We are pleased to feature all COVID-19-related articles published in JCI Insight.
The editors of JCI and JCI Insight have revised the editorial process to address the impact of the COVID-19 pandemic on the global research community. Highlights:
Immune and inflammatory responses to SARS-CoV-2 contribute to disease severity of COVID-19. However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity, and assessed type-I IFN-, type-II IFN-, and NF-κB-dependent whole blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and non-hematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type-I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients’ first samples revealed 12 biomarkers (CCL2, IL-15, sST2, NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and two additional biomarkers (lactoferrin, CXCL9) that were significantly associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.
Michael S. Abers, Ottavia M. Delmonte, Emily E. Ricotta, Jonathan Fintzi, Danielle Fink, Adriana A. de Jesus, Kol A. Zarember, Sara Alehashemi, Vasileios Oikonomou, Jigar V. Desai, Scott W. Canna, Bita Shakoory, Kerry Dobbs, Luisa Imberti, Alessandra Sottini, Eugenia Quiros-Roldan, Francesco Castelli, Camillo Rossi, Duilio Brugnoni, Andrea Biondi, Laura R. Bettini, Mariella D’Angio’, Paolo Bonfanti, Riccardo Castagnoli, Daniela Montagna, Amelia Licari, Gian Luigi Marseglia, Emily Gliniewicz, Elana R. Shaw, Dana Kahle, Andre T. Rastegar, Michael A Stack, Katherine Myint-Hpu, Susan L. Levinson, Mark J. DiNubile, Daniel W. Chertow, Peter Burbelo, Jeffrey I. Cohen, Katherine R. Calvo, John S. Tsang, Helen C. Su, John I. Gallin, Douglas B. Kuhns, Raphaela Goldbach-Mansky, Michail S Lionakis, Luigi D Notarangelo
We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in 2 animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg given daily) did not significantly benefit clinical outcome, nor did it reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment, neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these 2 preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19.
Kyle Rosenke, Michael A. Jarvis, Friederike Feldmann, Benjamin Schwarz, Atsushi Okumura, Jamie Lovaglio, Greg Saturday, Patrick W. Hanley, Kimberly Meade-White, Brandi N. Williamson, Frederick Hansen, Lizette Perez-Perez, Shanna Leventhal, Tsing-Lee Tang-Huau, Julie Callison, Elaine Haddock, Kaitlin A. Stromberg, Dana Scott, Graham Sewell, Catharine M. Bosio, David Hawman, Emmie de Wit, Heinz Feldmann
BACKGROUND Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) differ in the severity of disease. We hypothesized that characteristics of SARS-CoV-2–specific immunity correlate with disease severity.METHODS In this study, SARS-CoV-2–specific T cells and antibodies were characterized in uninfected controls and patients with different coronavirus disease 2019 (COVID-19) disease severity. SARS-CoV-2–specific T cells were flow cytometrically quantified after stimulation with SARS-CoV-2 peptide pools and analyzed for expression of cytokines (IFN-γ, IL-2, and TNF-α) and markers for activation, proliferation, and functional anergy. SARS-CoV-2–specific IgG and IgA antibodies were quantified using ELISA. Moreover, global characteristics of lymphocyte subpopulations were compared between patient groups and uninfected controls.RESULTS Despite severe lymphopenia affecting all major lymphocyte subpopulations, patients with severe disease mounted significantly higher levels of SARS-CoV-2–specific T cells as compared with convalescent individuals. SARS-CoV-2–specific CD4+ T cells dominated over CD8+ T cells and closely correlated with the number of plasmablasts and SARS-CoV-2–specific IgA and IgG levels. Unlike in convalescent patients, SARS-CoV-2–specific T cells in patients with severe disease showed marked alterations in phenotypical and functional properties, which also extended to CD4+ and CD8+ T cells in general.CONCLUSION Given the strong induction of specific immunity to control viral replication in patients with severe disease, the functionally altered characteristics may result from the need for contraction of specific and general immunity to counteract excessive immunopathology in the lung.FUNDING The study was supported by institutional funds to MS and in part by grants of Saarland University, the State of Saarland, and the Rolf M. Schwiete Stiftung.
