Autoantibodies in COVID-19 correlate with antiviral humoral responses and distinct immune signatures

Abstract

Background: Several autoimmune features occur during coronavirus disease 2019 (COVID-19), with possible implications for disease course, immunity, and autoimmune pathology. In this study, we longitudinally screened for clinically relevant systemic autoantibodies to assess their prevalence, temporal trajectory, and association with immunity, comorbidities, and severity of COVID-19.

Methods: We performed highly sensitive indirect immunofluorescence assays to detect antinuclear antibodies (ANA) and antineutrophil cytoplasmic antibodies (ANCA), along with serum proteomics and virome-wide serological profiling in a multicentric cohort of 175 COVID-19 patients followed up to 1 year after infection, eleven vaccinated individuals, and 41 unexposed controls.

Results: Compared with healthy controls, similar prevalence and patterns of ANA were present in patients during acute COVID-19 and recovery. However, the paired analysis revealed a subgroup of patients with transient presence of certain ANA patterns during acute COVID-19. Furthermore, patients with severe COVID-19 exhibited a high prevalence of ANCA during acute disease. These autoantibodies were quantitatively associated with higher SARS-CoV-2-specific antibody titers in COVID-19 patients and in vaccinated individuals, thus linking autoantibody production to increased antigen-specific humoral responses. Notably, the qualitative breadth of antibodies cross-reactive with other coronaviruses was comparable in ANA-positive and ANA-negative individuals during acute COVID-19. In autoantibody-positive patients, multiparametric characterization demonstrated an inflammatory signature during acute COVID-19 and alterations of the B-cell compartment after recovery.

Conclusion: Highly sensitive indirect immunofluorescence assays revealed transient autoantibody production during acute SARS-CoV-2 infection, while the presence of autoantibodies in COVID-19 patients correlated with increased antiviral humoral immune responses and inflammatory immune signatures.

Keywords: COVID-19; SARS-CoV-2; VirScan; antinuclear antibodies; autoantibodies.

FIGURE 1

Prevalence of autoantibodies in healthy controls and COVID‐19 patients during acute disease and follow‐up. (A) Study overview. (B–I) Prevalence of ANA titers (B–E) and ANCA (F–I) in healthy controls (n = 41) and COVID‐19 patients during acute disease (n = 175), 6 months (n = 116) and 1 year (n = 92) after symptom onset. (J–M) Venn diagrams depicting co‐occurrence of nuclear ANA, cytoplasmic ANA, and ANCA in healthy individuals (J; n = 17), acute COVID‐19 patients (K; n = 89), and COVID‐19 patients 6 months (L; n = 56) or 1 year (M; n = 42) after SARS‐CoV‐2 infection that presented with at least one type of autoantibody. p‐values indicate comparison of ANA (B–E) and ANCA (F–I) prevalence between mild and severe COVID‐19 patients using the Fisher's exact test

FIGURE 2

IIF pattern of autoantibodies in acute and recovered COVID‐19. (A–D) Intersection plots showing counts of the four most prevalent ANA patterns (horizontal bars) and counts of pattern combinations (vertical bars) as indicated by the dot matrix, for healthy controls (A), and COVID‐19 patients during acute disease (B), 6 months (C), and 1 year after symptom onset (D). (E–G) Example IIF pictures showing the most common nuclear, including fine‐granular (E) and nucleolar (F), and cytoplasmic, including speckled (G), ANA patterns observed in the study cohort. All images were recorded at a dilution of 1:320. y/o—years old. (H) IIF ANCA patterns observed in ANCA‐positive COVID‐19 patients during acute disease (n = 17) and 6 months after recovery (n = 3). (I) Anti‐MPO and anti‐PR3 antibodies during acute COVID‐19 (n = 175) in ANCA‐positive and ANCA‐negative individuals. Dashed lines indicate diagnostic cut‐off values

FIGURE 3

Paired longitudinal comparison indicates transient induction of autoantibodies in acute COVID‐19. (A–B) Temporal trajectory of ANA titers in mild (A, n = 85) and severe (B, n = 44) COVID‐19 patients, showing the first available follow‐up sample, i.e., at 6 months (n = 116) or 1 year (n = 13) after symptom onset. Colors indicate development of ANA status from acute disease to follow‐up. (C) Results from blinded, paired IIF picture analysis (n = 129). Patterns that were uniquely observed at one timepoint are colored. (D) Exemplary IIF pictures of three patients exhibiting transient ANA patterns during acute COVID‐19, with a transient nucleolar (left), cytoplasmic (middle) or mitotic (right) pattern. All pictures were recorded at a dilution of 1:320. y/o, years old. (E–F) Temporal trajectory of ANCA titers in mild (E, n = 85) and severe (F, n = 44) COVID‐19 patients. Colors indicate development of ANCA status from acute disease to follow‐up

