Paradoxical Sex-Specific Patterns of Autoantibodies Response to SARS-CoV-2 Infection (preprint)

BackgroundAmidst the millions of individuals affected directly by the pandemic, pronounced sex differences in the susceptibility and response to SARS-CoV-2 infection remain poorly understood. Emerging evidence has highlighted the potential importance of autoimmune activation in modulating not only the acute response but also recovery trajectories following SARS-CoV-2 exposure. Given that immune-inflammatory activity can be sex-biased in the setting of severe COVID-19 illness, we deliberately examined sex-specific autoimmune reactivity to SARS-CoV-2 in the absence of extreme clinical disease. MethodsWe used a bead-based array containing over 90 autoantigens previously linked to a range of classic autoimmune diseases to assess autoantibody (AAB) titers in 177 participants. All participants had confirmed evidence of prior SARS-CoV-2 infection based on presence of positive anti-nucleocapsid IgG serology results (Abbott Diagnostics, Abbott Park, Illinois). We used multivariate analysis to determine whether sex-bias was associated with increased rates of AABs reactivity and symptom burden after SARS-CoV2 infection. Results82.4% of AABs reactivity was associated with being male compared to 17.6% with female. We found a diversity of AABs responses that exhibited sex-specific patterns of frequency distribution as well as associations with symptomatology and symptom burden. ConclusionOur results reveal a remarkable sex-specific prevalence and selectivity of AAB responses to SARS-CoV-2. Further understanding of the nature of triggered and persistent AAB activation among men and women exposed to SARS-CoV-2 will be essential for developing effective interventions against immune-mediated sequelae of COVID-19.

A monoclonal antibody against staphylococcal enterotoxin B superantigen inhibits SARS-CoV-2 entry in vitro (preprint)

We recently discovered a superantigen-like motif, similar to Staphylococcal enterotoxin B (SEB), near the S1/S2 cleavage site of SARS-CoV-2 Spike protein, which might explain the multisystem-inflammatory syndrome (MIS-C) observed in children and cytokine storm in severe COVID-19 patients. We show here that an anti-SEB monoclonal antibody (mAb), 6D3, can bind this viral motif, and in particular its PRRA insert, to inhibit infection by blocking the access of host cell proteases, TMPRSS2 or furin, to the cleavage site. The high affinity of 6D3 for the furin-cleavage site originates from a poly-acidic segment at its heavy chain CDR2, a feature shared with SARS-CoV-2-neutralizing mAb 4A8. The affinity of 6D3 and 4A8 for this site points to their potential utility as therapeutics for treating COVID-19, MIS-C, or common cold caused by human coronaviruses (HCoVs) that possess a furin-like cleavage site.

Identification of a unique TCR repertoire, consistent with a superantigen selection process in Children with Multi-system Inflammatory Syndrome (preprint)

Multisystem Inflammatory Syndrome in Children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares many clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. The superantigen specificity for binding different Vbeta-chains results in Vbeta-skewing, whereby T cells with specific Vbeta-chains and diverse antigen specificity are overrepresented in the TCR repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCR Beta Variable gene (TRBV)11-2. Furthermore, TRBV11-2 skewing was remarkably correlated with MIS-C severity and serum cytokine levels. Further analysis of TRBJ gene usage and CDR3 length distribution of MIS-C expanding TRBV11-2 clones revealed extensive junctional diversity, indicating a superantigen-mediated selection process for TRBV expansion. In silico modelling indicates that polyacidic residues in TCR Vbeta11-2 engage in strong interactions with the superantigen-like motif of SARS-CoV-2 spike glycoprotein. Overall, our data indicate that the immune response in MIS-C is consistent with superantigenic activation.

Gain-of-function assay for SARS-CoV-2 Mpro inhibition in living cells (preprint)

The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here we demonstrate a quantitative reporter for Mpro function in living cells, in which protease inhibition by genetic or chemical methods results in strong eGFP fluorescence. This robust gain-of-function system readily distinguishes between inhibitor potencies and can be scaled-up to high-throughput platforms for drug testing.