David Schub, Verena Klemis, Sophie Schneitler, Janine Mihm, Philipp M. Lepper, Heinrike Wilkens, Robert Bals, Hermann Eichler, Barbara C. Gärtner, Sören L. Becker, Urban Sester, Martina Sester, Tina Schmidt
Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase (Gluc) or secreted nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque-reduction assay using an authentic, infectious SARS-CoV-2 strain. The assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms; patients included those in the intensive care unit (ICU), health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs, and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2 and demonstrates the efficacy of a potentially novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts.
Cong Zeng, John P. Evans, Rebecca Pearson, Panke Qu, Yi-Min Zheng, Richard T. Robinson, Luanne Hall-Stoodley, Jacob Yount, Sonal Pannu, Rama K. Mallampalli, Linda Saif, Eugene Oltz, Gerard Lozanski, Shan-Lu Liu
Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector–based assay.
Kento T. Abe, Zhijie Li, Reuben Samson, Payman Samavarchi-Tehrani, Emelissa J. Valcourt, Heidi Wood, Patrick Budylowski, Alan P. Dupuis II, Roxie C. Girardin, Bhavisha Rathod, Jenny H. Wang, Miriam Barrios-Rodiles, Karen Colwill, Allison J. McGeer, Samira Mubareka, Jennifer L. Gommerman, Yves Durocher, Mario Ostrowski, Kathleen A. McDonough, Michael A. Drebot, Steven J. Drews, James M. Rini, Anne-Claude Gingras
The emergence of SARS-CoV-2 has created an international health crisis, and small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection owing to low-affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here, we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promoter (K18). In contrast to nontransgenic mice, intranasal exposure of K18-hACE2 animals to 2 different doses of SARS-CoV-2 resulted in acute disease, including weight loss, lung injury, brain infection, and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals showed increases in transcripts involved in lung injury and inflammatory cytokines. In the low-dose challenge groups, there was a survival advantage in the female mice, with 60% surviving infection, whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared with female mice. To our knowledge, this is the first highly lethal murine infection model for SARS-CoV-2 and should be valuable for the study of SARS-CoV-2 pathogenesis and for the assessment of MCMs.
Joseph W. Golden, Curtis R. Cline, Xiankun Zeng, Aura R. Garrison, Brian D. Carey, Eric M. Mucker, Lauren E. White, Joshua D. Shamblin, Rebecca L. Brocato, Jun Liu, April M. Babka, Hypaitia B. Rauch, Jeffrey M. Smith, Bradley S. Hollidge, Collin Fitzpatrick, Catherine V. Badger, Jay W. Hooper
Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies against the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological ELISA using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 reverse transcription PCR–confirmed, SARS-CoV-2–hospitalized patients, as well as 464 healthy and non–COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies against the 2 different viral proteins showed a moderate correlation. Antibodies against N-protein were detected at a rate of 3.6% in healthy and non–COVID-19 sera collected during the pandemic in 2020, whereas 1.9% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD–binding antibodies exhibited neutralizing capacity, but only 74% of N-protein–positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein–binding antibodies does not always correlate with presence of S-RBD–neutralizing antibodies and caution against the extensive use of N-protein–based serology testing for determination of potential COVID-19 immunity.
