Emergence of multiple SARS-CoV-2 antibody escape variants in an immunocompromised host undergoing convalescent plasma treatment (preprint)

SARS-CoV-2 Variants of Concerns (VOC), e.g., B.1.351 (20H/501Y.V2) and P1 (20J/501Y.V3), harboring N-terminal domain (NTD) or the receptor-binding domain (RBD) (e.g., E484K) mutations, exhibit reduced in vitro susceptibility to convalescent serum, commercial antibody cocktails, and vaccine neutralization, and have been associated with reinfection. The accumulation of these mutations could be the consequence of intra-host viral evolution due to prolonged infection in immunocompromised hosts. In this study, we document the microevolution of SARS-CoV-2 recovered from sequential tracheal aspirates from an immunosuppressed patient on tacrolimus, steroids and convalescent plasma therapy, and identify the emergence of multiple NTD and RBD mutations associated with reduced antibody neutralization as early as three weeks after infection. SARS-CoV-2 genomes from the first swab (Day 0) and three tracheal aspirates (Day 7, 21 and 27) were compared at the sequence level. We identified five different S protein mutations at the NTD or RBD regions from the second tracheal aspirate sample (21 Day). The S:Q493R substitution and S:243-244LA deletion had ~70% frequency, while ORF1a:A138T, S:141-144LGVY deletion, S:E484K and S:Q493K substitutions demonstrated ~30%, ~30%, ~20% and ~10% mutation frequency, respectively. However, the third tracheal aspirate sample collected one week later (Day 27) was predominated by the haplotype of ORF1a:A138T, S:141-144LGVY deletion and S:E484K (> 95% mutation frequency). Notably, S protein deletions (141-144LGVY and 243-244LA deletions in NTD region) and substitutions (Q493K/R and E484K in the RBD region) previously showed reduced susceptibly to monoclonal antibody or convalescent plasma. The observation supports the hypothesis that VOCs can independently arise and that immunocompromised patients on convalescent plasma therapy are potential breeding grounds for immune-escape mutants.

A Novel Diagnostic Test to Screen SARS-CoV-2 Variants Containing E484K and N501Y Mutations (preprint)

Spike protein mutations E484K and N501Y carried by SARS-CoV-2 variants have been associated with concerning changes of the virus, including resistance to neutralizing antibodies and increased transmissibility. While the concerning variants are fast spreading in various geographical areas, identification and monitoring of these variants is lagging far behind, due in large part to the slow speed and insufficient capacity of viral sequencing. In response to the unmet need for a fast and efficient screening tool, we developed a single-tube duplex molecular assay for rapid and simultaneous identification of E484K and N501Y mutations from nasopharyngeal swab (NS) samples within 2.5 h from sample preparation to report. Using this tool, we screened a total of 435 clinical NS samples collected from COVID patients at 8 hospitals within the Hackensack Meridian Health network in New Jersey. While B.1.351 and P.1 variants were absent from the current study, our data revealed a dramatic increase in the frequency of E484K over time, underscoring the need for continuous epidemiological monitoring.

Systemic Tissue and Cellular Disruption from SARS-CoV-2 Infection revealed in COVID-19 Autopsies and Spatial Omics Tissue Maps (preprint)

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has infected over 115 million people and caused over 2.5 million deaths worldwide. Yet, the molecular mechanisms underlying the clinical manifestations of COVID-19, as well as what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome (ARDS), remains poorly understood. To address these challenges, we combined transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues, matched with spatial protein and expression profiling (GeoMx) across 357 tissue sections. These results define both body-wide and tissue-specific (heart, liver, lung, kidney, and lymph nodes) damage wrought by the SARS-CoV-2 infection, evident as a function of varying viral load (high vs. low) during the course of infection and specific, transcriptional dysregulation in splicing isoforms, T cell receptor expression, and cellular expression states. In particular, cardiac and lung tissues revealed the largest degree of splicing isoform switching and cell expression state loss. Overall, these findings reveal a systemic disruption of cellular and transcriptional pathways from COVID-19 across all tissues, which can inform subsequent studies to combat the mortality of COVID-19, as well to better understand the molecular dynamics of lethal SARS-CoV-2 infection and other viruses.

