The current pandemia of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a major global health challenge. The clinical spectrum of COVID-19 ranges from asymptomatic carriers to respiratory failure. A dysregulated immune response with evidence of hyperinflammation is observed in patients with severe COVID-19 (1). Siddiqi et al. suggested a disease stage model, where early disease is charaterized by infection and replication of the virus. Subsequently, pulmonary involvement and marked hyperinflammation occur during the next stage (2). The exact factors promoting disease progression are not yet known. However, sustained activation of the complement system (CS) induced by coronaviruses (CoV) seems to play a crucial role in this context.

The complement system (CS) is an integral part of the innate immune system and consists of a number of distinct plasma proteins that act as a first line of defense inducing an inflammatory response after opsonisation of pathogens and dying cells (3, 4). Inflammatory responses include the activation of macrophages, neutrophils, platelets and endothelial cells, and interactions with other plasmatic cascades. While the CS does not seem to be critical for controlling CoV replication, unregulated complement activation - induced by viruses including influenza and CoV - plays a crucial role in the pathogenesis of acute lung injury (ALI). Indeed, an animal model suggests that the CS mediates SARS-CoV-induced lung disease and regulates the proinflammatory response. Complement deficient mice infected with SARS-CoV were affected less severely and showed a reduced lung involvement and lower local and systemic cytokine levels compared to control mice (5). In line, inhibition of complement C5a signaling alleviated lung damage in animal infection models using MERS-CoV (6) and influenza H7N9 (7). Similar results were reported with inhibition of complement cascade C3a in an animal infection model using avian influenza (8). Gao et al. investigated the interaction of MERS-CoV, SARS-CoV, and SARS-CoV-2 with the lectin pathway of complement in more detail (9). They demonstrate an interaction of these highly pathogenic CoV with mannose-binding lectin associated serine protease-2 (MASP-2), the key activator of the lectin pathway of complement (10), leading to uncontrolled activation of the complement cascade. In line, MASP-2 knock-out mice and mice treated with MASP-2 inhibitors showed significantly milder symptoms in a virus protein mouse pneumonia model. Mannose-binding lectin, the pattern recognition molecule of the lectin pathway, that activates MASP-2 upon binding to pathogens, was found to bind to SARS-CoV spike glycoprotein (11). In COVID-19 patients, increased plasma levels of complement activation products C5a and soluble C5b-9 have been observed (12). Furthermore, autopsy findings from 5 patients with severe COVID-19 revealed excessive complement activation in lung tissue associated with complement-mediated microthrombotic disease (13).

Beside CS activation, involvement of the contact activation (CAS) and kallikrein-kinin system (KKS) in thromboinflammation, capillary leakage and subsequent pulmonary angioedema has been suspected. Angiotensin-converting enzyme 2 (ACE2), a cell membrane bound protein used by SARS-CoV-2 to enter cells, also inactivates bradykinin degradation products, and expression of ACE2 as well as its enzymatic activity is decreased by SARS-CoV (14). Hence, the interaction of SARS-CoV-2 with ACE2 may impair its function, leading to a relative abundance of bradykinin degradation products and local pulmonary edema.

The role of the CAS has not been elucidated in COVID-19. However, dysregulated coagulation is commonly observed in critically-ill patients with COVID-19 and thromboembolism has been reported more frequently compared to other diseases causing severe sepsis (15, 16). This may be a consequence of over-activation of CAS since sepsis and ARDS are prototypic states that strongly activate the CAS (17, 18). Consistent with thromboinflammation, microthrombi have been observed in autopsy series in COVID-19 (19).

C1 esterase inhibitor (C1INH) is a strong inhibitor of the CS, CAS, and KKS and interacts with stressed endothelial cells. C1INH treatment was associated with reduced capillary leakage after stem cell transplantation and organ damage in human sepsis (20–22). Recently, C1INH was identified among other inflammatory proteins to be upregulated in severe vs. non-severe COVID-19 in a proteomic and metabolomic characterization (23). In a mouse model of highly pathogenic CoV, C1INH treatment was able to block MASP-2 mediated overactivation of the complement system and subsequent lung injury and death (9). Lastly, when examining published interactomes of SARS-CoV-1 and−2, a significant interaction of C1INH with CoV-1 proteins was documented including proteins that are highly similar to their orthologous CoV-2 proteins (24).