Kathleen M. McAndrews, Dara P. Dowlatshahi, Jianli Dai, Lisa M. Becker, Janine Hensel, Laura M. Snowden, Jennifer M. Leveille, Michael R. Brunner, Kylie W. Holden, Nikolas S. Hopkins, Alexandria M. Harris, Jerusha Kumpati, Michael A. Whitt, J. Jack Lee, Luis L. Ostrosky-Zeichner, Ramesha Papanna, Valerie S. LeBleu, James P. Allison, Raghu Kalluri
BACKGROUND Elevated levels of inflammatory cytokines have been associated with poor outcomes among COVID-19 patients. It is unknown, however, how these levels compare with those observed in critically ill patients with acute respiratory distress syndrome (ARDS) or sepsis due to other causes.METHODS We used a Luminex assay to determine expression of 76 cytokines from plasma of hospitalized COVID-19 patients and banked plasma samples from ARDS and sepsis patients. Our analysis focused on detecting statistical differences in levels of 6 cytokines associated with cytokine storm (IL-1β, IL-1RA, IL-6, IL-8, IL-18, and TNF-α) between patients with moderate COVID-19, severe COVID-19, and ARDS or sepsis.RESULTS Fifteen hospitalized COVID-19 patients, 9 of whom were critically ill, were compared with critically ill patients with ARDS (n = 12) or sepsis (n = 16). There were no statistically significant differences in baseline levels of IL-1β, IL-1RA, IL-6, IL-8, IL-18, and TNF-α between patients with COVID-19 and critically ill controls with ARDS or sepsis.CONCLUSION Levels of inflammatory cytokines were not higher in severe COVID-19 patients than in moderate COVID-19 or critically ill patients with ARDS or sepsis 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 may be unwarranted.FUNDING Funding was received from NHLBI K23 HL125663 (AJR); The Bill and Melinda Gates Foundation OPP1113682 (AJR and CAB); Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases #1016687 NIH/NIAID U19AI057229-16; Stanford Maternal Child Health Research Institute; and Chan Zuckerberg Biohub (CAB).
Jennifer G. Wilson, Laura J. Simpson, Anne-Maud Ferreira, Arjun Rustagi, Jonasel Roque, Adijat Asuni, Thanmayi Ranganath, Philip M. Grant, Aruna Subramanian, Yael Rosenberg-Hasson, Holden T. Maecker, Susan P. Holmes, Joseph E. Levitt, Catherine A. Blish, Angela J. Rogers
COVID-19–associated morbidity and mortality have been attributed to a pathologic host response. Two divergent hypotheses have been proposed: hyperinflammatory cytokine storm; and failure of host protective immunity that results in unrestrained viral dissemination and organ injury. A key explanation for the inability to address this controversy has been the lack of diagnostic tools to evaluate immune function in COVID-19 infections. ELISpot, a highly sensitive, functional immunoassay, was employed in 27 patients with COVID-19, 51 patients with sepsis, 18 critically ill nonseptic (CINS) patients, and 27 healthy control volunteers to evaluate adaptive and innate immune status by quantitating T cell IFN-ɣ and monocyte TFN-α production. Circulating T cell subsets were profoundly reduced in COVID-19 patients. Additionally, stimulated blood mononuclear cells produced less than 40%–50% of the IFN-ɣ and TNF-α observed in septic and CINS patients, consistent with markedly impaired immune effector cell function. Approximately 25% of COVID-19 patients had increased IL-6 levels that were not associated with elevations in other canonical proinflammatory cytokines. Collectively, these findings support the hypothesis that COVID-19 suppresses host functional adaptive and innate immunity. Importantly, IL-7 administered ex vivo restored T cell IFN-ɣ production in COVID-19 patients. Thus, ELISpot may functionally characterize host immunity in COVID-19 and inform prospective therapies.
Kenneth E. Remy, Monty Mazer, David A. Striker, Ali H. Ellebedy, Andrew H. Walton, Jacqueline Unsinger, Teresa M. Blood, Philip A. Mudd, Daehan J. Yi, Daniel A. Mannion, Dale F. Osborne, R. Scott Martin, Nitin J. Anand, James P. Bosanquet, Jane Blood, Anne M. Drewry, Charles C. Caldwell, Isaiah R. Turnbull, Scott C. Brakenridge, Lyle L. Moldwawer, Richard S. Hotchkiss
BACKGROUND Reprogramming of host metabolism supports viral pathogenesis by fueling viral proliferation, by providing, for example, free amino acids and fatty acids as building blocks.METHODS To investigate metabolic effects of SARS-CoV-2 infection, we evaluated serum metabolites of patients with COVID-19 (n = 33; diagnosed by nucleic acid testing), as compared with COVID-19–negative controls (n = 16).RESULTS Targeted and untargeted metabolomics analyses identified altered tryptophan metabolism into the kynurenine pathway, which regulates inflammation and immunity. Indeed, these changes in tryptophan metabolism correlated with interleukin-6 (IL-6) levels. Widespread dysregulation of nitrogen metabolism was also seen in infected patients, with altered levels of most amino acids, along with increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and renal dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis. Interestingly, metabolite levels in these pathways correlated with clinical laboratory markers of inflammation (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen).CONCLUSION In conclusion, this initial observational study identified amino acid and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.FUNDING Boettcher Foundation Webb-Waring Biomedical Research Award; National Institute of General and Medical Sciences, NIH; and National Heart, Lung, and Blood Institute, NIH.
