Detection of Antinuclear Antibodies Targeting Intracellular Signal Transduction, Metabolism, Apoptotic Processes and Cell Death in Critical COVID-19 Patients

Abstract

Background and objectives: The heterogeneity of the coronavirus disease of 2019 (COVID-19) lies within its diverse symptoms and severity, ranging from mild to lethal. Acute respiratory distress syndrome (ARDS) is a leading cause of mortality in COVID-19 patients, characterized by a hyper cytokine storm. Autoimmunity is proposed to occur as a result of COVID-19, given the high similarity of the immune responses observed in COVID-19 and autoimmune diseases. Here, we investigate the level of autoimmune antibodies in COVID-19 patients with different severities.

Results: Initial screening for antinuclear antibodies (ANA) IgG using ELISA revealed that 1.58% (2/126) and 4% (5/126) of intensive care unit (ICU) COVID-19 cases expressed strong and moderate ANA levels, respectively. An additional sample was positive with immunofluorescence assays (IFA) screening. However, all the non-ICU cases (n=273) were ANA negative using both assays. Samples positive for ANA were further confirmed with large-scale autoantibody screening by phage immunoprecipitation-sequencing (PhIP-Seq). The majority of the ANA-positive samples showed "speckled" ANA pattern by microscopy and revealed autoantibody specificities that targeted proteins involved in intracellular signal transduction, metabolism, apoptotic processes, and cell death by PhIP-Seq; further denoting reactivity to nuclear and cytoplasmic antigens.

Conclusion: Our results further support the notion of routine screening for autoimmune responses in COVID-19 patients, which might help improve disease prognosis and patient management. Further, results provide compelling evidence that ANA-positive individuals should be excluded from being donors for convalescent plasma therapy in the context of COVID-19.

Keywords: ANA; Autoimmunity; COVID-19; Coronavirus; ICU.

Figure 1

ANA ELISA levels of the COVID-19 Patients. (A) Sera samples of ICU (n=126) and non-ICU (n=273) COVID-19 patients; and (B) sera samples of ICU COVID-19 patients (n=126). Samples were tested at 1:101 dilution.

Figure 2

Immunostaining of Hep2 Cells with ANA from COVID-19 Sera Samples. Sera samples of ICU patients (n=126) and an equivalent number of randomly selected non-ICU patients sera (n=121) were subjected to Indirect Fluorescent Antibody (IFA). HEp2 cells were used as a substrate to detect ANA antibodies in human serum. Samples were tested at 1:40 dilution. Note that # of days corresponds to the time of sample collection, assuming ICU admission is day 1.

Figure 3

Autoantibody profile of selected cases assessed by PhIP-Seq. (A). Principal component analysis of the peptide enrichment scores reflecting autoantibody-autoantigen interactions in ICU patients and asymptomatic COVID-19 cases. The color core indicates the ANA status. (B). Heatmap plot showing the binding profile of the 79 differentially enriched (DE) peptides in ICU cases versus asymptomatic cases, with hierarchical clustering. Each row indicates a human peptide (90mer, start position is indicated relative to the UniProtKB entry), and each column represents a sample. The color gradient for each cell of the heatmap plot represents the peptide enrichment score (−log₁₀(P) value) for a given antigenic peptide and sample. A -log₁₀(P) ≥ 2.3 was considered significantly enriched; * Represents known autoantigens with an entry in the human autoantigen database (AAgAtlas 1.0). (C). Stacked bar plot showing the results of a gene set enrichment analysis of the peptides shown in (B). The color code indicates the gene sets from the Molecular Signatures Database (MSigDB) for which at least one DE peptide was enriched (P-value < 10⁻⁵ and FDR q-value < 0.05). Samples are sorted as shown in (B) according to hierarchical clustering.