IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function

Abstract

BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).

Keywords: Autoimmunity; COVID-19; Rheumatology.

Figure 1. Flow diagram for identification of anti-ACE2 IgM antibodies.

Anti-ACE2 IgM antibodies were assayed by ELISA in serum from 66 patients with COVID-19, 52 COVID-19 patients with multiple bleeds available, 133 disease controls, and 30 healthy controls. The functional consequences of these antibodies were investigated with 3 different assays using IgM purified from anti-ACE2 IgM–positive sera. MDA5 DM, melanoma differentiation-associated 5 dermatomyositis.

Figure 2. Anti-ACE2 IgM antibodies are found in patients with COVID-19.

(A–C) Antibodies were assayed by ELISA in the combined COVID-19 cohort (n = 118 patients). (A) Number of patients with and without anti-ACE2 IgM antibodies shown grouped by disease severity: 27.2% of severe patients were anti-ACE2 positive compared with 3.8% with moderate COVID-19 (P = 0.0009; 2-tailed Fisher’s exact test). (B and C) Data from anti-ACE2 IgM (B) and IgG (C) ELISAs are presented as corrected OD 450 absorbance units. These data were obtained on all the patients with COVID-19 in A, as well as from 30 healthy controls. Red dots in the IgG panel denote IgG-positive samples that also have anti-ACE2 IgM antibodies. The horizontal line on each plot represents the cutoff for assigning a positive antibody status. (D) Anti-ACE2 IgM antibodies are detected in patients with COVID-19 but not in other infectious and autoimmune disease controls.

Figure 3. Clinical features of anti-ACE2 IgM–positive COVID-19 patients compared with those who do not have these antibodies.

(A) Age, (B) BMI, (C) sex, and levels of (D) temperature, (E) CRP, (F) D-dimer, and (G) neutrophils were compared between the anti-ACE2 IgM–positive and –negative COVID-19 patient groups. Red and blue colors denote anti-ACE2 IgM antibody–positive and –negative status, respectively. Box plots show median, 25th and 75th percentiles, and whiskers min to max. (D–G) Anti-ACE2 IgM-positive patients had higher average body temperature beginning early after hospital admission, followed by elevated CRP and D-dimer measurements. The IgM anti-ACE2–positive group had statistically significantly higher average temperatures, CRP, and D-dimer levels over the first 10 days of hospitalization than the IgM-negative group (P = 0.0001, 0.02, and 0.001, respectively). Average absolute neutrophil levels (G) were not statistically different between the 2 groups. Analyses in panels D–G use linear mixed effects model Wald test with 4 degrees of freedom (see Statistics). (H) Longitudinal analysis of anti-ACE2 IgM antibodies. For all those anti-ACE2 IgM–positive patients with multiple banked sera available (16/18), anti-ACE2 IgM and IgG antibodies were quantitated over time. Red and blue lines on each plot denote anti-ACE2 IgM and IgG antibodies, respectively. Solid black bars represent steroid treatment periods. Additional examples are shown in Supplemental Figure 4.

Figure 4. IgM isolated from SARS-CoV-2–infected patients, but not healthy donors, binds to ACE2.

Equilibrium binding titrations (A–C) and kinetic traces (D–K) of immobilized ACE2 and IgM purified from human serum, as measured by biolayer interferometry. (A–C) Normalized responses at the indicated concentrations of purified IgM from 3 healthy donors and 5 SARS-CoV-2–infected patients are plotted (data from 3 independent experiments). Equilibrium dissociation constants (K_D) calculated by fitting to a 4-parameter logistic regression model are provided. (D–K) The indicated percentages (color-coded on the right side of K) represent 2-fold dilutions of the 8 purified IgM preparations shown in A–C. Binding constants were determined via global fitting for each IgM sample using Octet Data Analysis software (assuming a 1:1 binding model). Quantitation of the data shown in A–K is provided in Supplemental Table 2.

Figure 5. Features of anti-ACE2 IgM antibodies.

(A) Anti-ACE2 IgM antibodies from patient CV-1 or control do not inhibit ACE2 activity. Positive and negative controls were ACE2 alone, and ACE2 plus ACE2 inhibitor, respectively (see Supplemental Figure 6A). (B) IgM antibodies to ACE2 activate complement. Purified IgMs from anti-ACE2 IgM–positive COVID-19 patients (n = 8) and healthy controls (n = 11) was used for C1q binding assays. Values are means from 2 independent experiments performed on different days. ***P < 0.0001, Mann-Whitney test. (C–G) Anti-ACE2 IgM affects the pulmonary endothelium. (C) Phase image of a pulmonary microvessel (top). Fluorescence images of microvessels exposed to anti-ACE2–negative IgM (HC) or anti-ACE2–positive IgM (CV) after perfusion with 10 kDa dextran (lower). Representative images across n = 3 to 6 independent experiments for each IgM condition are shown. (D) ACE2 and CD31 microvessel staining following 24-hour perfusion with IFN-α/γ. Representative images across n = 3 independent experiments are shown. (E) C3c staining after perfusion with IFN and anti-ACE2–positive or control IgM (3.33 μg/mL). Representative images across n = 3 independent experiments are shown. (F) Permeability of microvessels perfused with IFN and anti-ACE2–positive IgM (CV) or anti-ACE2 negative IgM (HC) (100 μg/mL). D.L., detection limit. A linear mixed effects model was used to test the effect of anti-ACE2–positive IgM on permeability. n = 3 to 5 for each IgM condition; each dot represents an independent replicate. (G) Inhibition of microvessel permeability to 10 kDa dextran in response to anti-ACE2 IgM is IFN dependent. Low CV-1: 3.33 μg/mL; affinity-purified anti-ACE2 IgM: 100 ng/mL. To compare each condition to control, a Kruskal-Wallis test followed by Dunn’s multiple-comparison test was performed. n = 4 to 6 for each IgM condition; each dot represents an independent replicate.