Tiffany Thomas, Davide Stefanoni, Julie A. Reisz, Travis Nemkov, Lorenzo Bertolone, Richard O. Francis, Krystalyn E. Hudson, James C. Zimring, Kirk C. Hansen, Eldad A. Hod, Steven L. Spitalnik, Angelo D’Alessandro
Coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in a global pandemic and a disruptive health crisis. COVID-19–related morbidity and mortality have been attributed to an exaggerated immune response. The role of complement activation and its contribution to illness severity is being increasingly recognized. Here, we summarize current knowledge about the interaction of coronaviruses with the complement system. We posit that (a) coronaviruses activate multiple complement pathways; (b) severe COVID-19 clinical features often resemble complementopathies; (c) the combined effects of complement activation, dysregulated neutrophilia, endothelial injury, and hypercoagulability appear to be intertwined to drive the severe features of COVID-19; (d) a subset of patients with COVID-19 may have a genetic predisposition associated with complement dysregulation; and (e) these observations create a basis for clinical trials of complement inhibitors in life-threatening illness.
Anuja Java, Anthony J. Apicelli, M. Kathryn Liszewski, Ariella Coler-Reilly, John P. Atkinson, Alfred H.J. Kim, Hrishikesh S. Kulkarni
BACKGROUND Fatal cases of COVID-19 are increasing globally. We retrospectively investigated the potential of immunologic parameters as early predictors of COVID-19.METHODS A total of 1018 patients with confirmed COVID-19 were enrolled in our 2-center retrospective study. Clinical feature, laboratory test, immunological test, radiological findings, and outcomes data were collected. Univariate and multivariable logistic regression analyses were performed to evaluate factors associated with in-hospital mortality. Receiver operator characteristic (ROC) curves and survival curves were plotted to evaluate their clinical utility.RESULTS The counts of all T lymphocyte subsets were markedly lower in nonsurvivors than in survivors, especially CD8+ T cells. Among all tested cytokines, IL-6 was elevated most significantly, with an upward trend of more than 10-fold. Using multivariate logistic regression analysis, IL-6 levels of more than 20 pg/mL and CD8+ T cell counts of less than 165 cells/μL were found to be associated with in-hospital mortality after adjusting for confounding factors. Groups with IL-6 levels of more than 20 pg/mL and CD8+ T cell counts of less than 165 cells/μL had a higher percentage of older and male patients as well as a higher proportion of patients with comorbidities, ventilation, intensive care unit admission, shock, and death. Furthermore, the receiver operating curve of the model combining IL-6 (>20 pg/mL) and CD8+ T cell counts (<165 cells/μL) displayed a more favorable discrimination than that of the CURB-65 score. The Hosmer-Lemeshow test showed a good fit of the model, with no statistical significance.CONCLUSION IL-6 (>20 pg/mL) and CD8+ T cell counts (<165 cells/μL) are 2 reliable prognostic indicators that accurately stratify patients into risk categories and predict COVID-19 mortality.Funding This work was supported by funding from the National Natural Science Foundation of China (no. 81772477 and 81201848).
