Background

B cell depleting therapies are highly effective treatments for multiple sclerosis (MS). B cells are more numerous in active than inactive lesions, and their intrathecal clonal expansion and oligoclonal band production are hallmarks of MS. B cells also present antigens to T cells and secrete inflammatory cytokines. The antigenic specificity of individual B cells in cerebrospinal fluid (CSF) obtained from patients with early MS may help further clarify the role of B cells in MS biology.

Objectives

To determine the viral and autoantigen repertoire of CSF-derived, class-switched B cells from untreated, early MS patients.

Methods

We performed single cell immunoglobulin sequencing on CSF plasma cells, plasmablasts, and class switched memory B cells from 9 untreated patients: five with relapsing remitting MS (RRMS) and four with clinically isolated syndrome (CIS). The interval between the first attack and lumbar puncture ranged from 1 - 222 days (median 67 days). Brain and spinal cord MRIs performed concurrently with lumbar punctures revealed 5/9 patients with gadolinium enhancing lesions.

Using paired heavy and light chain immunoglobulin sequences, we generated 75 monoclonal antibodies (mAbs) and screened them on a suite of unbiased antigen discovery platforms: 1) mouse brain tissue staining, 2) whole human proteome programmable phage display, 3) pan-viral programmable phage display, 4) mouse and human brain immunoprecipitation mass spectrometry.

Results

The mAbs showed diverse antigen specificities. Candidate antigens were primarily ubiquitously expressed, intracellular proteins; however, a minority were macromolecules associated with the plasma membrane and/or enriched in brain tissue. Shared antigenic targets were occasionally identified within subjects but were rarely identified across subjects, with the latter including cytoskeletal proteins. For two mAbs, high-confidence antigens with prima facie relevance to MS were identified: 1) a white matter-restricted lipid species, and 2) an Epstein-Barr virus-interacting host protein.

Conclusions

Using our panel of 75 mAbs derived from plasma cells, plasmablasts, and class-switched memory B cells found in the CSF of early, untreated RRMS/CIS patients, we identified a diverse repertoire of antigenic targets, with a majority comprised of intracellular host proteins.

Background

Coronavirus disease 19 (COVID-19) is the most globally impactful pandemic of the past century. The causative pathogen, SARS-CoV-2, infects ACE2-expressing cells and leads to pulmonary disease and a systemic immune response. In patients with severe COVID-19, a dysregulated immune response is associated with secondary extrapulmonary dysfunction, including neurological symptoms. Neurologic complications of SARS-CoV-2 infection are increasingly recognized, yet it is unknown to what degree humoral autoimmunity is a feature of neurological impairment in COVID-19.

Objectives

To perform an unbiased survey of peripheral and central humoral autoimmunity in COVID-19 patients with neurologic dysfunction.

Methods

Paired cerebrospinal fluid (CSF) and plasma biospecimens were collected from nasopharyngeal (NP) SARS-CoV-2 PCR positive patients with comorbid neurologic impairment (n = 5). Additional unpaired CSF biospecimens were collected from neurologically impaired NP PCR positive patients (n = 3). All COVID-19 patients were PCR negative for SARS-CoV-2 in the CSF. CSF and plasma were also collected from SARS-CoV-2 uninfected healthy control volunteers. Neurologic syndromes were diverse and included myositis, seizures, and encephalopathy. Biospecimens were screened in replicate by mouse brain immunostaining, immunoprecipitation mass spectrometry (IP-MS), and human peptidome phage display immunoprecipitation sequencing (PhIP-Seq). IP-MS spectra were analyzed by both spectral counting and MS1 peak area. For PhIP-Seq, proteins with overlapping peptides that were enriched at least 10-fold above control samples, or single peptides enriched 100-fold above controls were considered candidate autoantigens. Candidate autoantigens identified by at least two of three methods (PhIP-Seq, spectral counting, and peak area) were carried forward for validation.

Results

Unexpectedly, seven of eight COVID-19 CSF samples had evidence of humoral autoimmunity by tissue staining (n = 7), and IP-MS (n = 6), PhIP-Seq (n = 7), or both (n = 6). By IP-MS, significantly more candidate autoantigens were identified in COVID-19 biospecimens than in uninfected controls. PhIP-Seq identified twice as many candidate autoantigens in COVID-19 biospecimens than in controls. Notably, COVID-19 biospecimens were enriched for clinically relevant candidate autoantigens including those associated with dermatomyositis, and myasthenia gravis (none known to be pre-existing comorbidities). Additionally, COVID-19 biospecimens were enriched for candidate autoantigens with prima facie clinical relevance as they targeted proteins enriched in skeletal muscle, endothelial cells, and at the synapse. One candidate autoantibody targeted a ciliary protein implicated in syndromic anosmia.

