown in-vitro (Fishelson Z, Horstmann RD, Müller-Eberhard HJ. Regulation of the alternative pathway of complement by pH. J Immunol. 1987 May 15;138(10):3392-5).

6. Considering that complement and thrombosis converge in the author´s research, they should cite Bont CM, et al., NETosis, complement, and coagulation: a triangular relationship. Cell Mol Immunol. 2019; 16 (1): 19-27. doi: 10.1038 / s41423-018-0024-0.

Reviewer #2: In this paper, the authors describe the use of eculizumab at the onset of COVID-19 related ARDS as reflected by the need for CPAP. Eculizumab, a monoclonal antibody against complement protein C5, was used in 10 patients with COVID (each patient received two doses 7-10 days apart) and compared them to a cohort of COVID-19 patients who did not receive eculizumab, cared for by the same team. They measured plasma C5b-9, generated ‘in vitro’ C5b-9, and thrombus formation from patient samples, at various time points. The authors describe that the eculizumab group showed improvement in the respiratory rate (primary outcome) at 1 and 2 weeks compared to their baseline, while the control group did not have a similar change. Additionally, they noticed the eculizumab respiratory rate correlated with the ex vivo C5b-9 deposits. This report is clinically relevant in highlighting the role of complement in COVID-19 pathophysiology, especially since a targetable drug such as eculizumab is already being used for other diseases.

1. From a methodology perspective, the manuscript has been written confusingly about this being a prospective and a retrospective study (line 141). Additionally, the patients who received eculizumab have been reported to be ‘every 6th or 7th patient’ with COVID. At the same time, the manuscript also reports that it was the treating physician's discretion as to who gets eculizumab (lines 104 and 107). These discrepancies concern how this study was performed and how the data was analyzed. There should be a better justification to why no sample size calculation was performed while primary and secondary objectives were defined.

2. The described study and details of the NCT04288713 study on clinicaltrials.gov do not match. The latter is registered in the United States, and reports of eculizumab dosing every seven days, and the endpoints are also different. The authors should explain this discrepancy and why this deviation in the protocol was made.

3. The methods section should include the time period of this study, as there have been several studies looking at complement blockade in COVID-19. Some studies and case reports have demonstrated increased use of higher doses of eculizumab or more frequent dosing in COVID. These patients with COVID have been reported to have a higher level of complement activation than aHUS or PNH. Was complement blockade examined in the treated patients, along with other complement activation markers?

4. Can the authors comment on why the in vitro C5b-9 at baseline in the control group (Fig 2b) was higher than their plasma sC5b-9 values (as shown in figure 2A)?

5. The two groups have different levels of inflammation even though the p-value wasn’t significant. The group which received eculizumab had higher sC5b-9, higher PaO2, lower LDH, creatinine, GFR and D-dimer. This trend with the eculizumab group being slightly milder at the onset and reportedly had a better response to eculizumab. Even though there was a smaller ‘n’, it may be helpful to match and perform sub-analyses of the Ecu group with ‘selected patients’ from the non-Ecu group with similar respiratory and laboratory profiles.

6. The control group had elevated D-Dimer levels, and this high level has been shown to be associated with poor outcomes in several COVID-19 studies. Can the reported increase in ex vivo thrombus formation in the control group be attributed to this high baseline D-dimer with or without evidence of pulmonary embolus, contributing to the poorer respiratory outcome? The ‘n’ of 3 and 2 in the control group for thrombus formation assays is severely underpowered and questions the validity of the data.

7. Authors should report if patients in either group required mechanical ventilation, renal replacement therapy, cardiac dysfunction, systemic thrombosis, antiviral medications, among other outcomes. This would help ascertain if other endpoints were or were not different between these two groups.

8. Can authors also report which patients received hydroxychloroquine and remdesivir and how it may have contributed to their response to COVID-19 infection.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

27 Oct 2021

Additional Editor Comments:

1) As a non-expert reader of his manuscript, I found the description of the study protocols and patient populations very confusing. It seems this is a retrospective study but written as a prospective study.