Investigating a regulator of the CS, CAS, and KKS for the first time in severe COVID-19, we report the experience of early administration of conestat alfa, a recombinant human C1INH, in non-critically ill patients to prevent deterioration.

The treatments occurred at the University Hospital Basel from April 2, 2020, to April 28, 2020 and were approved by the Ethics Committee of Northwest and Central Switzerland (EKNZ 2020-1013). All participants consented to the compassionate use of conestat alfa and the acquisition of health-related personal data and biological material.

Five patients (4 males, median age 60 years, range 53–85 years) were treated with conestat alfa. Four had preexisting medical conditions (Table 1). A CT scan of the chest demonstrated moderate to severe pneumonia with involvement of lung parenchyma ranging from 11 to 39% (Figure 1A). All patients received hydroxychloroquine and lopinavir/ritonavir (LPV/r) as per local treatment guideline at the time. Conestat alfa was administered a median of 1 day (range 1–7 days) after admission for 48 h and was well-tolerated. No treatment associated serious adverse events were recorded.

A range of strategies have been proposed and are being evaluated to dampen hyper-inflammation in COVID-19 such as corticosteroid therapies, antibodies against IL-1 or IL-6, treatments interfering with the interferon pathway and anti-tumor necrosis factor therapies (28). In particular, the CS has gained attention as a potential therapeutic target in COVID-19 patients (29, 30), and first experiences targeting C3 and C5 of the complement system have been published (31, 32). However, in this case series, we investigated – to the best of our knowledge – for the first time a strategy of interfering with 3 plasmatic cascades including the CS (i.e., CS, CAS, and KKS) in 5 non-critically ill patients with severe COVID-19. Treatment with conestat alfa over 48 h was well-tolerated. No signal of impaired viral clearance emerged from our case series.

The clinical condition improved in all but 1 patient, reflected by immediate defervescence, improvement in oxygen requirements, and a decline in systemic inflammation and markers of complement activation. Notably, 4/5 patients treated early did not require mechanical ventilation despite the presence of risk factors such as markedly elevated inflammatory proteins. In the majority of patients (4/5) IL-6 levels were markedly elevated (>80 ng/ml) during the disease course, a finding shown to predict respiratory failure and mechanical ventilation, previously (33).

Similar to previous studies in sepsis (20), C1INH protein levels were elevated in COVID-19 patients consistent with an acute phase response. However, elevated C1INH levels may not be sufficient to block ongoing extensive CS, CAS, and KKS activation in COVID-19. An increased amount of modified (cleaved) inactive C1INH in patients with severe sepsis was documented previously, which may indicate a relative C1INH-deficient state (34). Of note, concentration of complement activation products C4d and C5a decreased following conestat alfa administration. Early supplementation of C1INH with conestat alfa is thus a plausible treatment option in selected patients with COVID-19, thereby inhibiting upstream complement proteases and its associated over-activation of the CS in COVID-19 (13, 29).

In this uncontrolled case series of 5 non-critically ill patients with severe COVID-19 pneumonia, administration of conestat alfa over 48 h to inhibit the CS, CAS and KKS was well-tolerated and associated with improvement in the clinical condition of 4 patients. Consequently, we have initiated a randomized controlled trial to investigate this promising approach (ClinicalTrials.gov, number NCT04414631).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethics Committee of Northwest and Central Switzerland (EKNZ 2020-1013). The patients/participants provided their written informed consent to participate in this study.

SM, BG, MT, and MO designed the study. MO, PU, SM, PC, IH, MR, PS, GS, MT, and SB performed the study, collected, analyzed, and interpreted the data. PU and MO drafted the manuscript. All authors critically revised the manuscript.

BG reports being employed by Pharming Group NV. MT reports receiving grants from the Swiss National Science Foundation, Roche, Novartis, and Idorsia outside of the submitted work. MO reports receiving consulting fees from Pharming Biotechnologies B.V. during the conduct of the study and grants from Pharming Biotechnologies B.V. outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.