Conclusions

We identified evidence of an increased burden of humoral autoimmunity in COVID-19 patients with comorbid neurologic dysfunction.

Background

Multiple Sclerosis (MS) is a complex and heterogeneous disease. Investigating the biological pathways and cell types involved in MS pathophysiology as represented by protein biomarker expression can help inform the development of tools to monitor disease activity, disease progression, identify early evidence of relapse, and monitor treatment response.

Objectives

To develop a blood based multiplex proteomic assay that associates with clinical and radiographic endpoints in patients with MS. These endpoints include the presence of gadolinium-enhanced (Gd+) lesions, Annualized Relapse Rate (ARR) and clinically defined relapse status (active versus stable).

Methods

Serum samples (n=690 in total) from multiple deeply-phenotyped cohorts (ACP, CLIMB and EPIC) were tested in immunoassays for the measurement of 1196 proteins using Proximity Extension Assays (PEA) from Olink^TM and for 215 proteins using xMAP^TM immunoassays from Myriad RBM, Inc. (RBM). Associated radiographic and clinical endpoints at the time of the blood draw were correlated with the protein levels. Twenty-one proteins were selected for inclusion in a custom assay based on their performance in univariate and multivariate statistical models, and replication across independent cohorts. Biological pathway modeling and network analysis were performed to ensure comprehensive representation of MS neurophysiology. Area under the curve (AUC) was selected as the key metric for model performance evaluation.

Results

Multivariate statistical ensembles restricted to the expression levels of the biomarkers selected for the custom assay achieved AUC performance of 0.827 for classification of the presence of Gd+ lesions, 0.802 for classification of clinically defined relapse status, and 0.930 for the classification of patients with Low ARR (≤0.2 relapses) vs High ARR (≥1.0 relapses). A multivariate model utilizing shifts in biomarker expression in longitudinally paired samples achieved the highest observed performance of 0.950 for classification of Gd+ lesion presence. In each case, the multivariate models significantly outperformed (p-value <0.05) the AUC of the highest performing univariate biomarker.

Conclusions

Multivariate models restricted to the 21 selected proteins effectively classified several radiographic and clinical endpoints with stronger performance than any single biomarker. A 21-plex custom assay panel is being developed for further investigation and validation using additional cohorts.

Background

Progressive multifocal leukoencephalopathy (PML) is an often fatal white matter disease caused by the polyomavirus JC virus that affects patients with various types of immunodeficiencies. Some multiple sclerosis disease modifying therapies, including natalizumab, dimethyl fumarate and fingolimod, increase the risk for PML. Successful PML treatment and survival is critically dependent on early recognition and immune reconstitution; unfortunately, rapid immune reconstitution is not always achievable. New therapies are being investigated, including a pilot study conducted at NIH exploring ex vivo T cells generated from polyomavirus-specific partially matched first degree relative donors of PML patients (NCT02694783). Anti-viral specific cell products were generated following 14-day culture with 15mer polyomavirus peptide libraries.

Objectives

To perform T cell immune repertoire sequencing on donor polyomavirus-specific T cell populations with the goal of identifying T cell receptors (TCRs) that might be used to create a new “off the shelf” designer T cell therapy for PML.

Methods

T cells from twelve first degree relative donors and four PML patients from the NIH study were obtained, including donor T cells pre- and post-polyomavirus antigen stimulation. Using immune repertoire sequencing of CD4 and CD8 T cells, V(D)J sequences from post-stimulation donor cells were analyzed to identify clonally expanded TCRs relative to the pre-stimulation, same donor controls. Clonally expanded TCRs were also compared to PML patient V(D)J sequences to look for sequence similarity. Once candidate TCRs are identified, we will perform functional assays to determine their effectiveness against JC virus-infected cells.

Results

Candidate TCRs and their antigen specificity will be presented at the meeting in the hopes that the most effective candidate TCRs can serve as therapeutics for HLA-matched patients with PML.

Conclusions

Candidate TCRs and their antigen specificity will be presented at the meeting in the hopes that the most effective candidate TCRs can serve as therapeutics for HLA-matched patients with PML.