We acknowledge that the study design was complex, but this was due to reasons that were largely beyond our control. As soon as we got the approval by the Ethical Committee we used all supplied medication to prospectively treat all approachable patients (from February 2020 to April 2020) in the context of the FDA program of eculizumab off-label compassionate use for the treatment of non-intubated patients with COVID-19. The compassionate use implied that patients could not be treated in the context of a controlled study. When the last eculizumab-treated patient had completed the treatment period (and therefore the phase of compassionate therapy had been completed) we identified patients who required CPAP ventilator support at the same institution and during the same period (from February 2020 to April 2020), and fulfilled the same eligibility criteria for eculizumab therapy but for practical reasons could not access to eculizumab (see below). These patients served as controls. Then, baseline and outcome data from eculizumab-treated patients and from non-eculizumab treated controls were recorded and analysed in the context of a retrospective controlled study. Thus, eculizumab patients were prospectively treated whereas data from eculizumab patients and non-eculizumab controls were retrospectively recorded in the context of a controlled study. We clarified this important issue in the revised version of the manuscript (Page 5, lines 94-96).

As for the description of the study populations it must be taken into consideration that the study was performed during the first, dramatic wave of the COVID-19 pandemic in Lombardy (Italy). In actual facts, during that emergency period it was virtually impossible to treat with eculizumab and adequately monitor all consecutive patients who required CPAP ventilator support. Thus, the selection of eculizumab recipients was at the discretion of the treating physician and influenced by logistic reasons. As clarified in the Methods of the manuscript, “to avoid overlaps of drug administrations and specific laboratory tests to monitor treatment effects in different patients, and prevent possible interference with clinical patient management, we administered eculizumab to one patient every six to seven consecutive potential candidates”. Then, we identified patients who required CPAP ventilator support during the same inclusion period but who could not access to eculizumab. We finally included into the study eculizumab treated patients and non-eculizumab treated controls. This is why there was no sample size calculation for this study. In the revised manuscript we better clarified this important issue (Page 5, lines 94-96).

2) The authors conclude: "Eculizumab safely improved respiratory dysfunction and long-term outcomes of patients with severe COVID-19" (Abstract and a similar statement in the discussion). Considering this is a non-blinded study, with only 10 patients receiving the in the drug of interest, in an extremely complex disease, the authors should downplay this conclusion. This includes being specific regarding their findings and staying away from statements like "long-term outcomes" which can easily be misinterpreted by readers. After reading this manuscript, my conclusion would be that this data support the safety and rationalizes a more extensive, blinded, multi-center trial for the use of Eculizumab in this patient population, or something of that nature.

We agree with the comment of the Reviewer that we have to tone down the emphasis on our conclusions because they are based on data that were obtained from only 10 treated patients and that were analysed in the context of a non-blinded study. As suggested in the conclusions of the abstract and discussion of the revised manuscript we emphasized that our findings need confirmation in a prospective randomized clinical trial. We also replaced “long-term outcome” with “combined endpoint of mortality and discharge with chronic complications” (Page 3, lines 57-59; Page 25, lines 461-465).

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We have amended our manuscript according to PLOS ONE's style requirements.

2. Thank you for including your ethics statement: "The compassionate treatment protocol and the controlled study were both approved by the local Ethical Committee. Participants provided written informed consent. Local Ethical Committee approved the study. "

a) Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

In the revised version of the manuscript, we have specified that the Ethical Committee of Bergamo approved the study (Page 5, lines 97-98).

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

We have amended the “Ethics Statement” field of the submission form as required.

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

We ensure to have provided the same grant information in the ‘Funding Information’ and ‘Financial Disclosure’ sections.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Please consider that Brembo SpA (Curno, Bergamo, Italy) partially covered study costs by a liberal grant. Thus, there was not a grant number.

4. Thank you for stating in your Funding Statement: " The ASST Papa Giovanni XXIII in Bergamo (Italy) sponsored the trial, Brembo SpA (Curno, Bergamo, Italy) partially covered study costs by a liberal grant under the initiative "Progetto TrexUno" and Alexion Pharma Italy S.R.L. (Milan, Italy) freely supplied the study drug. Neither the sponsor nor the companies had any role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. All authors had full access to all the data in the study and accept responsibility to submit for publication. "

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

We have included the amendment Funding Statement within our cover letter.