Virucidal efficacy of different formulations for hand and surface disinfection targeting SARS CoV-2 (preprint)

In the ongoing SARS CoV-2 pandemic effective measures are needed, and guidance based on the methodological framework of the European committee for standardization (CEN) can help to choose effective disinfectants on an immediate basis. This study demonstrates that two commercially available formulations for surface disinfection and one formulation for hand disinfection claiming virucidal activity against enveloped viruses are effectively inactivating SARS-CoV-2. This study emphasizes that chemical disinfectants claiming virucidal activity against enveloped viruses are an effective choice to target enveloped SARS-CoV-2 as a preventive measure.

Short Term Metformin Intervention Inhibits IL-6 and IL-1β Secretion and Prevents Acute Respiratory Distress Syndrome-Implications for COVID-19 (preprint)

Acute respiratory distress syndrome (ARDS) is a leading cause of COVID-19 death. Long-term metformin usage decreases COVID-19 mortality through an obscure mechanism unrelated to glycemic control. It is unclear whether and how short-term metformin-based intervention can block acute inflammation. We show that metformin inhibits IL-6 and IL-1β secretion from LPS or SARS-CoV-2 Spike protein primed macrophages undergoing NLRP3 inflammasome activation, a key to ARDS initiation. Reduced IL-6 production correlates with blunted NFAT and C/EBPβ/NFIL-6 activation, whereas inhibition of IL-1β secretion reflects interference with NLRP3 inflammasome activation. By targeting electron transfer chain complex 1, but independently of AMPK and mitophagy, metformin inhibits ATP-dependent mitochondrial DNA synthesis and blocks generation of oxidized mitochondrial DNA, a trigger of inflammasome assembly. Correspondingly, short-term metformin treatment abrogates LPS-induced ARDS, lung macrophage recruitment, damage and mortality. We suggest that metformin, a cheap and safe drug, can be used to prevent ARDS onset in COVID-19 patients.Funding: The UCSD Tissue Technology Shared Resource supported by a National Cancer Institute Cancer Center Support Grant (CCSG P30CA23100). M.A. was supported by NIH (R01AI072726). E.S-L. was supported by NIAMS (K01AR077111). Research was supported by NIH grant awards to M.K. and M.A. (U54CA260591), and M.K. (R01A1043477, P42ES010337), who is an American Cancer Research Society Professor and holds the Ben and Wanda Hildyard Chair for Mitochondrial and Metabolic Diseases. Conflict of Interest: M.K. is a founder of Elgia Pharmaceuticals and receives research support from Gossamer Bio and Jansen Pharmaceuticals. All other authors declare no competing interests.Ethical Approval: All mouse studies were conducted in accordance with UCSD and NIH guidelines and regulations for the housing and treatment of laboratory animals using protocols approved by the UCSD Institutional Animal Care and Use Committee.

SARS-CoV-2 Seroprevalence in Relation to Timing of Symptoms (preprint)

Of individuals with SARS-CoV-2 IgG antibody testing performed, those who contemporaneously experienced a cluster of Covid-19 relevant symptoms in the 1-2 months preceding the antibody assay were more likely to test positive whereas those who experienced the symptom clustering in the prior 3-6 months were more likely to test negative. These findings suggest that antibodies likely wane over a period of months, particularly in relation to the timing of symptoms.

