key: cord-0877578-qrcz7yzq authors: Henry, Brandon Michael; Szergyuk, Ivan; de Oliveira, Maria Helena Santos; Lippi, Giuseppe; Benoit, Justin L.; Vikse, Jens; Benoit, Stefanie W. title: Complement levels at admission as a reflection of coronavirus disease 2019 (COVID‐19) severity state date: 2021-05-19 journal: J Med Virol DOI: 10.1002/jmv.27077 sha: 5f700cb538f16bc341b33df40fe2949484ce82c2 doc_id: 877578 cord_uid: qrcz7yzq Complement system hyperactivation has been proposed as a potential driver of adverse outcomes in severe acute respiratory syndrome coronavirus 2 infected patients, given prior research of complement deposits found in tissue and blood samples, as well as evidence of clinical improvement with anticomplement therapy. Its role in augmenting thrombotic microangiopathy mediated organ damage has also been implicated in coronavirus disease 2019 (COVID‐19). This study aimed to examine associations between complement parameters and progression to severe COVID‐19 illness, as well as correlations with other systems. Blood samples of COVID‐19 patients presenting to the emergency department (ED) were analyzed for a wide panel of complement and inflammatory biomarkers. The primary outcome was COVID‐19 severity at index ED visit, while the secondary outcome was peak disease severity over the course of illness. Fifty‐two COVID‐19 patients were enrolled. C3a (p = 0.018), C3a/C3 ratio (p = 0.002), and sC5b‐9/C3 ratio (p = 0.021) were significantly elevated in with severe disease at ED presentation. Over the course of illness, C3a (p = 0.028) and C3a/C3 ratio (p = 0.003) were highest in the moderate severity group. In multivariate regression controlled for confounders, complement hyperactivation failed to predict progression to severe disease. C3a, C3a/C3 ratio, and sC5b‐9/C3 ratio were correlated positively with numerous inflammatory biomarkers, fibrinogen, and VWF:Ag, and negatively with plasminogen and ADAMTS13 activity. We found evidence of complement hyperactivation in COVID‐19, associated with hyperinflammation and thrombotic microangiopathy. Complement inhibition should be further investigated for potential benefit in patients displaying a hyperinflammatory and microangiopathic phenotype. Evidence has emerged that multiorgan injury from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is propagated by a maladaptive, dysregulated host immune response, including complement hyperactivation. [1] [2] [3] The complement system plays a central role within the innate immune system, responding to invading pathogens via the classical, alternative, and lectin pathways, which all converge at component 3 (C3), a core constituent in this pathway. 4 This common product results in enzymatically-driven formation of many activation products (C3a, C5a), and culminates in formation of the membrane attack complex (C5b-9), which kills invading pathogens via generation of membrane pores. 4 Complement not only plays a key role in the first line of defense against infectious agents, but also acts to bridge innate and adaptive immune responses through activation of T and B cells, and creation of immunologic memory. 5 While complement is essential in defense against viral infections, its hyperactivation and dysregulation can result in widespread systemic multiorgan damage. 6 Moreover, complement mediates vascular injury in various thrombotic microangiopathies (TMAs), such as atypical hemolytic uremic syndrome and antiphospholipid syndrome. 7, 8 In keeping with this previous evidence, complement has been implicated in the pathogenesis of microvascular thrombosis frequently observed in patients with coronavirus disease 2019 . 9 In five COVID-19 patients with pulmonary and dermatologic signs of microvascular thrombosis, Magro et al. identified terminal complement (C5d-9), C4d, and mannose-binding protein-associated serine protease 2 (MASP-2) deposits in biopsies of lung and skin tissue, thus highlighting a potential contribution of the complement pathway to the process of thrombotic microvascular injury via endothelial damage and subsequent activation of the coagulation cascade. 9 Gao et al. 10 performed lung biopsies of patients who died from COVID-19, and also reported evidence of complement activation, including deposits of mannose binding lectin, MASP-2, C4a, C3, and C5b-9. Additionally, patients with severe COVID-19 illness were found to have significantly increased serum C5a, a potent anaphylatoxin which triggers the release of a myriad of inflammatory cytokines from leukocytes. 2,10 Holter and colleagues observed increased values of all markers of complement early in the course of SARS-CoV-2 infection, as well as an association of C4d and C5b-9 with respiratory failure and systemic inflammation. 11 In accordance with these findings, small case series using anti-C5a and anti-C5 monoclonal antibodies (BDB-0001 and eculizumab) in patients with COVID-19 reported decreases in systemic inflammation and improvement in pulmonary function. 