5. Thank you for stating the following financial disclosure: "The ASST Papa Giovanni XXIII in Bergamo (Italy) sponsored the trial, Brembo SpA (Curno, Bergamo, Italy) partially covered study costs by a liberal grant under the initiative "Progetto TrexUno" and Alexion Pharma Italy S.R.L. (Milan, Italy) freely supplied the study drug. Neither the sponsor nor the companies had any role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. All authors had full access to all the data in the study and accept responsibility to submit for publication."

We note that one or more of the authors is affiliated with the funding organization, indicating the funder may have had some role in the design, data collection, analysis or preparation of your manuscript for publication; in other words, the funder played an indirect role through the participation of the co-authors. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please do the following:

a. Review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. These amendments should be made in the online form.

We ensure to have specifically and accurately indicated the roles that the authors affiliated to the ASST Papa Giovanni XXIII in Bergamo (Italy) had in our study.

b. Confirm in your cover letter that you agree with the following statement, and we will change the online submission form on your behalf:

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section."

We confirm our agreement with the above sentence, that was included within the cover letter.

6. Thank you for stating the following in the Competing Interests section: "M.G. reported grants Omeros Corporation, Alexion Pharmaceutical, F. Hoffman-La Roche Ltd and Novartis Pharma AG (payments were made to her institution); M.N. reported grants from Omeros Corporation, Novartis Pharma AG, Roche and BioCryst Pharmaceutical (payments were made to her institution) as well as personal fees from Inception Sciences and BioCryst Pharmaceutical. A.B. reported personal fees from Akebia Pharmaceuticals, Alexion Pharmaceutical, BioCryst Pharmaceutical, Janssen Research & Development LLC, as well as speaker honorarium/travel reimbursements from Alnylam, Boehringer Ingelheim and Inception Science Canada. G.R. reported personal fees from Akebia Pharmaceuticals, Alexion Pharmaceutical, BioCryst Pharmaceutical and Janssen Research & Development LLC, as well as speaker honorarium/travel reimbursements from Alnylam, Boehringer Ingelheim and Inception Science Canada. All the other authors have nothing to disclose."

We note that you received funding from a commercial source: Omeros Corporation, Alexion Pharmaceutical, F. Hoffman-La Roche Ltd, Novartis Pharma AG, Novartis Pharma AG, Roche and BioCryst Pharmaceutical, Akebia Pharmaceuticals, and Janssen Research & Development LLC.

Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.

Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf.

We have included the amended Competing Interests Statement within our cover letter.

7. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

As required, we have deleted the Ethics Statement from the Abstract section of the revised manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a good, comprehensive retrospective cohort study using Eculizumab in patients with severe coronavirus disease 2019 (COVID-19). However, some omissions should be addressed.

We thank the Reviewer for the rewarding comments on our work. Please find below all our point-by-point answers to the comments of the Reviewer.

1. Authors should explain why the standard dose of LMWH may be sufficient or insufficient, as in some reported cases (Stattin K, et al. Inadequate prophylactic effect of low-molecular weight heparin in critically ill COVID-19 patients. J Crit Care. 2020 Dec; 60:249-252). Taking into account that LMWH significantly suppresses the cleavage of C3 (Amara U, et al., Molecular intercommunication between the complement and coagulation systems. J Immunol. 2010 Nov 1;185(9):5628-36), and therefore the levels of sC5b-9.

The Reviewer raised an important point. Actually, there is a cross-link between coagulation and the complement system. Heparin and LMWH have been shown to inhibit complement activation in vitro, however this effect is observed only with supratherapeutic concentrations of the drug. For instance, in the J Immunol paper by Amara et al. 25 micrograms/ml fondaparinux were required to significantly reduce C5a generation in vitro in human serum, a concentration much higher than therapeutic Cmax levels of the drug (0.3-0.7 micrograms/ml). In addition, in a previously published study we found that addition of 10 U/ml heparin to serum from patients with atypical hemolytic uremic syndrome (aHUS) before incubation with HMEC-1 did not reduce the intensity of C5b-9 staining (Bettoni S et al J Immunol 2017; 199:1021-1040).

