Microbial Dynamics and Pulmonary Immune Responses in COVID-19 Secondary Bacterial Pneumonia (preprint)

Secondary bacterial pneumonia (2°BP) is associated with significant morbidity following respiratory viral infection, yet mechanistically remains incompletely understood. In a prospective cohort of 112 critically ill adults intubated for COVID-19, we comparatively assessed longitudinal airway microbiome dynamics and studied the pulmonary transcriptome of patients who developed 2°BP versus controls who did not. We found that 2°BP was significantly associated with both mortality and corticosteroid treatment. The pulmonary microbiome in 2°BP was characterized by increased bacterial RNA load, dominance of culture-confirmed pathogens, and lower alpha diversity. Bacterial pathogens were detectable days prior to 2°BP clinical diagnosis, and in most cases were also present in nasal swabs. Pathogen antimicrobial resistance genes were also detectable in both the lower airway and nasal samples, and in some cases were identified prior to 2°BP clinical diagnosis. Assessment of the pulmonary transcriptome revealed suppressed TNFa signaling via NF-kB in patients who developed 2°BP, and a sub-analysis suggested that this finding was mediated by corticosteroid treatment. Within the 2°BP group, we observed a striking inverse correlation between innate and adaptive immune gene expression and bacterial RNA load. Together, our findings provide fresh insights into the microbial dynamics and host immune features of COVID-19-associated 2°BP.

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling (preprint)

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections, including with SARS-CoV-2. Here, we identified a gene program, defined by correlation with EN-RAGE (S100A12) gene expression, which was up-regulated in airway and blood myeloid cells from COVID-19 patients. The EN-RAGE program was expressed in 7 cohorts and observed in patients with both COVID-19 and acute respiratory distress syndrome (ARDS) from other causes. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGE+ myeloid cells express features consistent with suppressor cell functionality, with low HLA-DR and high PD-L1 surface expression and higher expression of T cell-suppressive genes. Sustained EN-RAGE signature expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell exhaustion markers, such as PD-1. IL-6 treatment of monocytes in vitro upregulated many of the severity-associated genes in the EN-RAGE gene program, along with potential mediators of T cell suppression, such as IL-10. Blockade of IL-6 signaling by tocilizumab in a placebo-controlled clinical trial led to a rapid normalization of ENRAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

Immediate myeloid depot for SARS-CoV-2 in the human lung (preprint)

In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic1, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model2,3 to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.

A 2-Gene Host Signature for Improved Accuracy of COVID-19 Diagnosis Agnostic to Viral Variants (preprint)

The continued emergence of SARS-CoV-2 variants is one of several factors that may cause false negative viral PCR test results. Such tests are also susceptible to false positive results due to trace contamination from high viral titer samples. Host immune response markers provide an orthogonal indication of infection that can mitigate these concerns when combined with direct viral detection. Here, we leverage nasopharyngeal swab RNA-seq data from patients with COVID-19, other viral acute respiratory illnesses and non-viral conditions (n=318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to predict COVID-19. Optimal classifiers achieve an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort (n=553). We show that a classifier relying on a single interferon-stimulated gene, such as IFI6 or IFI44, measured in RT-qPCR assays (n=144) achieves AUC values as high as 0.88. Addition of a second gene, such as GBP5, significantly improves the specificity compared to other respiratory viruses. The performance of a clinically practical 2-gene RT-qPCR classifier is robust across common SARS-CoV-2 variants, including Omicron, and is unaffected by cross-contamination, demonstrating its utility for improving accuracy of COVID-19 diagnostics.