10, 12 Hence, in this study we have carried out an assessment of a complete complement panel in patients with COVID-19, using blood samples collected at initial presentation at the Emergency Department (ED). Our aim was to analyze the relationship between complement parameters and COVID-19 severity, as well as explore correlations between the complement cascade and other systems, to help elucidate the pathophysiology of SARS-CoV-2 infection. Adult patients with reverse transcriptase polymerase chain reaction confirmed SARS-CoV-2 infection presenting at the ED of the University of Cincinnati Medical Center\ between April and May 2020 were enrolled in this prospective, observational study. Blood samples were taken as part of routine blood draws in the ED, and analyzed at the Clinical Nephrology Lab of the Cincinnati Children's Hospital Medical Center, a national referral center for complement testing. This study was approved by the Institutional Review Board of the University of Cincinnati and received a waiver of informed consent. This study was conducted in compliance with the Declaration of Helsinki, under the terms of relevant local and national legislation. Serum levels of 50% hemolytic complement activity (CH50, representing total complement activity, MicroVue; Quidel Corporation), alternative pathway activity and lectin pathway activity (Wieslab, SVAR) were assessed using enzyme-linked immunosorbent assays (ELISA). Quidel MicroVue ELISA kits were also used to measure complement components, including C3a, C4a, C5a, sC5b-9, Bb, and C1 inhibitor activity. C3, C4, and C1 inhibitor antigen were Analysis System (Brea). All assays were run according to manufacturer instructions; ELISA assays were run on either the DS2 or DSX automated ELISA processing systems (DYNEX Technologies). The primary outcome was COVID-19 disease severity at index ED visit, while the secondary outcome was peak disease severity over the course of illness. Patients were stratified into three severity level classes, according to the World Health Organization R&D Blueprint for COVID-19 severity scale, 13 as follows: mild (ambulatory), moderate (hospitalized), severe (requiring intensive care unit admission). Categorical variables were reported as frequencies (%), whilst continuous data was described using median and interquartile ranges (IQR). Statistically significant differences in laboratory values among groups were identified using the Kruskal-Wallis test, followed by Dunn test for multiple comparisons (when necessary). The relationship between complement parameters and inflammatory biomarkers was tested using Spearman's correlation coefficient. C3a/C3 and sC5b-9/C3 ratios were calculated to assess potential alternative pathway and terminal pathway hyperactivation, respectively. Multivariable logistic regression was used to identify complement variables independently predicting disease severity after adjusting for age, sex, and comorbidities, with calculation of odds ratios and their 95% confidence intervals (95% confidence interval). Variable selection was based on univariate analysis and Stepwise algorithm. All statistical analyses were performed using R software (version 4.0.2; R Foundation for Statistical Computing), with p < 0.05 being considered statistically significant. A total number of 52 adult patients with laboratory-confirmed SARS-CoV-2 infection were enrolled in the study. The median age was 50.5 (IQR: 39.3-66.0) years and 30 (57.7%) were males. Patient demographics and outcomes are summarized in Table 1 . At ED presentation, 6 patients (12%) were severe, while over the course of illness 16 (31%) patients reached a peak severe disease status. The values of complement parameters stratified according to COVID-19 disease severity at ED presentation and peak are summarized in Table 2 . Statistically significant elevations were observed in C3a (p = 0.018) over the normal range, as well as C3a/C3 ratio (p = 0.002) and sC5b-9/C3 ratio (p = 0.021) in those with severe disease at ED presentation ( Figure 1 ). For peak disease, only C3a (p = 0.028) and the C3a/C3 ratio (p = 0.003) were significantly different between severity groups, with moderate group displaying the highest values. C5a and sC5b-9 had nonsignificant trends toward higher levels those with more severe disease both at ED presentation and at peak, although levels were relatively within normal range. When adjusted for age and comorbidities, no complement variable measured at ED presentation was predictive of progression to severe disease during illness. Spearman's correlation coefficients representing association between C3a, C3a/C3, as well as sC5b-9/C3 and a panel of inflammatory markers are presented in Table 3 . Levels of the individual inflammatory markers measured at ED presentation are summarized in Table S1 . In- In this prospective study, we observed evidence of complement hyperactivation in patients presenting to the ED with severe disease, as reflected by significant increase in C3a above the norm, as well as elevations in the C3a/C3 and sC5b-9/C3 ratios, which are suggestive of alternative and terminal pathway hyperactivation, respectively. C3a is an anaphylatoxin that induces inflammation, endothelial activation, 14 as well as coagulation via binding of the C3a receptor (C3aR) on platelets, 9, 15 in turn aggravating microthrombosis and contributing to a TMA-like phenomenon. 