A causal link between autoantibodies and neurological symptoms in long COVID

Abstract

Acute SARS-CoV-2 infection triggers the generation of diverse and functional autoantibodies (AABs), even after mild cases. Persistently elevated autoantibodies have been found in some individuals with long COVID (LC). Using a >21,000 human protein array, we identified diverse AAB targets in LC patients that correlated with their symptoms. Elevated AABs to proteins in the nervous system were found in LC patients with neurocognitive and neurological symptoms. Purified Immunoglobulin G (IgG) samples from these individuals reacted with human pons tissue and were cross-reactive with mouse sciatic nerves, spinal cord, and meninges. Antibody reactivity to sciatic nerves and meninges correlated with patient-reported headache and disorientation. Passive transfer of IgG from patients to mice led to increased sensitivity and pain, mirroring patient-reported symptoms. Similarly, mice injected with IgG showed loss of balance and coordination, reflecting donor-reported dizziness. Our findings suggest that targeting AABs could benefit some LC patients.

Keywords: Autoantibodies; Chronic Pain; Long COVID; SARS-CoV-2.

Figure 1.. Purified IgG from participants with Long COVID reacts with CNS and PNS tissues.

A-C. Demographics (gender, age and symptoms) of biospecimens analyzed from MY-LC cohort. D-H. Confocal microscopy showing human and mouse tissues immunostained with human total IgG (green) purified from Long COVID, healthy or convalescent controls, as indicated, and nuclear DNA stain (DAPI, blue). D. Experiment schematic. C. Representative images of human pons immunostaining and Mean of fluorescence intensity. F. Representative images of mouse sciatic nerve immunostaining and mean of fluorescence. G. Representative images of mouse meninges immunostaining and mean of fluorescence. H. Representative images of mouse meninges immunostaining and mean of fluorescence. Scale bar described in the image. Insets indicate a higher magnification of a region indicated (red rectangle). Each dot in the figure represents the value obtained from an individual participant. Data are presented as the mean. Significant p-values are described in the image, as determined by One-way ANOVA with Tukey multiple comparisons test.

Figure 2.. IgG from participants with long COVID that reacts to meninges and sciatic nerve are associated with symptoms.

A. Mouse meninges stain divided by symptoms displayed by the participants, as headache, memory, weakness and disorientation. B. Confocal microscopy showing meninge from NG2-dsRED mouse stained with total IgG from Long COVID participant (green) and CD31 (yellow), NG2-dsRED stains in red, nuclear DNA stain in blue (Dapi). Line scan analysis showing fluorescent intensity for hIgG, CD31 and NG2-dsRED. White line indicates the scan analysis in the graph. Scale bar described in the image. C. Mouse sciatic nerve stain divided by symptoms displayed by the participants, as tinnitus. D. Heatmap showing positive stain pattern across different tissues between participants, each collum is a different participant, each row is a different tissue. Each dot in the figure represents the value obtained from an individual participant. Data are presented as the mean. Significant p-values are described in the image, as determined by One-way ANOVA with Tukey multiple comparisons test.

Figure 3.. Individuals with Long COVID demonstrate elevated autoantibody reactivity including to CNS tissues.

A. Plasma from participants were incubated with HuProt microarray and probed with a secondary antibody against human IgG, positive targets were identified using an microarray scanner. B. Number of HuProt-derived auto-reactivities for each person within each group starting at a HuProt intensity threshold of >3x the mean of controls. Model was run using negative binomial (NB) regression with longlink function. Dispersion-adjusted incidence rate ratio for each group was compared to the overall mean using analysis of Means (ANOM), with significance adjustment using the Nelson method. Each point denotes the count of HuProt positive hits for an individual. C. Heatmap depicting the incidence rate ratios derived from negative binomial regression for each group at HuProt thresholds between 2–3x the signal intensity of the control mean, as in A. Asterisks denote significance from the adjusted overall mean using NB-derived ANOM with significance adjustment using the Nelson method. D. Heatmap depicting HuProt autoantibody reactivity to CNS starting at HuProt scores of >=3x. Each column represents a single participant and each row represents a single protein. E. Heatmap depicting the incidence rate ratio difference between each respective group and the mean of all groups using Poisson Regression with ANOM analysis followed by Nelson correction. Asterisks denote significance. F. Heatmap showing unsupervised cluster analysis for HuProt autoantibody reactivity. G. Heatmap depicting HuProt autoantibody reactivity to common antigens by group starting at HuProt scores of >=2.8x. H-I. ELISA and Area Uder Curve (AUC) analysis for top hit targets identified by Huprot.

Figure 4.. IgG from individuals with LC induces symptoms in mice.

A A dose of 38 mg/kg of total IgG purified from healthy, convalescent controls and Long COVID participants were administered to 6–8 weeks-old C57BL/6 female mice by intraperitoneal (IP) injection, which were followed up for 5 days. B Quantification of human IgG by ELISA in brain homogenate and serum at day 5 post-injection. C Hot plate test performed on day 3 post injection. D Hot plate test divided by status of chronic pain displayed by the participants. E Frequency of mice with pain at the hot plate test and the patients with diagnosed chronic pain. F-H Hot plate test divided by how the participants described their pain sensation. I Hot plate test divided by participants that displayed weakness. J Hot plate test divided by participants that displayed dysautonomia. K Grip strength test performed on day 3 post injection. L Grip strength test divided by participants that displayed tinnitus. M Grip strength test divided by participants that displayed headache. N Rotarod test performed on day 2 post injection. O Rotarod test divided by participants that displayed dizziness. P Longitudinal Two-photon imaging of Scn10a-Cre::td Tomato reporter mice reveals collagen fibers by second harmonic generation (SHG) as well as nociceptor axons and the perpendicular intraepidermal nerve fibers (IENF; white/pink). Quantification of total IENF volume and number of fibers crossing the dermis boundary after passive transfer. Scale bars 50μm. Each dot in the figure represents the value obtained from an individual mouse. Each mouse received antibodies isolated from a single human participant. Data are presented as the mean. Significant p values are described in the image, as determined by T-test for two groups comparison or one-way ANOVA corrected for multiple comparisons with Tukey test for multiple groups comparison.