Colin R. Zamecnik

The timing of the biological onset of multiple sclerosis (MS) is unclear. We used high-throughput discovery proteomics and samples from presymptomatic patients with MS and matched healthy controls to define the biological neurological onset and characterize the mechanisms involved. Remarkably, evidence of myelin injury was seen ~7 years before the symptomatic onset and preceded evidence of axonal injury by ~1 year. By contrast, astrocyte involvement became evident only at clinical onset. Numerous changes in the serum proteome indicate the involvement of interleukin 3 and nuclear factor kappa B pathways during the presymptomatic stage. Furthermore, people with MS with a previously reported distinct autoantibody signature showed increased immune cell activity compared to those without. We propose a protein biomarker panel that may help distinguish presymptomatic patients with MS from healthy controls, pending validation in future studies. Our findings can help understand the pathophysiology of MS as well as the cascade of central nervous system injury and might facilitate early detection of MS in high-risk people.

Tight junctions regulate epithelial barrier function and have been shown to be influenced by multiple classes of proteins. Apical integrins have been identified as potential regulators of epithelial barrier function; however, only indirect approaches have been used to measure integrin regulation of the epithelial barrier. Here, we used polymeric nanowires conjugated with anti-integrin β1 antibodies to specifically target apically localized integrins in either their closed or open conformation. Barrier regulation by apical integrins was found to be conformation specific. Nanowires targeting integrins in the closed conformation increased epithelial permeability and caused zonula occludens-1 (ZO-1, also known as TJP1) to change from a linear to a ruffled morphology. Claudin-2 and claudin-4 colocalized with ZO-1 and were also ruffled; however, claudin-1 and claudin-7 remained linear. Ruffling was dependent on myosin light chain kinases (MLCKs) and Rho kinases (ROCKs). Conversely, targeting integrins in the open conformation decreased epithelial permeability and made junctions more linearized. Anti-integrin β1 nanowires differentially affected actin and talin (analyzed using pan-talin antibodies), depending on whether they contained activating or inhibitory antibodies. Thus, apical integrins can act as a conformation-sensitive switch that regulates epithelial barrier function.

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection^1,2, yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of samples from patients with MIS-C to identify a distinct set of host proteins targeted by patient autoantibodies including a particular autoreactive epitope within SNX8, a protein involved in regulating an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed antibody responses from patients with MIS-C to the complete SARS-CoV-2 proteome and found enriched reactivity against a distinct domain of the SARS-CoV-2 nucleocapsid protein. The immunogenic regions of the viral nucleocapsid and host SNX8 proteins bear remarkable sequence similarity. Consequently, we found that many children with anti-SNX8 autoantibodies also have cross-reactive T cells engaging both the SNX8 and the SARS-CoV-2 nucleocapsid protein epitopes. Together, these findings suggest that patients with MIS-C develop a characteristic immune response to the SARS-CoV-2 nucleocapsid protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link between the infection and the inflammatory syndrome, with implications for better understanding a range of post-infectious autoinflammatory diseases.

Vitamin B12 is critical for hematopoiesis and myelination. Deficiency can cause neurologic deficits including loss of coordination and cognitive decline. However, diagnosis relies on measurement of vitamin B12 in the blood, which may not accurately reflect the concentration in the brain. Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Anti-CD320 impaired cellular uptake of cobalamin (B12) in vitro by depleting its target from the cell surface. Despite a normal serum concentration, B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic B12 supplementation were associated with increased B12 in the CSF and clinical improvement. Optofluidic screening enabled isolation of a patient-derived monoclonal antibody that impaired B12 transport across an in vitro model of the blood-brain barrier (BBB). Autoantibodies targeting the same epitope of CD320 were identified in seven other patients with neurologic deficits of unknown etiology, 6% of healthy controls, and 21.4% of a cohort of patients with neuropsychiatric lupus. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. However, these individuals did not display any hematologic signs of B12 deficiency despite systemic CD320 impairment. Using a genome-wide CRISPR screen, we found that the low-density lipoprotein receptor serves as an alternative B12 uptake pathway in hematopoietic cells. These findings dissect the tissue specificity of B12 transport and elucidate an autoimmune neurologic condition that may be amenable to immunomodulatory treatment and nutritional supplementation.