A 2-Gene Host Signature for Improved Accuracy of COVID-19 Diagnosis Agnostic to Viral Variants (preprint)

The continued emergence of SARS-CoV-2 variants is one of several factors that may cause false negative viral PCR test results. Such tests are also susceptible to false positive results due to trace contamination from high viral titer samples. Host immune response markers provide an orthogonal indication of infection that can mitigate these concerns when combined with direct viral detection. Here, we leverage nasopharyngeal swab RNA-seq data from patients with COVID-19, other viral acute respiratory illnesses and non-viral conditions (n=318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to predict COVID-19. Optimal classifiers achieve an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort (n=553). We show that a classifier relying on a single interferon-stimulated gene, such as IFI6 or IFI44, measured in RT-qPCR assays (n=144) achieves AUC values as high as 0.88. Addition of a second gene, such as GBP5, significantly improves the specificity compared to other respiratory viruses. The performance of a clinically practical 2-gene RT-qPCR classifier is robust across common SARS-CoV-2 variants, including Omicron, and is unaffected by cross-contamination, demonstrating its utility for improving accuracy of COVID-19 diagnostics.

Upper airway gene expression reveals a more robust innate and adaptive immune response to SARS-CoV-2 in children compared with older adults (preprint)

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults and only rarely in children. To investigate whether differences in the upper airway immune response could contribute to this disparity, we compared nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes (ISGs) was robustly activated in both children and adults with SARS-CoV-2 compared to the respective non-viral groups, with only relatively subtle distinctions. Children, however, demonstrated markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including TNF, IFN{gamma}, IL-2 and IL-4 production. Cell type deconvolution confirmed greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibited a decrease in proportions of ciliated cells, the primary target for SARS-CoV-2, upon infection with the virus. These findings demonstrate that children elicit a more robust innate and adaptive immune response to SARS-CoV-2 infection in the upper airway that likely contributes to their protection from severe disease in the lower airway.

SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator (preprint)

SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2- positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.

Impaired immune signaling and changes in the lung microbiome precede secondary bacterial pneumonia in COVID-19 (preprint)

Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing (scRNA-seq) we assessed lower respiratory tract immune responses and microbiome dynamics in 28 COVID-19 patients, 15 of whom developed VAP, and eight critically ill uninfected controls. Two days before VAP onset we observed a transcriptional signature of bacterial infection. Two weeks prior to VAP onset, following intubation, we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset.

COVID-19 ARDS is characterized by a dysregulated host response that differs from cytokine storm and is modified by dexamethasone (preprint)

We performed comparative lower respiratory tract transcriptional profiling of 52 critically ill patients with the acute respiratory distress syndrome (ARDS) from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a cytokine storm, we observed reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS was characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity that were predicted to be modulated by dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 was characterized by impaired interferon-stimulated gene expression (ISG). We found that the relationship between SARS-CoV-2 viral load and expression of ISGs was decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients with COVID-19 ARDS did not demonstrate cytokine storm but instead revealed a unique and dysregulated host response predicted to be modified by dexamethasone.

Protective Effects of STI-2020 Antibody Delivered Post-Infection by the Intranasal or Intravenous Route in a Syrian Golden Hamster COVID-19 Model (preprint)

We have previously reported that the SARS-CoV-2 neutralizing antibody, STI-2020, potently inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. We now have extended our in vivo studies of STI-2020 to include disease treatment efficacy, profiling of biodistribution of STI-2020 in mice when antibody is delivered intranasally (IN) or intravenously (IV), as well as pharmacokinetics in mice following IN antibody administration. Importantly, SARS-CoV-2-infected hamsters were treated with STI-2020 using these routes, and treatment effects on severity and duration of COVID-19-like disease in this model were evaluated. In SARS-CoV-2 infected hamsters, treatment with STI-2020 12 hours post-infection using the IN route led to a decrease in severity of clinical disease signs and a more robust recovery during 9 days of infection as compared to animals treated with an isotype control antibody. Treatment via the IV route using the same dose and timing regimen resulted in a decrease in the average number of consecutive days that infected animals experienced weight loss, shortening the duration of disease and allowing recovery to begin more rapidly in STI-2020 treated animals. Following IN administration in mice, STI-2020 was detected within 10 minutes in both lung tissue and lung lavage. The half-life of STI-2020 in lung tissue is approximately 25 hours. We are currently investigating the minimum effective dose of IN-delivered STI-2020 in the hamster model as well as establishing the relative benefit of delivering neutralizing antibodies by both IV and IN routes.

Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables fast and accurate outbreak analysis (preprint)

Current transmission rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still increasing and many countries are facing second waves of infections. Rapid SARS-CoV-2 whole genome sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring and determining transmission links. Here we report a novel reverse complement polymerase chain reaction (RC-PCR) technology for WGS of SARS-CoV-2. This technique is unique as it enables library preparation in a single PCR saving time, resources and enables high throughput screening. A total of 173 samples tested positive for SARS-CoV-2 between March and September 2020 were included. RC-PCR WGS applicability for outbreak analysis in public health service and hospital settings was tested on six predefined clusters containing samples of healthcare workers and patients. RC-PCR resulted in WGS data for 146 samples. It showed a genome coverage of up to 98,2% for samples with a maximum Ct value of 32. Three out of six suspected clusters were fully confirmed, while in other clusters four healthcare workers were not associated. Importantly, a previously unknown chain of transmission was confirmed in the public health service samples. These findings confirm the reliability and applicability of the RC-PCR technology for SARS-CoV-2 sequencing in outbreak analysis and surveillance.

Global Absence and Targeting of Protective Immune States in Severe COVID-19. (preprint)

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense. One Sentence SummaryIn severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.

Global Absence and Targeting of Protective Immune States in Severe COVID-19. (preprint)

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a wholeblood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferonstimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and autodirected antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.

Genetic and non-genetic factors affecting the expression of COVID-19 relevant genes in the large airway epithelium (preprint)

Particular host and environmental factors influence susceptibility to severe COVID-19. We analyzed RNA-sequencing data from bronchial epithelial brushings - a relevant tissue for SARS-CoV-2 infection - obtained from three cohorts of uninfected individuals, and investigated how non-genetic and genetic factors affect the regulation of host genes implicated in COVID-19. We found that ACE2 expression was higher in relation to active smoking, obesity, and hypertension that are known risk factors of COVID-19 severity, while an association with interferon-related inflammation was driven by the truncated, non-binding ACE2 isoform. We discovered that expression patterns of a suppressed airway immune response to early SARS-CoV-2 infection, compared to other viruses, are similar to patterns associated with obesity, hypertension, and cardiovascular disease, which may thus contribute to a COVID-19-susceptible airway environment. eQTL mapping identified regulatory variants for genes implicated in COVID-19, some of which had pheWAS evidence for their potential role in respiratory infections. These data provide evidence that clinically relevant variation in the expression of COVID-19-related genes is associated with host factors, environmental exposures, and likely host genetic variation.

Direct detection of SARS-CoV-2 using CRISPR-Cas13a and a mobile phone (preprint)

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic and pre- symptomatic carriers of the virus. CRISPR-based diagnostics that utilize RNA and DNA-targeting enzymes can augment gold-standard PCR-based testing if they can be made rapid, portable and accurate. Here we report the development of an amplification-free CRISPR-Cas13a-based mobile phone assay for direct detection of SARS-CoV-2 from nasal swab RNA extracts. The assay achieved ~100 copies/L sensitivity in under 30 minutes and accurately detected a set of positive clinical samples in under 5 minutes. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity, and we directly quantified viral load using enzyme kinetics. Combined with mobile phone-based quantification, this assay can provide rapid, low-cost, point-of-care screening to aid in the control of SARS-CoV-2.

Nasopharyngeal SARS-CoV2 viral loads in young children do not differ significantly from those in older children and adults (preprint)

The role of children in the spread of the SARS-CoV2 coronavirus has become a matter of urgent debate as societies in the US and abroad consider how to safely reopen schools. Small studies have suggested higher viral loads in young children. Here we present a multicenter investigation on over five thousand SARS-CoV-2 cases confirmed by real-time reverse transcription (RT) PCR assay. Notably, we found no discernable difference in amount of viral nucleic acid among young children and adults.

