Biochemical, Biophysical, and Immunological Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We find the percent solids and protein content are greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibit heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observe increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor-stimulated gene-6 (TSG6) staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicate that increases in HA and DNA in COVID-19 respiratory secretion samples correlate with enhanced inflammatory burden and suggest that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Detection of bacterial co-infections and prediction of fatal outcomes in COVID-19 patients presenting to the emergency department using a 29 mRNA host response classifier.

ObjectiveClinicians in the emergency department (ED) face challenges in concurrently assessing patients with suspected COVID-19 infection, detecting bacterial co-infection, and determining illness severity since current practices require separate workflows. Here we explore the accuracy of the IMX-BVN-3/IMX-SEV-3 29 mRNA host response classifiers in simultaneously detecting SARS-CoV-2 infection, bacterial co-infections, and predicting clinical severity of COVID-19. Methods161 patients with PCR-confirmed COVID-19 (52.2% female, median age 50.0 years, 51% hospitalized, 5.6% deaths) were enrolled at the Stanford Hospital ED. RNA was extracted (2.5 mL whole blood in PAXgene Blood RNA) and 29 host mRNAs in response to the infection were quantified using Nanostring nCounter. ResultsThe IMX-BVN-3 classifier identified SARS-CoV-2 infection in 151 patients with a sensitivity of 93.8%. Six of 10 patients undetected by the classifier had positive COVID tests more than 9 days prior to enrolment and the remaining oscillated between positive and negative results in subsequent tests. The classifier also predicted that 6 (3.7%) patients had a bacterial co-infection. Clinical adjudication confirmed that 5/6 (83.3%) of the patients had bacterial infections, i.e. Clostridioides difficile colitis (n=1), urinary tract infection (n=1), and clinically diagnosed bacterial infections (n=3) for a specificity of 99.4%. 2/101 (2.8%) patients in the IMX-SEV-3 Low and 7/60 (11.7%) in the Moderate severity classifications died within thirty days of enrollment. ConclusionsIMX-BVN-3/IMX-SEV-3 classifiers accurately identified patients with COVID-19, bacterial co-infections, and predicted patients risk of death. A point-of-care version of these classifiers, under development, could improve ED patient management including more accurate treatment decisions and optimized resource utilization.

Association Between SARS-CoV-2 RNAemia and Post-Acute Sequelae of COVID-19.

Determinants of Post-Acute Sequelae of COVID-19 are not known. Here we show that 75% of patients with viral RNA in blood (RNAemia) at presentation were symptomatic in the post-acute phase. RNAemia at presentation successfully predicted PASC, independent of patient demographics, initial disease severity, and length of symptoms.

Intramuscular SARS-CoV-2 vaccines elicit varying degrees of plasma and salivary antibody responses as compared to natural infection

Vaccination induced antibody and T-cell immune responses are important for systemic protection from COVID-19. Because SARS-CoV-2 infects and is transmitted by oral-pharyngeal mucosa, we wished to test mucosal antibodies elicited by natural infection or intramuscular vaccine injection. In a non-randomized observational study, we measured antibodies against the SARS-CoV-2 RBD in plasma and saliva from convalescent or vaccinated individuals and tested their neutralizing potential using a replication competent rVSV-eGFP-SARS-CoV-2. We found IgG and IgA anti-RBD antibodies as well as neutralizing activity in convalescent plasma and saliva. Two doses of mRNA vaccination (BNT162b2 or mRNA-1273) induced high levels of IgG anti-RBD in saliva, a subset of whom also had IgA, and significant neutralizing activity. We detected anti-RBD IgG and IgA with significant neutralizing potential in the plasma of single dose Ad26.COV2.S vaccinated individuals, and we detected slight amounts of anti-RBD antibodies in matched saliva. The role of salivary antibodies in protection against SARS-CoV-2 infection is unknown and merits further investigation. This study was not designed to, nor did it study the full kinetics of the antibody response or protection from infection, nor did it address variants of SARS-CoV-2.

Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19

Our understanding of protective vs. pathologic immune responses to SARS-CoV-2, the virus that causes Coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses reveal widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, with the most profound disturbances including a prominent neutrophil hyperactivation signature and monocytes with anti-inflammatory features. We further demonstrate that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention. One Sentence SummarySingle-cell profiling demonstrates multifarious dysregulation of innate immune phenotype associated with COVID-19 severity.

SARS-CoV-2 RNAaemia predicts clinical deterioration and extrapulmonary complications from COVID-19.