Altogether, the above findings indicate that doses of LMWH given to COVID-19 patients are not enough to control the burst of complement activation caused by SARS-CoV-2 infection. We briefly addressed this important point in the Discussion of the revised manuscript (Page 24, lines 434-436).

2. The authors need to explain in Table 2, during the follow-up of the study group (Baseline, Week 1 and Week 2) with Eculizumab; why the levels of D-Dimer increase slightly, however, the thrombus formation decreases, at least during the week1?

D-dimer test reflects progressive reabsorption of preformed intravascular thrombi. The ex vivo test, on the contrary, assesses the formation of thrombi on the surface of HMEC-1 lines induced by patient sera. Thus the two tests describe two different phenomena. The D-Dimer test measures the consequence of an event that has already occurred or that is occurring. Thus the D-Dimer test increase follows or parallels thrombi formation. The ex vivo test measures an activity that is present in patient sera that induces (and therefore precedes) thrombus formation. Thus, it is expected that inhibition of serum thrombogenic activity (documented by the ex vivo test) precedes the inhibition of thrombi formation which in turn precedes the decrease in the D-Dimer test. Thus, normalisation in ex vivo thrombus formation is expected to precede D-Dimer test normalisation. This could explain why at week 2 ex vivo thrombus formation was inhibited whereas the D-Dimer test was still increased.

3. In addition, Table 2 could include the neutrophils to lymphocytes ratio and the platelets to lymphocytes ratio in order to discuss and correlate them with laboratory parameters.

As suggested by the Reviewer, in the revised version of the manuscript we included neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio at baseline (Table 1) and during follow-up (Table 2) according to study group. In the main text we specified that baseline characteristics were similar between eculizumab-treated patients and controls (Table 1), with the exception of lower platelet count and platelet-to-lymphocyte ratio, and more frequent use of renin-angiotensin system (RAS) blockers in controls (Page 17, lines 294-296).

At baseline, thrombus formation directly correlated with neutrophil-to-lymphocyte ratio (n=9, r=0.675, p=0.046) and with platelet-to-lymphocyte ratio (n=9, r=0.807, p=0.009). Changes in thrombus formation at one week follow-up positively correlated with changes in neutrophil-to-lymphocyte ratio (n=5, r=0.884, p=0.046) and platelet-to-lymphocyte (n=5, r=0.908, p=0.033) over the same time period. We thank the Reviewer for the suggestion to perform these analyses and briefly mentioned the results in the revised version of the manuscript (Pages 20-21, lines 368-372).

4. The authors mention in lines 322-324 “Mortality rate, however, did not significantly differ between groups, even after adjustment for age and sex, and further adjustment for baseline serum creatinine (Fig 4B)”; but they should explain this.

We speculate that eculizumab induced terminal complement inhibition translates into reduced inflammation and thrombogenesis and that both effects are expected to translate into reduced morbidity and mortality. However, in our present study the number of fatal events (that were numerically but remarkably less frequent in eculizumab treated patients than in non- eculizumab treated controls), was too small to provide the statistical analyses with adequate power to detect a significant effect on patient mortality considered as a single end point. We briefly discussed this important issue in the revised version of the manuscript (Page 22, lines 392-394).

5. The authors should include the pH of the patients in Table 2. Taking into consideration that there could be some correlation with complement activation in patients with severe COVI-19 since a relationship has been shown in-vitro (Fishelson Z, Horstmann RD, Müller-Eberhard HJ. Regulation of the alternative pathway of complement by pH. J Immunol. 1987 May 15;138(10):3392-5).

As suggested by the Reviewer, in the revised version of the manuscript we included arterial pH of the patients at baseline (Table 1) and during follow-up (Table 2) according to study group. At baseline, arterial pH did not correlate with plasma sC5b-9 levels, serum-induced C5b-9 deposits or thrombus formation. Similarly, changes in arterial pH at one week and two weeks of follow-up did not correlate with concomitant changes in plasma sC5b-9 levels, serum-induced C5b-9 deposits or thrombus formation.