Miao Luo, Jing Liu, Weiling Jiang, Shuang Yue, Huiguo Liu, Shuang Wei
Yogendra Kanthi, Jason S. Knight, Yu Zuo, David J. Pinsky
BACKGROUND. Identifying immune correlates of COVID-19 disease severity is an urgent need for clinical management, vaccine evaluation, and drug development. Here, we present a temporal analysis of key immune mediators, cytokines, and chemokines in blood of hospitalized COVID-19 patients from serial sampling and follow-up over 4 weeks. METHODS. A total of 71 patients with laboratory-confirmed COVID-19 admitted to Beijing You’an Hospital in China with either mild (53 patients) or severe (18 patients) disease were enrolled with 18 healthy volunteers. We measured 34 immune mediators, cytokines, and chemokines in peripheral blood every 4–7 days over 1 month per patient using a bioplex multiplex immunoassay. RESULTS. We found that the chemokine RANTES (CCL5) was significantly elevated, from an early stage of the infection, in patients with mild but not severe disease. We also found that early production of inhibitory mediators including IL-10 and IL-1RA were significantly associated with disease severity, and a combination of CCL5, IL-1 receptor antagonist (IL-1RA), and IL-10 at week 1 may predict patient outcomes. The majority of cytokines that are known to be associated with the cytokine storm in virus infections such as IL-6 and IFN-γ were only significantly elevated in the late stage of severe COVID-19 illness. TNF-α and GM-CSF showed no significant differences between severe and mild cases. CONCLUSION. Together, our data suggest that early intervention to increase expression of CCL5 may prevent patients from developing severe illness. Our data also suggest that measurement of levels of CCL5, as well as IL-1RA and IL-10 in blood individually and in combination, might be useful prognostic biomarkers to guide treatment strategies.
Yan Zhao, Ling Qin, Ping Zhang, Kang Li, Lianchun Liang, Jianping Sun, Bin Xu, Yanchao Dai, Xuemei Li, Chi Zhang, Yanchun Peng, Yingmei Feng, Ang Li, Zhongjie Hu, Haiping Xiang, Graham Ogg, Ling-Pei Ho, Andrew McMichael, Ronghua Jin, Julian C. Knight, Tao Dong, Yonghong Zhang
BACKGROUND. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a pandemic. This study addresses the clinical and immunopathological characteristics of severe COVID-19. METHODS. Sixty-nine patients with COVID-19 were classified into severe and nonsevere groups to analyze their clinical and laboratory characteristics. A panel of blood cytokines was quantified over time. Biopsy specimens from 2 deceased cases were obtained for immunopathological, ultrastructural, and in situ hybridization examinations. RESULTS. Circulating cytokines, including IL-8, IL-6, TNF-α, IP10, MCP1, and RANTES, were significantly elevated in patients with severe COVID-19. Dynamic IL-6 and IL-8 were associated with disease progression. SARS-CoV-2 was demonstrated to infect type II and type I pneumocytes and endothelial cells, leading to severe lung damage through cell pyroptosis and apoptosis. In severe cases, lymphopenia, neutrophilia, depletion of CD4+ and CD8+ T lymphocytes, and massive macrophage and neutrophil infiltrates were observed in both blood and lung tissues. CONCLUSIONS. A panel of circulating cytokines could be used to predict disease deterioration and inform clinical interventions. Severe pulmonary damage was predominantly attributed to both cytopathy caused by SARS-CoV-2 and immunopathologic damage. Strategies that prohibit pulmonary recruitment and overactivation of inflammatory cells by suppressing cytokine storm might improve the outcomes of patients with severe COVID-19.
Shaohua Li, Lina Jiang, Xi Li, Fang Lin, Yijin Wang, Boan Li, Tianjun Jiang, Weimin An, Shuhong Liu, Hongyang Liu, Pengfei Xu, Lihua Zhao, Lixin Zhang, Jinsong Mu, Hongwei Wang, Jiarui Kang, Yan Li, Lei Huang, Caizhong Zhu, Shousong Zhao, Jiangyang Lu, Junsheng Ji, Jingmin Zhao
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti–SARS-CoV spike protein antibody and a mouse monoclonal anti–SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.