BackgroundThe determinants of COVID-19 disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterise the relationships between SARS-CoV-2 RNAaemia and disease severity, clinical deterioration, and specific EPCs. MethodsWe used quantitative (qPCR) and digital (dPCR) PCR to quantify SARS-CoV-2 RNA from nasopharyngeal swabs and plasma in 191 patients presenting to the Emergency Department (ED) with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterised the role of RNAaemia in predicting clinical severity and EPCs using elastic net regression. Findings23{middle dot}0% (44/191) of SARS-CoV-2 positive patients had viral RNA detected in plasma by dPCR, compared to 1{middle dot}4% (2/147) by qPCR. Most patients with serial measurements had undetectable RNAaemia 10 days after onset of symptoms, but took 16 days to reach maximum severity, and 33 days for symptoms to resolve. Initially RNAaemic patients were more likely to manifest severe disease (OR 6{middle dot}72 [95% CI, 2{middle dot}45 - 19{middle dot}79]), worsening of disease severity (OR 2{middle dot}43 [95% CI, 1{middle dot}07 - 5{middle dot}38]), and EPCs (OR 2{middle dot}81 [95% CI, 1{middle dot}26 - 6{middle dot}36]). RNA load correlated with maximum severity (r = 0{middle dot}47 [95% CI, 0{middle dot}20 - 0{middle dot}67]). InterpretationdPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAaemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Since many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAaemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate. FundingNIH/NIAID (Grants R01A153133, R01AI137272, and 3U19AI057229 - 17W1 COVID SUPP #2) and a donation from Eva Grove. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe varied clinical manifestations of COVID-19 have directed attention to the distribution of SARS-CoV-2 in the body. Although most concentrated and tested for in the nasopharynx, SARS-CoV-2 RNA has been found in blood, stool, and numerous tissues, raising questions about dissemination of viral RNA throughout the body, and the role of this process in disease severity and extrapulmonary complications. Recent studies have detected low levels of SARS-CoV-2 RNA in blood using either quantitative reverse transcriptase real-time PCR (qPCR) or droplet digital PCR (dPCR), and have associated RNAaemia with disease severity and biomarkers of dysregulated immune response. Added value of this studyWe quantified SARS-CoV-2 RNA in the nasopharynx and plasma of patients presenting to the Emergency Department with COVID-19, and found an array-based dPCR platform to be markedly more sensitive than qPCR for detection of SARS-CoV-2 RNA, with a simplified workflow well-suited to clinical adoption. We collected serial plasma samples during patients course of illness, and showed that SARS-CoV-2 RNAaemia peaks early, while clinical condition often continues to worsen. Our findings confirm the association between RNAaemia and disease severity, and additionally demonstrate a role for RNAaemia in predicting future deterioration and specific extrapulmonary complications. Implications of all the available evidenceVariation in SARS-CoV-2 RNAaemia may help explain disparities in disease severity and extrapulmonary complications from COVID-19. Testing for RNAaemia with dPCR early in the course of illness may help guide patient triage and management.

Additional analyses exploring the hypothesized transdifferentiation of plasmablasts to developing neutrophils in severe COVID-19

We thank Alquicira-Hernandez et al. for their reanalysis of our single-cell transcriptomic dataset profiling peripheral immune responses to severe COVID-19. We agree that careful analysis of single-cell sequencing data is important for generating cogent hypotheses but find several aspects of their criticism of our analysis to be problematic. Here we respond briefly to misunderstandings and inaccuracies in their commentary that may have led to misinformed interpretation of our results.

Integrated analysis of multimodal single-cell data

The simultaneous measurement of multiple modalities, known as multimodal analysis, represents an exciting frontier for single-cell genomics and necessitates new computational methods that can define cellular states based on multiple data types. Here, we introduce weighted-nearest neighbor analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of hundreds of thousands of human white blood cells alongside a panel of 228 antibodies to construct a multimodal reference atlas of the circulating immune system. We demonstrate that integrative analysis substantially improves our ability to resolve cell states and validate the presence of previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets, and to interpret immune responses to vaccination and COVID-19. Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets, including paired measurements of RNA and chromatin state, and to look beyond the transcriptome towards a unified and multimodal definition of cellular identity. AvailabilityInstallation instructions, documentation, tutorials, and CITE-seq datasets are available at http://www.satijalab.org/seurat

Hyaluronan is abundant in COVID-19 respiratory secretions

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percent solids and protein content are all greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis (CF), a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. These results highlight the dramatic biophysical properties of COVID-19 respiratory secretions and suggest that DNA and hyaluronan may be viable therapeutic targets in COVID-19 infection.

SARS-CoV-2 Antibody Responses Correlate with Resolution of RNAemia But Are Short-Lived in Patients with Mild Illness

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived.

Human B cell clonal expansion and convergent antibody responses to SARS-CoV-2

During virus infection B cells are critical for the production of antibodies and protective immunity. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify extensive convergence of antibody sequences across SARS-CoV-2 patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.

Cytokine profile in plasma of severe COVID-19 does not differ from ARDS and sepsis

RationaleElevated levels of inflammatory cytokines have been associated with poor outcomes among COVID-19 patients. It is unknown, however, how these levels compare to those observed in critically ill patients with ARDS or sepsis due to other causes. ObjectivesTo directly compare plasma levels of inflammatory cytokines, with a focus on 6 cytokines associated with cytokine storm (IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNF), between hospitalized COVID-19 patients and banked plasma samples from ARDS and sepsis patients from prior to the COVID-19 pandemic. Findings15 hospitalized COVID-19 patients, 9 of whom were critically ill, were compared to 28 critically ill patients with ARDS or sepsis. There were no statistically significant differences in baseline levels of IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNF between patients with severe COVID-19 and critically ill controls with ARDS or sepsis. ConclusionsLevels of inflammatory cytokines IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNF were not higher in critically ill COVID-19 patients than in critically ill patients admitted with ARDS or sepsis due to other causes in this small cohort. Broad use of immunosuppressive therapies in ARDS has failed in numerous Phase 3 studies; use of these therapies in unselected patients with COVID-19 is likely unwarranted.

A single-cell atlas of the peripheral immune response to severe COVID-19

There is an urgent need to better understand the pathophysiology of Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2. Here, we apply single-cell RNA sequencing (scRNA-seq) to peripheral blood mononuclear cells (PBMCs) of 7 patients hospitalized with confirmed COVID-19 and 6 healthy controls. We identify substantial reconfiguration of peripheral immune cell phenotype in COVID-19, including a heterogeneous interferon-stimulated gene (ISG) signature, HLA class II downregulation, and a novel B cell-derived granulocyte population appearing in patients with acute respiratory failure requiring mechanical ventilation. Importantly, peripheral monocytes and lymphocytes do not express substantial amounts of pro-inflammatory cytokines, suggesting that circulating leukocytes do not significantly contribute to the potential COVID-19 cytokine storm. Collectively, we provide the most thorough cell atlas to date of the peripheral immune response to severe COVID-19.