Life-threatening “breakthrough” cases of critical COVID-19 are attributed to poor or waning antibody (Ab) response to SARS-CoV-2 vaccines in individuals already at risk. Preexisting auto-Abs neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; their contribution to hypoxemic breakthrough cases in vaccinated people is unknown. We studied a cohort of 48 individuals (aged 20 to 86 years) who received two doses of a messenger RNA (mRNA) vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Ab levels to the vaccine, neutralization of the virus, and auto-Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal Ab response to the vaccine. Among them, 10 (24%) had auto-Abs neutralizing type I IFNs (aged 43 to 86 years). Eight of these 10 patients had auto-Abs neutralizing both IFN-α2 and IFN-ω, whereas two neutralized IFN-ω only. No patient neutralized IFN-β. Seven neutralized type I IFNs at 10 ng/ml and three at 100 pg/ml only. Seven patients neutralized SARS-CoV-2 D614G and Delta efficiently, whereas one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only type I IFNs at 100 pg/ml neutralized both D614G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating Abs capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a notable proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production ( Rag2 ^–/– and μMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor–restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn ^–/– IgD ^+/– mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire ^–/– mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Vitamin B12 is critical for hematopoiesis and myelination. ¹ Deficiency can cause neurologic deficits including loss of coordination, spasticity, and cognitive decline. ^2,3,4 However, diagnosis relies on vitamin B12 measurement in the blood which may not accurately reflect levels in the brain. Here, we discovered an autoimmune cause of vitamin B12 deficiency restricted to the central nervous system (CNS), termed autoimmune B12 central deficiency (ABCD). Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Patient immunoglobulins impaired cellular uptake of vitamin B12 in vitro . Despite normal serum levels, vitamin B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic vitamin B12 supplementation were associated with increased CSF B12 levels and clinical improvement. Autoantibodies targeting the same epitope of CD320 were identified in 7 other patients with neurologic deficits of unknown etiology and in 6 percent of healthy controls. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. These findings elucidate a new autoimmune cause of metabolic neurologic disease that may be amenable to immunomodulatory treatment and/or nutritional supplementation.

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection, yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of MIS-C patient samples (n=199) to identify a distinct set of host proteins that are differentially targeted by patient autoantibodies relative to matched controls. We identified an autoreactive epitope within SNX8, a protein expressed primarily in immune cells which regulates an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed the SARS-CoV-2 proteome-wide MIS-C patient antibody response and found it to be differentially reactive to a distinct domain of the SARS-CoV-2 nucleocapsid (N) protein relative to controls. This viral N region and the mapped SNX8 epitope bear remarkable biochemical similarity. Furthermore, we find that many children with anti-SNX8 autoantibodies also have T-cells cross-reactive to both SNX8 and this distinct domain of the SARS-CoV-2 N protein. Together, these findings suggest that MIS-C patients develop a distinct immune response against the SARS-CoV-2 N protein that is associated with cross reactivity to the self-protein SNX8, demonstrating a link from the infection to the inflammatory syndrome.

Although B cells are implicated in multiple sclerosis (MS) pathophysiology, a predictive or diagnostic autoantibody remains elusive. Here, the Department of Defense Serum Repository (DoDSR), a cohort of over 10 million individuals, was used to generate whole-proteome autoantibody profiles of hundreds of patients with MS (PwMS) years before and subsequently after MS onset. This analysis defines a unique cluster of PwMS that share an autoantibody signature against a common motif that has similarity with many human pathogens. These patients exhibit antibody reactivity years before developing MS symptoms and have higher levels of serum neurofilament light (sNfL) compared to other PwMS. Furthermore, this profile is preserved over time, providing molecular evidence for an immunologically active prodromal period years before clinical onset. This autoantibody reactivity was validated in samples from a separate incident MS cohort in both cerebrospinal fluid (CSF) and serum, where it is highly specific for patients eventually diagnosed with MS. This signature is a starting point for further immunological characterization of this MS patient subset and may be clinically useful as an antigen-specific biomarker for high-risk patients with clinically- or radiologically-isolated neuroinflammatory syndromes.