16 Interestingly though, emerging evidence also proposes an Table S1 . Abbreviations: ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; CRP, C reactive protein; IL-6, interleukin-6; LDH, lactate dehydrogenase; TNF-α, tumor necrosis factor-α; VWF:Ag, von Willebrand Factor antigen; WBC, white blood cells. measured at time of ED presentation, C5a and sC5b-9 displayed a nonsignificant trend toward higher levels among patients with moderate and severe disease severity both in the ED and at peak during hospitalization. Moreover, the trend for sC5b-9 is in part supported by significant elevations in the sC5b-9/C3 ratio in patients with severe disease on admission. Absence of such considerable elevations in C5a and sC5b-9 in our study may be attributed to the differences in severity of patients at time of sampling and the nonsignificance may be explained by differences in sample sizes, whereby the smaller cohort in our study compared to the study by Cugno et al. may have limited the statistical power to identify these differences, or by heterogeneity related to underlying baseline characteristics of included patients in both studies. 20 Additionally, C5a has a much shorter half-life than relatively more stable C3a, as it quickly binds to high affinity C5a receptors (C5aR) on neutrophils, 21 complicating the ability to detect accurate levels of C5a in blood samples. In multivariable regression, complement hyperactivation did not predict progression to severe disease in patients with mild or moderate illness at ED presentation. This suggests that complement levels may only reflect acute disease status, becoming hyperactivated following other insults during progression to more critical illness. This finding is in opposition to a recent report suggesting that complement overactivation on admission was associated with development of respiratory failure. 11 We suspect that this heterogeneity between studies may also be attributed to the differences in severity of patients at time of sampling. bearing a deficient C3 gene had significantly less respiratory dysfunction than those with an intact complement system, despite equivalent viral loads. 22 In COVID-19, Ramlall et al. noted that none of the patients with complement deficiency who would normally be at higher risk of developing severe infection required invasive oxygen support or died. 23 Moreover, the role of complement in driving COVID-19 severity is reflected in small clinical trials demonstrating reduced system inflammation and lung injury with anti-C5, anti-C5a, and anti-C3 monoclonal antibodies. 10, 12, 24 Our results also show that C3a and the C3a/C3 and sC5b-9/C3 ratios were correlated with an array of inflammatory biomarkers, thus suggesting a close interplay between different systems. Elevated C3a was correlated with fibrinogen levels, which is in accordance with extensive cross-talk between complement and coagulation systems. 25 20 These findings should thus be corroborated by additional multi-national studies. However, finding statistically significant differences in C3a level, and the C3a/C3 and sC5b-9/C3 ratios in a design with high probability of a false negative error, reinforces our confidence that the results are valid. Additionally, our study is strengthened by many multi-system biomarkers analyzed, enabling assessment of potential interaction between complement and other systems. Finally, as this study was exploratory, we did not control for multiple comparisons. In conclusion, our results suggest that complement hyperactivation may be associated with hyperinflammation and thrombotic microangiopathy in COVID-19. As complement activation may propagate immune-mediated and thrombotic organ damage, further studies should be planned to address whether complement inhibition may have potential therapeutic benefits in selected categories of patients with a hyperinflammatory and microangiopathic phenotype. This study was funded by the University of Cincinnati College of Program. The authors declare that there are no conflict of interests. The peer review history for this article is available at https://publons. com/publon/10.1002/jmv.27077 The data that support the findings of this study are available from the corresponding author upon reasonable request. This study was approved by the Institutional Review Board (IRB) of the University of Cincinnati and received a waiver of informed consent on the basis of no more than minimal risk. 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