key: cord-0943025-5x2eqjyu authors: Laurence, Jeffrey; Mulvey, J. Justin; Seshadri, Madhav; Racanelli, Alexandra; Harp, Joanna; Schenck, Edward J.; Zappetti, Dana; Horn, Evelyn M.; Magro, Cynthia M. title: Anti-complement C5 therapy with eculizumab in three cases of critical COVID-19 date: 2020-08-06 journal: Clin Immunol DOI: 10.1016/j.clim.2020.108555 sha: 4b166e095e40c4d3a618d678b66117a312c8861d doc_id: 943025 cord_uid: 5x2eqjyu Respiratory failure and acute kidney injury (AKI) are associated with high mortality in SARS-CoV-2-associated Coronavirus disease 2019 (COVID-19). These manifestations are linked to a hypercoaguable, pro-inflammatory state with persistent, systemic complement activation. Three critical COVID-19 patients recalcitrant to multiple interventions had skin biopsies documenting deposition of the terminal complement component C5b-9, the lectin complement pathway enzyme MASP2, and C4d in microvascular endothelium. Administration of anti-C5 monoclonal antibody eculizumab led to a marked decline in D-dimers and neutrophil counts in all three cases, and normalization of liver functions and creatinine in two. One patient with severe heart failure and AKI had a complete remission. The other two individuals had partial remissions, one with resolution of his AKI but ultimately succumbing to respiratory failure, and another with a significant decline in FiO(2) requirements, but persistent renal failure. In conclusion, anti-complement therapy may be beneficial in at least some patients with critical COVID-19. absolute neutrophil count (ANC) acute kidney injury (AKI) SARS-CoV-2-associated Coronavirus disease 2019 left ventricular ejection fraction (LVEF) mannose binding lectin (MBL)-associated serine protease (MASP2) Progressive respiratory failure and acute kidney injury (AKI) are associated with the highest risk of mortality in SARS-CoV-2-associated Coronavirus disease 2019 (COVID-19) [1] [2] [3] . Organ dysfunction is linked to a systemic microvascular thrombosis in the context of complement activation and a persistent inflammatory state [4] . Increasing D-dimers and high levels of proinflammatory cytokines, particularly interleukin (IL)-6 and C-reactive protein (CRP), are significant prognostic indicators of in-hospital mortality in COVID-19 [3] [4] [5] [6] . In terms of the complement cascade, early COVID-19 lung involvement is characterized by a thrombogenic vasculopathy with prominent deposits of terminal complement component C5b-9, C4d, and the lectin pathway of complement enzyme mannose binding lectin (MBL)-associated serine protease (MASP)2 [4] . This pattern of pulmonary vascular damage with complement deposition is also a feature of the cutaneous [4] and renal [7] microvasculature in severe COVID-19, findings consistent with systemic complement activation. Neither initial nor peak viral loads of SARS-CoV-2 in saliva or nasopharyngeal samples distinguish mild from severe or critical COVID-19, nor do they correlate with known comorbidities for COVID-19 severity such as diabetes mellitus and obesity [8] . These data suggest that, possibly apart from initiation of SARS-CoV-2 antiviral drugs very early after infection, such agents alone will be insufficient to control COVID-19 progression. Indeed, infection of wild-type mice with mouse-adapted SARS-CoV led to marked weight loss and respiratory dysfunction, and Our group [4] , and others [11, 12] , have proposed that complement blockade might be of benefit in severe COVID-19. Very recently, four patients with COVID-19 characterized by respiratory failure were reported to have a complete recovery following administration of the anti-C5 humanized monoclonal antibody (mAb) eculizumab [13] . However, these patients did not have laboratory findings which correlate with extended intensive care unit hospitalization or increased mortality, did not have an AKI, and one of the four had normal D-dimer levels, the others minimally elevated values. A report of a moderate COVID-19 patient treated with the cyclic peptide inhibitor of complement C3, AMY-101, is also of interest, but again this did not involve a severe or critical case [14] . We describe three patients with critical COVID-19 who experienced complete or partial remissions following eculizumab administration. These cases represent the first three patients who met the criteria described below in consideration of the use of anti-complement therapy. They were among the first 14 consecutive cases considered over a six week period. Cutaneous biopsies were requested by ICU staff responsible for the care of those 14 patients in order to assess for possible intervention with anticomplement therapy, based on the fact that clinical conditions were