SARS-CoV-2 Seroprevalence Across a Diverse Cohort of Healthcare Workers (preprint)

Importance: Antibody testing is important for understanding patterns of exposure and potential immunity to SARS-CoV-2. Prior data on seroprevalence have been subject to variations in selection of individuals and nature as well as timing of testing in relation to exposures. Objective: We sought to determine the extent of SARS-CoV-2 seroprevalance and the factors associated with seroprevelance across a diverse cohort of healthcare workers. Design: Observational cohort study of healthcare workers, including SARS-CoV-2 serology testing and participant questionaires. Participants: A diverse and unselected population of adults (n=6,062) employed in a multi-site healthcare delivery system located in Los Angeles County, including individuals with direct patient contact and others with non-patient-oriented work functions. Exposure: Exposure and infection with the SARS-CoV-2 virus, as determined by seropositivity. Main Outcomes: Using Bayesian and multi-variate analyses, we estimated seroprevalence and factors associated with seropositivity and antibody titers, including pre-existing demographic and clinical characteristics; potential Covid-19 illness related exposures; and, symptoms consistent with Covid-19 infection. Results: We observed a seroprevalence rate of 4.1%, with anosmia as the most prominently associated self-reported symptom in addition to fever, dry cough, anorexia, and myalgias. After adjusting for potential confounders, pre-existing medical conditions were not associated with antibody positivity. However, seroprevalence was associated with younger age, Hispanic ethnicity, and African-American race, as well as presence of either a personal or household member having a prior diagnosis of Covid-19. Importantly, African American race and Hispanic ethnicity were associated with antibody positivity even after adjusting for personal Covid-19 diagnosis status, suggesting the contribution of unmeasured structural or societally factors. Notably, number of people, or children, in the home was not associated with antibody positivity. Conclusion and Relevance: The demographic factors associated with SARS-CoV-2 seroprevalence among our healthcare workers underscore the importance of exposure sources beyond the workplace. The size and diversity of our study population, combined with robust survey and modeling techniques, provide a vibrant picture of the demographic factors, exposures, and symptoms that can identify individuals with susceptibility as well as potential to mount an immune response to Covid-19.

Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19 (preprint)

Development of antibody protection during SARS-CoV-2 (CoV-2) infection is a pressing question for public health and for vaccine development. We developed highly sensitive CoV-2-specific antibody and neutralization assays. CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n=87) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. CoV-2 neutralization was determined in COVID-19 and convalescent plasma up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which was also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Further, subjects who donated plasma further out from the diagnosis of COVID-19 appeared to have lower titers. Interestingly, some COVID-19 patients also contained NAbs against SARS Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

An insertion unique to SARS-CoV-2 exhibits superantigenic character strengthened by recent mutations (preprint)

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with Coronavirus Disease 2019 (COVID-19) is a newly recognized condition in which children with recent SARS-CoV-2 infection present with a constellation of symptoms including hypotension, multiorgan involvement, and elevated inflammatory markers. These symptoms and the associated laboratory values strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to MHCII molecules and T cell receptors (TCRs). Here, we used structure-based computational models to demonstrate that the SARS-CoV-2 spike (S) exhibits a high-affinity motif for binding TCR, interacting closely with both the - and {beta}-chains variable domains complementarity-determining regions. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in any other SARS coronavirus), which is highly similar in both sequence and structure to bacterial superantigens. Further examination revealed that this interaction between the virus and human T cells is strengthened in the context of a recently reported rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from a motif shared between the SARS viruses from the 2003 and 2019 pandemics, which has intracellular adhesion molecule (ICAM)-like character. These data suggest that the SARS-CoV-2 S may act as a superantigen to drive the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches. SignificanceAlthough children have been largely spared from severe COVID-19 disease, a rare hyperinflammatory syndrome has been described in Europe and the East Coast of the United States, termed Multisystem Inflammatory Syndrome in Children (MISC). The symptoms and diagnostic lab values of MIS-C resemble those of toxic shock, typically caused by pathogenic superantigens stimulating excessive activation of the adaptive immune system. We show that SARS-CoV-2 spike has a sequence and structure motif highly similar to those of bacterial superantigens, and may directly bind to the T cell receptors. This sequence motif, not present in other coronaviruses, may explain the unique potential for SARS-CoV-2 to cause both MIS-C and the cytokine storm observed in adult COVID-19 patients.