Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection

Abstract

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers: Washington University School of Medicine (n=134) and Yale School of Medicine (n=49). We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza (n=54), and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV, n=22). We demonstrate that circulating markers of complement activation are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., angiopoietin-2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials.

Fig. 1. Markers of complement activation are higher in COVID-19 compared to non-COVID-19 respiratory failure.

Plasma for determination of circulating markers of complement activation was obtained in patients with COVID-19 (n=134) and influenza (n=54) at Barnes-Jewish Hospital (BJH)/Washington University School of Medicine (WUSM). (A) CONSORT flow diagram showing patient enrollment, allocation and outcomes in the COVID-19 cohort. The CONSORT diagram for the influenza and non-COVID acute respiratory failure cohorts are in Figure S1. Violin plots of differences in sC5b-9 between (B) the influenza (EDFLU, n=54) and COVID-19 (n=124) cohorts, (C) the non-COVID acute respiratory failure (Immunity in Pneumonia and Sepsis, IPS, n=22) and the COVID-19 cohorts (n=124), and (D) restricting both the cohorts from Fig. 1C to those who died (non-COVID, IPS, n=8 vs COVID-19, n=30). Statistical significance is determined using Mann-Whitney U test.

Fig. 2. Complement activation is associated with worse outcomes in COVID-19 in two independent cohorts.

Markers of complement activation were quantified in the plasma at WUSM and Yale University School of Medicine (Yale). Violin plots of sC5b-9 levels in the WUSM COVID-19 cohort in (A) patients requiring ICU admission (n=72) versus those who did not (n=62), (B) patients requiring invasive mechanical ventilation (IMV, n=29) versus those who did not (n=105), and (C) patients who died (n=30) versus those who survived (n=104). (D) A linear regression line shows the relationship between plasma levels of sC5b-9 and C5a. The spline chart demonstrates the mean with 95% confidence intervals. R² represents the goodness-of-fit. The degree of correlation is assessed using Spearman’s rank correlation coefficient test (ρ=0.4909, 95% CI 0.2321 – 0.6848, N=48). In the Yale cross-sectional cohort, concurrently measured C5a levels are utilized to compare (E) patients requiring ICU admission (n=40) versus those who did not (n=9), and (F) patients requiring IMV (n=26) versus those who did not (n=23). Statistical significance is determined using Mann-Whitney U test.

Fig. 3. Alternative pathway activation is associated with worse outcomes in COVID-19.

Comparisons in the levels of components involved in the alternative pathway (AP) in plasma of patients requiring ICU admission (n=26) versus those who did not (n=22), in the WUSM COVID-19 cohort, are presented using violin plots - (A) iC3b: C3 ratio, (B) Factor B, and (D) Ba. (C) A linear regression line shows the relationship between plasma levels of sC5b-9 and Factor B. The spline chart demonstrates the mean with 95% confidence intervals. R² represents the goodness-of-fit. The degree of correlation is assessed using Spearman’s rank correlation coefficient test (ρ=0.4768, 95% CI 0.2146 – 0.6749, N=48). (E) Plasma Ba levels are compared in patients who survived [1,301.0 (966.0 – 2250.0), N=29] versus those who did not [3,266 (2,368 – 6236), N=19], as are the plasma levels of Factor D (F). Statistical significance is determined using Mann-Whitney U test.

Fig. 4. Complement activation is associated with markers of endothelial injury and a prothrombotic state in patients with COVID-19.

A linear regression line shows the relationship between plasma levels of Factor D and (A) angiopoietin-2 (Ang2), (B) thrombomodulin, and (C) von Willebrand factor antigen (vWF:Ag) in the Yale cross-sectional cohort. The spline chart demonstrates the mean with 95% confidence intervals. R² represents the goodness-of-fit. The degree of correlation is assessed using Spearman’s rank correlation coefficient test between Factor D and (a) Ang2 (ρ=0.5095, 95% CI 0.2585 – 0.6960, n=49), (b) thrombomodulin (ρ=0.6050, 95% CI 0.3829 – 0.7609, n=49), and (c) vWF:Ag (ρ=0.3367, 95% CI 0.04612 – 0.5747, n=47). Violin plots are utilized for comparing the levels of (D) Ang2, (E) thrombomodulin, and (F) vWF:Ag in plasma of patients requiring invasive mechanical ventilation (IMV) versus those who did not. Statistical significance is determined using Mann-Whitney U test.