deteriorating despite use of corticosteroids, hydroxychloroquine, the antiviral remdesivir, and the anti-IL-6 receptor mAb tocilizumab, and reports of sustained complement activation in critical COVID-19 [4] . The coding system for these three patients-Cases 5, 6 and 9--corresponds to a dermatopathologic study of those patients pre-eculizumab treatment, along with 11 other severe or critical cases (submitted for publication). On hospital admission, all three patients were given hydroxychloroquine, dosed as 600mg every 12 hours for one day, then 400mg every 12 hours for 4 days, for a total of 5 days. Tocilizumab was initiated and dosed as described in the Case reports (below). Cases 6 and 9 also received remdesivir, 5mg/kg intravenously daily for 10 days, with the timing of drug initiation decided by the treating physician. Intermittent moderate doses of steroids were used at variable intervals in all three patients. Anticoagulation regimens, as outlined in the Case reports and illustrated in Fig IHC assessment for the deposition of C5b-9, MASP2, and C4d via a diaminobenzidene (DAB) technique utilized commercial antibodies (Agilent, Santa Clara CA, M077701-5 for C5b-9 and C4d, and Sigma, St. Louis, MO, HPA029313 for MASP2) applied to formalin-fixed, paraffinembedded sections of skin. Heat-mediated antigen retrieval with Tris-EDTA buffer (pH = 9, epitope retrieval solution 2) was performed for 20 min, followed by incubation with each antibody for 15 min. Details have been described elsewhere [4] . Hematoxylin and eosin counterstain was used and mounted with Leica Micromount. Peak lactate dehydrogenase (LDH) levels >550 U/L and absolute neutrophil counts (ANC) >11.6/mm 3 are predictive of ICU admission and in-hospital mortality for COVID-19 [3, 6] , and these levels were seen in all three cases ( Fig. 1A-C) . Characteristics pre-disposing to, or indicative of, a hypercoaguable state in COVID-19, include evidence of liver damage by elevation of aspartate transaminase (AST) [15] , and skeletal muscle damage assessed by creatine kinase (CK) [16] , and these were also present in all three cases ( Additional 1200mg doses of eculizumab were given on days 13 and 20 (Fig. 1A) to >12,000bg/ml (Fig. 1B) , with marked increases in ANC, LDH, and AST (Fig. 1B) shortly thereafter. CK rose to 912 U/L. Given elevated CRP levels to 32.4mg/dL, and development of an AKI, an 800mg intravenous infusion of tocilizumab was administered and remdesivir initiated on day 6, along with a change in anticoagulation regimen to therapeutic enoxaparin. By day 14 there was a stabilization of his D-dimers and decline in LDH, but this was followed by a dramatic rise in ANC and AST, and a continued rise in serum creatinine (Fig. 1B) . was vaccinated for N. meningitides, maintained on prophylactic antibiotics, and given a 900mg intravenous infusion of eculizumab on day 21, followed by 1200mg doses on days 28 and 42. His course has been complicated by Pseudomonas aeruginosa sepsis and P. aeruginosa and Candida albicans lung abscesses. D-dimer levels have stabilized at about 1800ng/ml, and his ANC and LDH have normalized. His atrial fibrillation and acral livedoid rashes have resolved. His FiO2 requirements have markedly decreased from peaks of 100% to 45% with low positive endexpiratory pressure. He remains in non-oliguric renal failure and is on intermittent hemodialysis, but is being prepared for discharge to a rehabilitation service. Administration of the anti-C5 monoclonal antibody eculizumab led to a rapid and marked decline in biomarkers for systemic clotting and inflammation in all three of our critical COVID-19 patients, accompanied by resolution of livedoid rashes, indicative of a systemic thrombotic vasculopathy [4] , in the two cases receiving multiple doses of eculizumab. There was also variable improvement in functions of the lung, heart and kidney, with a complete response in one patient. This is noteworthy as eculizumab was instituted only after failure of these critically ill individuals to respond to multiple interventions, including steroids, remdesivir, and tociluzimab. Advanced COVID-19 is characterized by a hyper-inflammatory and hypercoaguable state. This is accompanied by systemic thrombotic vascular injury and activation of complement cascades [4] . However, the sequence of these events, and the precise mechanisms of their induction, is unclear. SARS-CoV-2 may directly activate the LP of complement through binding to MBL via its glycoprotein envelope spikes, as has been documented for SARS-CoV [9] , and suggested by the demonstration by our group of CoV-2 envelope spike protein binding to MASP2 [4] . Multiple positive feedback loops involving complement receptor-mediated