Upper airway gene expression differentiates COVID-19 from other acute respiratory illnesses and reveals suppression of innate immune responses by SARS-CoV-2 (preprint)

We studied the host transcriptional response to SARS-CoV-2 by performing metagenomic sequencing of upper airway samples in 238 patients with COVID-19, other viral or non-viral acute respiratory illnesses (ARIs). Compared to other viral ARIs, COVID-19 was characterized by a diminished innate immune response, with reduced expression of genes involved in toll-like receptor and interleukin signaling, chemokine binding, neutrophil degranulation and interactions with lymphoid cells. Patients with COVID-19 also exhibited significantly reduced proportions of neutrophils and macrophages, and increased proportions of goblet, dendritic and B-cells, compared to other viral ARIs. Using machine learning, we built 26-, 10- and 3-gene classifiers that differentiated COVID-19 from other acute respiratory illnesses with AUCs of 0.980, 0.950 and 0.871, respectively. Classifier performance was stable at low viral loads, suggesting utility in settings where direct detection of viral nucleic acid may be unsuccessful. Taken together, our results illuminate unique aspects of the host transcriptional response to SARS-CoV-2 in comparison to other respiratory viruses and demonstrate the feasibility of COVID-19 diagnostics based on patient gene expression.

Clinical features, diagnostics, and outcomes of patients presenting with acute respiratory illness: a comparison of patients with and without COVID-19 (preprint)

BackgroundEmerging data on the clinical presentation, diagnostics, and outcomes of patients with COVID-19 have largely been presented as case series. Few studies have compared these clinical features and outcomes of COVID-19 to other acute respiratory illnesses. MethodsWe examined all patients presenting to an emergency department in San Francisco, California between February 3 and March 31, 2020 with an acute respiratory illness who were tested for SARS-CoV-2. We determined COVID-19 status by PCR and metagenomic next generation sequencing (mNGS). We compared demographics, comorbidities, symptoms, vital signs, and laboratory results including viral diagnostics using PCR and mNGS. Among those hospitalized, we determined differences in treatment (antibiotics, antivirals, respiratory support) and outcomes (ICU admission, ICU interventions, acute respiratory distress syndrome, cardiac injury). FindingsIn a cohort of 316 patients, 33 (10%) tested positive for SARS-CoV-2; 31 patients, all without COVID-19, tested positive for another respiratory virus (16%). Among patients with additional viral testing, no co-infections with SARS-CoV-2 were identified by PCR or mNGS. Patients with COVID-19 reported longer symptoms duration (median 7 vs. 3 days) and were more likely to report fever (82% vs. 44%) fatigue (85% vs. 50%) and myalgias (61% vs 27%); p<0.001 for all comparisons. Lymphopenia (55% vs 34%, p=0.018) and bilateral opacities on initial chest radiograph (55% vs. 24%, p=0.001) were more common in patients with COVID-19. Patients with COVID-19 were more often hospitalized (79% vs. 56%, p=0.014). Of 186 hospitalized patients, patients with COVID-19 had longer hospitalizations (median 10.7d vs. 4.7d, p<0.001) and were more likely to develop ARDS (23% vs. 3%, p<0.001). Most comorbidities, home medications, signs and symptoms, vital signs, laboratory results, treatment, and outcomes did not differ by COVID-19 status. InterpretationWhile we found differences in clinical features of COVID-19 compared to other acute respiratory illnesses, there was significant overlap in presentation and comorbidities. Patients with COVID-19 were more likely to be admitted to the hospital, have longer hospitalizations and develop ARDS, and were unlikely to have co-existent viral infections. These findings enhance understanding of the clinical characteristics of COVID-19 in comparison to other acute respiratory illnesses.