Host genome analysis of structural variations by Optical Genome Mapping provides clinically valuable insights into genes implicated in critical immune, viral infection, and viral replication pathways in patients with severe COVID-19. (preprint)

BackgroundThe varied clinical manifestations and outcomes in patients with SARS-CoV-2 infections implicate a role of host-genetics in the predisposition to disease severity. This is supported by evidence that is now emerging, where initial reports identify common risk factors and rare genetic variants associated with high risk for severe/ life-threatening COVID-19. Impressive global efforts have focused on either identifying common genetic factors utilizing short-read sequencing data in Genome-Wide Association Studies (GWAS) or whole-exome and genome studies to interrogate the human genome at the level of detecting single nucleotide variants (SNVs) and short indels. However, these studies lack the sensitivity to accurately detect several classes of variants, especially large structural variants (SVs) including copy number variants (CNVs), which account for a substantial proportion of variation among individuals. Thus, we investigated the host genomes of individuals with severe/life-threatening COVID-19 at the level of large SVs (500bp-Mb level) to identify events that might provide insight into the inter-individual clinical variability in clinical course and outcomes of COVID-19 patients. MethodsOptical genome mapping using Bionanos Saphyr(R) system was performed on thirty-seven severely ill COVID-19 patients admitted to intensive care units (ICU). To extract candidate SVs, three distinct analyses were undertaken. First, an unbiased whole-genome analysis of SVs was performed to identify rare/unique genic SVs in these patients that did not appear in population datasets to determine candidate loci as decisive predisposing factors associated with severe COVID-19. Second, common SVs with a population frequency filter was interrogated for possible association with severe COVID-19 based on literature surveys. Third, genome-wide SV enrichment in severely ill patients versus the general population was investigated by calculating odds ratios to identify top-ranked genes/loci. Candidate SVs were confirmed using qPCR and an independent bioinformatics tool (FaNDOM). ResultsOur patient-centric investigation identified 11 SVs involving 38 genes implicated in three key host-viral interaction pathways: (1) innate immunity and inflammatory response, (2) airway resistance to pathogens, and (3) viral replication, spread, and RNA editing. These included seven rare/unique SVs (not present in the control dataset), identified in 24.3% (9/37) of patients, impacting up to 31 genes, of which STK26 and DPP4 are the most promising candidates. A duplication partially overlapping STK26 was corroborated with data showing upregulation of this gene in severely ill patients. Further, using a population frequency filter of less than 20% in the Bionano control dataset, four SVs involving seven genes were identified in 56.7% (21/37) of patients. ConclusionThis study is the first to systematically assess and highlight SVs potential role in the pathogenesis of COVID-19 severity. The genes implicated here identify novel SVs, especially STK26, and extend previous reports involving innate immunity and type I interferon response in the pathogenesis of COVID-19. Our study also shows that optical genome mapping can be a powerful tool to identify large SVs impacting disease outcomes with split survival and add valuable genomic information to the existing sequencing-based technology databases to understand the inter-individual variability associated with SARS-CoV-2 infections and COVID-19 mortality.

Type 2 and interferon inflammation strongly regulate SARS-CoV-2 related gene expression in the airway epithelium (preprint)

Coronavirus disease 2019 (COVID-19) outcomes vary from asymptomatic infection to death. This disparity may reflect different airway levels of the SARS-CoV-2 receptor, ACE2, and the spike protein activator, TMPRSS2. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci (eQTL) for both ACE2 and TMPRSS2, that vary in frequency across world populations. Importantly, we find TMPRSS2 is part of a mucus secretory network, highly upregulated by T2 inflammation through the action of interleukin-13, and that interferon response to respiratory viruses highly upregulates ACE2 expression. Finally, we define airway responses to coronavirus infections in children, finding that these infections upregulate IL6 while also stimulating a more pronounced cytotoxic immune response relative to other respiratory viruses. Our results reveal mechanisms likely influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.