inflammatory cytokine release and interactions among leukocytes, platelets, and endothelial cells have been documented in a variety of pathologic settings [18] . Significant deposits of complement components C5b-9, C4d, and MASP2 in the pulmonary, renal, and cutaneous microvasculature of SARS-CoV-2-infected patients [4, 7] , accompanied by microthrombosis, are consistent with systemic activation of complement and related pathology. Complement components C3a and C5a activate platelets, and can initiate the coagulation cascade with J o u r n a l P r e -p r o o f Journal Pre-proof thrombin generation via a variety of mechanisms, including tissue factor expression elicited by pro-inflammatory cytokines from monocytes and endothelial cells [19, 20] . Although complement levels were within the normal range or only slightly elevated in our cases, this may represent consumption of complement proteins at sites of tissue injury, as is classic for certain acute autoimmune disorders [21] . Indeed, total hemolytic complement levels for Case 5 were 0, despite extensive deposition of C5b-9, C4d, and MASP2 in her microvasculature. Plasma levels of C3a and C5a would more accurately reflect complementdriven disease activity than standard measures of serum C3 and C4 [21] . C5a and soluble C5b-9 are elevated in plasmas of patients with severe COVID-19 [22] . One caveat in the interpretation of plasma levels of complement components is variations in clearance, with soluble C5b-9 less rapidly cleared than other factors. Our cases extend the observations on use of eculizumab and an anti-C3 agent in much less severe COVID-19 cases [13, 14] . As complement and inflammatory cytokines can interact in positive feedback loops [4, 23] , dual intervention with eculizumab and the JAK1/2 inhibitor ruxolitinib was proposed, and clinical improvement using this combination in seven cases of COVID-19 recently reported [24] . However, the moderate FiO 2 requirements (median 50%), minimal elevation in D-dimers (median 138ng/ml), and absence of AKI in these patients [24] suggest the need for evaluation of such strategies in more severe/critical cases. There are limitations to attributing the responses observed in our cases to eculizumab. First, a small number of patients were studied, and a complete response obtained in only one of the two cases receiving multiple doses of eculizumab. Second, delayed effects of other therapeutics are possible. A prominent impact from remdesivir is unlikely, given its lack of effect on mortality in at least one large study in severe COVID-19 [25] . All three patients also received tocilizumab. IL-6 inhibition was unlikely to have influenced Cases 5 or 6, where only a single dose was given and little change in ANC or serum creatinine was noted, but it could have influenced responses in Case 9. For example, tocilizumab can interfere with complement signaling pathways to suppress C5aR1 expression [26] , and therefore may have additive or synergistic effects with anti-complement therapies. Third, the doses of eculizumab administered initially, 900mg, which are standard in the treatment of atypical hemolytic-uremic syndrome (aHUS) [27] , as well as its increase to 1200mg in subsequent infusions, may have been Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in the New York City area Features of 20 133 UK patients in hospital with covid-19 using the ISARIC WHO Clinical Characterization Protocol: prospective observational cohort study Kidney disease is associated with in-hospital death of patients with COVID-19 Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases COVID-19 and its implications for thrombosis and anticoagulation Hematologic parameters in patients with COVID-19 infection Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis. mBio Blockade of the C5a-C5aR axis alleviates lung damage in hDPP4-tranasgenic mice infected with MERS-CoV Complement as a target in COVID-19? 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Complement activation in patients with COVID-19: a novel therapeutic target COVID-19: Immunology and treatment options Combination of ruxolitinib and eculizumab for treatment of severe SARS-CoV-2-related acute respiratory distress syndrome: a controlled study Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicenter trial IL-6 receptor inhibition by tocilizumab attenuated expression of C5a receptor 1 and 2 in non-ST-elevation myocardial infarction Defining treatment duration in atypical hemolytic uremic syndrome in adults: a clinical and pathological approach Dr. Laurence is a Senior Scientist for Programs at amfAR, The Foundation for AIDS Research, and received funding from the Angelo Donghia Foundation in support of this study.