Sequencing B Cell Repertoires to Elucidate Autoantibodies and the Role of EBV in PASC

Introduction: This RECOVER proposal performed B cell repertoire sequencing and identified B cell-encoded Mabs that bind autoantigens and thereby define clinical endotypes of post-acute sequelae of SARS-CoV-2 (PASC), with a focus on connective tissue PASC, lung PASC, and neurocognitive PASC. We integrated use of our laboratory's EBV single cell sequencing methods and EBV antigen arrays to further define the role of EBV in PASC.

Objective: To define the role of SARS-CoV-2-induced autoimmunity and EBV in connective tissue PASC, lung PASC, and neurocognitive PASC.

Methods: Aim 1 performed scRNA-Seq analysis of the B cell/antibody repertoire and EBV gene expression in clinical subsets of PASC patients enrolled in RECOVER. Aim 1.1 used 10X Genomics VDJ with CITE-seq + EBV primers to sequence the B cell repertoires in the following subtypes of PASC with autoimmune features: ANA-positive connective tissue PASC; neurocognitive PASC; lung PASC; healthy comparators. Aim 1.2 used informatics to characterize PASC clinical subsets, dysregulated B cell subsets, and the role of EBV in dysregulated B cells. Aim 2 expressed PASC B cell Mabs and define their antigen targets using antigen arrays and Aim 3 expanded the identified targets to PASC antigen arrays.

Results: We expressed 150 PASC plasmablast and EBV+ B cell mAbs, and identified 3 EBV+ B cells derived neuro-PASC (Brain fog PASC) that bound human brain in immunostaining. Our findings suggest that a subset of neuro-PASC are mediated by autoimmunity, and specifically by EBV-infected anti-neuronal B cells. We further characterized EBV+ B cell mAbs from the other subsets of PASC that we sequenced and identified several targets on human proteome arrays.

Conclusion/Discussion: Our finds demonstrated that in PASC EBV infects and activates B cells. Our findings suggest that EBV+ B cells serve as APCs that might mediate an autoimmune response. Further, our findings suggest that therapeutic targeting of these EBV+ B cells and/or autoimmune response may provide therapeutic benefit to patients with PASC patients. Further, SARS-CoV-2 may activate TLRs to mediate EBV reactivation, which may promote EBV+ B cell-mediated anti-neuronal autoimmunity in neuro-PASC.

Key Topics:

• Assay and in vitro studies to gain mechanistic insights
• Biomarker, in-depth phenotyping assays and in vitro studies using tissue and other biospecimens
• Comparative studies of Long COVID with other post-viral and post-infectious syndromes
• Long COVID and other chronic conditions