6. Considering that complement and thrombosis converge in the author´s research, they should cite Bont CM, et al., NETosis, complement, and coagulation: a triangular relationship. Cell Mol Immunol. 2019; 16 (1): 19-27. doi: 10.1038 / s41423-018-0024-0.

As properly suggested by the Reviewer we quoted the mentioned reference in the revised version of the manuscript (Page 4, line 430).

Reviewer #2: In this paper, the authors describe the use of eculizumab at the onset of COVID-19 related ARDS as reflected by the need for CPAP. Eculizumab, a monoclonal antibody against complement protein C5, was used in 10 patients with COVID (each patient received two doses 7-10 days apart) and compared them to a cohort of COVID-19 patients who did not receive eculizumab, cared for by the same team. They measured plasma C5b-9, generated ‘in vitro’ C5b-9, and thrombus formation from patient samples, at various time points. The authors describe that the eculizumab group showed improvement in the respiratory rate (primary outcome) at 1 and 2 weeks compared to their baseline, while the control group did not have a similar change. Additionally, they noticed the eculizumab respiratory rate correlated with the ex vivo C5b-9 deposits. This report is clinically relevant in highlighting the role of complement in COVID-19 pathophysiology, especially since a targetable drug such as eculizumab is already being used for other diseases.

We thank the Reviewer for the rewarding comments on our work.

1. From a methodology perspective, the manuscript has been written confusingly about this being a prospective and a retrospective study (line 141). Additionally, the patients who received eculizumab have been reported to be ‘every 6th or 7th patient’ with COVID. At the same time, the manuscript also reports that it was the treating physician's discretion as to who gets eculizumab (lines 104 and 107). These discrepancies concern how this study was performed and how the data was analyzed. There should be a better justification to why no sample size calculation was performed while primary and secondary objectives were defined.

We were authorized by our Ethical Committee to administer eculizumab to our patients with severe COVID-19 in the context of the FDA program of eculizumab off-label compassionate use for the treatment of non-intubated patients with COVID-19. The drug was freely supplied by the manufacturer (Alexion Pharma Italy S.R.L., Milan). As soon as we got the approval by the Ethical Committee we used all supplied medication to prospectively treat all approachable patients from February 2020 to April 2020, that is during the first, dramatic wave of the COVID-19 pandemic. Because of the compassionate use of eculizumab these patients could not be included in a prospective controlled trial. We aimed to treat all patients in need. In actual facts, during that emergency period it was virtually impossible to treat and adequately monitor all consecutive patients who required CPAP ventilator support. Thus, the selection of eculizumab recipients was at the discretion of the treating physician and influenced by logistic reasons. Indeed, as clarified in the Methods of the manuscript, “to avoid overlaps of drug administrations and specific laboratory tests to monitor treatment effects in different patients, and prevent possible interference with clinical patient management, we administered eculizumab to one patient every six to seven consecutive potential candidates”. Then, we identified patients who required CPAP ventilator support during the same inclusion period but who could not access to eculizumab (for the reasons described above). Thus we included into the study eculizumab treated patients and non-eculizumab treated controls. This is why there was no sample size calculation for this study. In the revised manuscript we better clarified this important issue. We pointed out that treatment with eculizumab was prospective but that the controlled study that recorded and analysed data from eculizumab-treated patients and non-eculizumab controls was retrospective in nature (Page 5, lines 94-98).

2. The described study and details of the NCT04288713 study on clinicaltrials.gov do not match. The latter is registered in the United States, and reports of eculizumab dosing every seven days, and the endpoints are also different. The authors should explain this discrepancy and why this deviation in the protocol was made.

The described study and details of the NCT04288713 study on clinicaltrials.gov do not match with our study because the NCT04288713 registration number refers to another study by another group.