FIGURE 4

Presence of autoantibodies is associated with an increased virus‐specific humoral response after SARS‐CoV‐2 infection and vaccination. (A–B) S1‐specific IgA and IgG in ANA‐positive and ANA‐negative COVID‐19 patients during acute disease (n = 175) and 6 months after recovery (n = 104). (B) p‐values indicate significance of the correlation of ANA positivity as an independent parameter in a multiple linear regression model accounting for age, disease severity and sampling time point (Table S3). (C) S1‐specific IgA and IgG in ANCA‐positive and ANCA‐negative COVID‐19 patients during acute disease (n = 175). (D) ANA prevalence and titers in previously unexposed individuals (n = 11) before and after vaccination with BNT162b2 at indicated time points. The p‐value was calculated using the chi‐squared test of independence. (E) S1‐specific IgA and IgG before and after COVID‐19 vaccination (n = 11). Red vertical lines indicate the time points of first and second vaccination with BNT162b2. (F) S1‐specific IgA and IgG in ANA‐positive and ANA‐negative participants following COVID‐19 vaccination with BNT162b2, combining data from 10–13 days after the first (n = 11) and 1–3 days after the second (n = 10) vaccination

FIGURE 5

Comprehensive serological profiling (VirScan) in ANA‐positive and ANA‐negative individuals during acute COVID‐19. (A) Principal component analysis (PCA) of 112 viral species, including data of 18 healthy individuals and 96 acute COVID‐19 patients, grouped by time point of sample collection after symptom onset. Each dot represents an individual participant. (B) Loadings of PCA depicted in (A), with each viral species shown as individual dots (Table S4). Colors indicate participant groups with higher mean epitope hits per species. Viral species with significant difference (p < .005) between COVID‐19 patients and healthy controls are shown as large colored dots. Black crosses indicate insignificant differences of coronaviruses (p > .005). (C) Temporal association of summed epitope hits of six coronaviruses after symptom onset, shown for acute COVID‐19 patients (n = 97) and healthy controls (n = 18). Horizontal green bars represent means of healthy controls. (D) Percentage of healthy controls and COVID‐19 patients with positive results for epitopes of six coronavirus species. Significantly enriched epitopes (p < .05) of spike and nucleocapsid are indicated accordingly. (E–F) Summed hits for cross‐reactive epitopes, comparing healthy controls and patients with mild and severe COVID‐19 (E) or COVID‐19 patients with or without ANA (F). (G) Percentage of ANA‐positive and ANA‐negative COVID‐19 patients with positive results for cross‐reactive and non‐cross‐reactive epitopes of six coronavirus species. Dashed lines mark significance threshold at p < .05. (H) Percentage of ANA‐positive and ANA‐negative study participants (n = 115) with positive results, shown for all available epitopes. Significantly enriched epitopes (p < .005) are colored. EBV—Epstein–Barr virus; HSV‐2—herpes simplex virus 2; VZV—varizella‐zoster virus; other—other viruses comprising Aichivirus A and Mamastrovirus 1. (I) Summed epitope hits per individual including all available epitopes, comparing ANA‐negative and ANA‐positive participants (top; n = 115) and as a function of age (bottom)

FIGURE 6

ANA‐positive COVID‐19 patients exhibit a proinflammatory signature. (A) PCA accounting for 130 parameters (Table S5) including data of healthy controls (n = 28) and acute COVID‐19 patients (n = 146). Participants with missing values were excluded from this analysis. 95% confidence ellipses (t‐distributed) are shown for healthy controls and severe COVID‐19 patients. (B) Loadings (variable coordinates) of the PCA depicted in (A), with each parameter shown as an individual dot. Colors indicate the group of COVID‐19 patients with higher mean for each parameter, and parameters with significant differences (p < .05) are represented as large dots, and selected parameters are annotated (Table S5). (C–E) Comparison of ANA‐negative and ANA‐positive individuals among healthy controls or acute COVID‐19 patients. (C) Patient characteristics, including duration of hospitalization (n = 174) and age (n = 216). (D) Inflammation markers, including CRP (n = 209) and IL‐6 (n = 215). (E) T‐cell activation, including sIL‐2Rα (n = 215), and CD38⁺HLA‐DR⁺ CD4⁺ (n = 210) and CD8⁺ (n = 209) T cells. (F–G) PCA (F) and loadings (G) accounting for 43 parameters (Table S6) including data of COVID‐19 patients 6 months after recovery (n = 107). Participants with missing values were excluded from this analysis. (H–I) Comparison of ANA‐negative and ANA‐positive COVID‐19 patients 6 months after recovery. (H) Concentration of total Ig subclasses in serum (n = 116). (I) Frequency of B‐cell subsets, including IgD⁺CD27⁻ naïve, IgD⁺CD27⁺ nonswitched memory and IgD⁻CD27⁺ switched memory B cells (n = 114)