Role of the Superantigen- and Neurotoxin-like Motifs in SARS-CoV2 Spike in Post-acute Sequelae of SARS-CoV-2 Infection (PASC)

Introduction: We have previously identified Superantigen motifs in Spike protein of SARS-CoV-2 (Cheng et al, PNAS). Indeed, superantigen-like T cell expansion was identified in MISC and COVID-19 - associated with hyperinflammation syndromes (Cheng et al, PNAS and Porritt et al, JCI). S1 can mediate opening of the blood-brain barrier (BBB), and multiple protease cleavage sites in S1 likely results in smaller fragments that may cross into the central nervous system (CNS).

Objective: We hypothesize that circulating S1 or S1 protein fragments (containing neurotoxin motifs) may cross the BBB and directly impair neurological activity, eliciting neurological symptoms observed in MIS-C and individuals recovering from COVID infections.

Methods: We collected peripheral blood mononuclear cells (PBMCs) and stimulated them with Spike S1 (1 microgram/ml) for 3 hours. We observed that S1 stimulation induces innate immune responses and cytokine production (IL-1beta, IL-6, TNFa and IL-8). We tested in vitro the capacity of SARS-CoV-2 Spike 1 (S1) protein and neurotoxin-like peptide (NP)3 and NP4 to induce the production of pro-inflammatory mediators by IPSC-induced human macrophages (mΦ).

Results: While NP3 stimulation induced a modest increase in the production of IL-1β, TNF-α and IL-6, S1 and NP4 stimulation strongly increased the release of IL-1β, TNF-α, IL-6, and CCL7 by IPSC-induced human mΦ. We assessed how S1 and spike neurotoxin-like peptide (NP) alter the spontaneous activity of neurons in vitro. Baseline recording of spontaneous activity of D30 midbrain dopaminergic neural cultures was measured 15 minutes before stimulation with either S1 spike protein (5µM), NP3 (10µM) or its scrambled peptide (SCR3; 10µM) or NP4 (10µM) or its scrambled peptide (SCR4; 10µM). Compared with their scrambled peptide (SCR), neural spontaneous activity was significantly reduced at 24 hours post-stimulation with NP3 or NP4. Next, to identify gene expression changes in neural cells induced by NP4 stimulation, we performed RNA sequencing on neural cells stimulated with either NP4 or SCR4.

Conclusion/Discussion: We observed upregulation by NP4 stimulation of genes associated with membrane repolarization, and notably an increased expression of two K+ channel genes, KCNQ1 and KCNE1B. KCNE1B and KCNQ1 form a voltage-gated K+ channel present in neurons and cardiomyocytes. Some neurotoxins block potassium ion transport across the plasma membrane by interacting directly with and blocking K+ channels. If SARS-CoV-2 neurotoxin-like peptides act similarly, the increased expression of these K+ channels may be a compensatory upregulation mechanism of blocked channels.

Key Topics:

• Assay and in vitro studies to gain mechanistic insights
• Long COVID and other chronic conditions