3. The methods section should include the time period of this study, as there have been several studies looking at complement blockade in COVID-19. Some studies and case reports have demonstrated increased use of higher doses of eculizumab or more frequent dosing in COVID. These patients with COVID have been reported to have a higher level of complement activation than aHUS or PNH. Was complement blockade examined in the treated patients, along with other complement activation markers?

As reported in the original version of the manuscript, study participants were included from February 2020 to April 2020 (Page 2, line 36; Page 10, line 170).

The Reviewer is right. In a non-randomized proof-of-concept study (Annane D et al. EClinicalMedicine 2020; 28:100590) patients with severe COVID-19 and admitted to ICU were treated with standard care or with standard care plus eculizumab. The initial regimen in the first 10 patients consisted of 900 mg of eculizumab at day 1, 8, 15 and 22 of ICU admission. This dosage resulted in transient and incomplete inhibition of the terminal pathway, which led to protocol amendment and the subsequent patients received higher and more frequent doses of the drug.

In our study, we did not measure the degree of circulating C5 blockade by eculizumab through CH50 or similar assays. However, finding that ex vivo serum-induced C5b-9 deposition on HMEC-1 fully normalized in COVID-19 patients after eculizumab, would support an effective degree of C5 inhibition as we previously documented in patients with aHUS (Galbusera M et al. Am J Kidney Dis 2019; 74:56-72) We added this piece of information in the Limitations paragraph of the revised version of the manuscript (Page 25, lines 456-459).

4. Can the authors comment on why the in vitro C5b-9 at baseline in the control group (Fig 2b) was higher than their plasma sC5b-9 values (as shown in figure 2A)?

The two assays have different meanings. Plasma sC5b-9 values reflect the activation of terminal complement pathway in fluid phase. Soluble C5b-9 formed in the circulation is complexed with Vitronectin (S Protein) and fails to insert into membranes. At variance, the test of serum-induced C5b-9 formation on HMEC-1 is an ex vivo index of terminal complement activation on cell surface with the insertion of lytic membrane attack complex. Thus, values of the two parameters cannot be compared.

5. The two groups have different levels of inflammation even though the p-value wasn’t significant. The group which received eculizumab had higher sC5b-9, higher PaO2, lower LDH, creatinine, GFR and D-dimer. This trend with the eculizumab group being slightly milder at the onset and reportedly had a better response to eculizumab. Even though there was a smaller ‘n’, it may be helpful to match and perform sub-analyses of the Ecu group with ‘selected patients’ from the non-Ecu group with similar respiratory and laboratory profiles.

We wish to point out that none of the differences mentioned by the Reviewer was statistically significant. However, we acknowledge that the lack of significance could be explained by the relatively small number of patients and it cannot be definitely ruled out the possibility that some of the mentioned differences might have some biological relevance, even if statistically non-significant. Thus, to address the suggestion of the Reviewer we performed sensitivity analyses by matching cases and controls (1:3) by all the parameters mentioned by the Reviewer with the exception of sC5b9 that was available in only 3 controls. The results of these additional analyses were consistent with the original results of the study. In particular, four of the ten eculizumab-treated patients died or were discharged with chronic complications as compared to 24 of the 30 matched controls (80%). As previously reported in the whole study population, the event rate was significantly lower in eculizumab-treated patients than in the 30 controls [HR (95CI): 0.28 (0.11-0.70), p=0.006]. Two of the eculizumab-treated patients vs 14 controls (47%) died. Consistently with data in the whole study group, mortality rate considered as a single endpoint did not significantly differ between groups [HR (95CI): 0.33 (0.08-1.28), p=0.109]. Finally, six eculizumab treated patients and six controls (20%) were discharged without chronic complications. Again, consistently with results of original analyses, the event rate was higher in eculizumab-treated patients than in controls [HR (95CI). 3.07 (0.99-9.54), p=0.053], even if the difference was only borderline significant because of the reduced sample size. We thank the Reviewer for the suggestion to perform these additional analyses that provided additional evidence of the robustness of our findings.