Jun Liu, April M. Babka, Brian J. Kearney, Sheli R. Radoshitzky, Jens H. Kuhn, Xiankun Zeng
BACKGROUND The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a novel viral pneumonia (COVID-19), which is rapidly spreading throughout the world. The positive result of nucleic acid test is a golden criterion to confirm SARS-CoV-2 infection, but the detection features remain unclear.METHODS We performed a retrospective analysis in 5630 high-risk individuals receiving SARS-CoV-2 nucleic acid tests in Wuhan, China, and investigated their characteristics and diagnosis rates.RESULTS The overall diagnosis rate was 34.7% (1952/5630). Male (P = 0.025) and older populations (P = 2.525 × 10–39) were at significantly higher risk of SARS-CoV-2 infection. People were generally susceptible, and most cases concentrated in people of 30–79 years. Furthermore, we investigated the association between diagnosis rate and the amount of testing in 501 subjects. Results revealed a 1.27-fold improvement (from 27.9% to 35.5%) of diagnosis rate from testing once to twice (P = 5.847 × 10–9) and a 1.43-fold improvement (from 27.9% to 39.9%) from testing once to 3 times (P = 7.797 × 10–14). More than 3 testing administrations was not helpful for further improvement. However, this improvement was not observed in subjects with pneumonia (P = 0.097).CONCLUSION All populations are susceptible to SARS-CoV-2 infection, and male and older-aged populations are at significantly higher risk. Increasing the amount of testing could significantly improve diagnosis rates, except for subjects with pneumonia. It is recommended to test twice in those high-risk individuals whose results are negative the first time, and performing 3 tests is better, if possible.FUNDING This work was supported by National Mega Project on Major Infectious Disease Prevention (no. 2017ZX10103005-007) and National Key Research and Development Program of China (no. 2018YFE0204500).
Na Shen, Yaowu Zhu, Xiong Wang, Jing Peng, Weiyong Liu, Feng Wang, Yanjun Lu, Liming Cheng, Ziyong Sun
In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have the potential to propagate inflammation and microvascular thrombosis — including in the lungs of patients with acute respiratory distress syndrome. We now report that sera from patients with COVID-19 have elevated levels of cell-free DNA, myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3); the latter 2 are specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute-phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.
Yu Zuo, Srilakshmi Yalavarthi, Hui Shi, Kelsey Gockman, Melanie Zuo, Jacqueline A. Madison, Christopher Blair, Andrew Weber, Betsy J. Barnes, Mikala Egeblad, Robert J. Woods, Yogendra Kanthi, Jason S. Knight
BACKGROUND The coronavirus disease 2019 (COVID-19), infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a severe outbreak throughout the world. The host immunity of COVID-19 patients is unknown.METHODS The routine laboratory tests and host immunity in COVID-19 patients with different severity of illness were compared after patient admission.RESULTS A total of 65 SARS-CoV-2–positive patients were classified as having mild (n = 30), severe (n = 20), and extremely severe (n = 15) illness. Many routine laboratory tests, such as ferritin, lactate dehydrogenase, and D-dimer, were increased in severe and extremely severe patients. The absolute numbers of CD4+ T cells, CD8+ T cells, and B cells were gradually decreased with increased severity of illness. The activation markers such as HLA-DR and CD45RO expressed on CD4+ and CD8+ T cells were increased in severe and extremely severe patients compared with mild patients. The costimulatory molecule CD28 had opposite results. The percentage of natural Tregs was decreased in extremely severe patients. The percentage of IFN-γ–producing CD8+ T cells was increased in both severe and extremely severe patients compared with mild patients. The percentage of IFN-γ–producing CD4+ T cells was increased in extremely severe patients. IL-2R, IL-6, and IL-10 were all increased in extremely severe patients. The activation of DC and B cells was decreased in extremely severe patients.CONCLUSION The number and function of T cells are inconsistent in COVID-19 patients. The hyperfunction of CD4+ and CD8+ T cells is associated with the pathogenesis of extremely severe SARS-CoV-2 infection.FUNDING This work was funded by the National Mega Project on Major Infectious Disease Prevention (2017ZX10103005-007) and the Fundamental Research Funds for the Central Universities (2019kfyRCPY098).
Feng Wang, Hongyan Hou, Ying Luo, Guoxing Tang, Shiji Wu, Min Huang, Weiyong Liu, Yaowu Zhu, Qun Lin, Liyan Mao, Minghao Fang, Huilan Zhang, Ziyong Sun
John P. Mills, Keith S. Kaye, Lona Mody
Lessons from history underline the importance of having direct lines of communication to and from public health officials, who must remain free from policital bias in times of crisis.
Kathleen L. Collins, Howard Markel, Andrew P. Lieberman