6. The control group had elevated D-Dimer levels, and this high level has been shown to be associated with poor outcomes in several COVID-19 studies. Can the reported increase in ex vivo thrombus formation in the control group be attributed to this high baseline D-dimer with or without evidence of pulmonary embolus, contributing to the poorer respiratory outcome? The ‘n’ of 3 and 2 in the control group for thrombus formation assays is severely underpowered and questions the validity of the data.

Baseline D-dimer levels did not differ in the eculizumab and the control group (Table 2 baseline), but thereafter increased at week 1 and week 2 only in the control group, likely reflecting worsening of the disease as compared with the eculizumab group.

Very unlikely D-dimer levels would have influenced significantly the results of ex vivo thrombus formation assay, since patients’ serum was washed out from the HMEC-1 monolayer after the preincubation step, then thrombus formation was assessed by flowing the cells with whole blood from healthy volunteers.

We recognize that the numerosity in the control group is low due to limited quantity of blood that could be taken from so sick patients. However, data were very comparably elevated in the 3 patients analyzed at baseline and at 1 week, and were normal in both patients analyzed at recovery.

7. Authors should report if patients in either group required mechanical ventilation, renal replacement therapy, cardiac dysfunction, systemic thrombosis, antiviral medications, among other outcomes. This would help ascertain if other endpoints were or were not different between these two groups.

In the revised version of Table 1 we have reported that 7 (70%) eculizumab-treated patients and 32 (49%) controls received antiviral therapy (darunavir and cobicistat combination).

During the observation period, 5 (50%) eculizumab-treated patients and 21 (32.3%) controls required mechanical ventilation (p=0.301). There were six (9.2%) cardiovascular events (two cardiogenic shocks, one myocardial infarction, one atrial fibrillation, one atrioventricular block and one supraventricular tachycardia) and six (9.2%) thromboembolic events in controls versus none in eculizumab-treated patients. No patient in either group required renal replacement therapy. We reported these endpoints in the “Other endpoints” paragraph that we have added in the revised version of the manuscript (Pages 19-20, lines 342-348).

8. Can authors also report which patients received hydroxychloroquine and remdesivir and how it may have contributed to their response to COVID-19 infection.

In the revised version of Table 1 we have reported that 9 (90%) eculizumab-treated patients and 42 (65%) controls received hydroxychloroquine (p=0.154). In the original version of the manuscript, we have accidentally indicated that most patients received remdesivir. Instead, none of the eculizumab-treated or control patients was given remdesivir, since during the first wave of COVID-19 the combination of darunavir and cobicistat was considered as first-line antiviral therapy at our institution. We thank the Reviewer for bringing to our attention this issue, that we have addressed in the revised version of the manuscript (Page 10, lines 180-181) and Table 1. In actual fact, 7 (70%) eculizumab-treated patients and 32 (49%) controls were given antiviral therapy (darunvir and cobicistat combination) (p=0.313).

Study findings were unlikely confounded by concomitant treatment with hydroxychloroquine or the combination of darunvir and cobicistat since the proportion of patients given these medications did not differ appreciably between the two study groups. Moreover, available data do not support the use of hydroxychloroquine or the combination of darunvir and cobicistat for the treatment of hospitalized patients with COVID-19 (JAMA 2020; 324:2165-2176; Front Med (Lausanne) 2021; 8:639970).

Submitted filename: PONE-D-21-17700 Answers to reviewers.docx

Click here for additional data file.

25 Nov 2021

Eculizumab in patients with severe coronavirus disease 2019 (COVID-19) requiring continuous positive airway pressure ventilator support: retrospective cohort study

PONE-D-21-17700R1

Dear Dr. Remuzzi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ruud AW Veldhuizen

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript is technically understandable, well written, and the data support the conclusions, it is recommended to accept.

Reviewer #2: The authors have satisfactorily addressed the reviewer's comments and this have strengthened the quality of this paper.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

9 Dec 2021

PONE-D-21-17700R1

Eculizumab in patients with severe coronavirus disease 2019 (COVID-19) requiring continuous positive airway pressure ventilator support: retrospective cohort study

Dear Dr. Remuzzi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Ruud AW Veldhuizen

Academic Editor

PLOS ONE