Background: Patients infected with 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 COVID-19 related 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 (IFNγ, IL-2 and TNFα) 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 to 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 convalescents, 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. Trial registration: n.a. Funding: The study was supported by institutional funds by M.S., and in part by grants of Saarland University (to M.S. and. R.B), the State of Saarland, and the Dr. Rolf M. Schwiete Stiftung to R.B.
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
The emergence of SARS-CoV-2 has created an international health crisis. Small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection due 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 promotor. In contrast to non-transgenic mice, intranasal exposure of K18-hACE2 animals to two 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 revealed increases in transcripts involved in lung injury and inflammatory cytokines. In the lower 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 to female mice. This is the first highly lethal murine infection model for SARS-CoV-2. The K18-hACE2 murine model will be valuable for the study of SARS-CoV-2 pathogenesis and 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 to 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 enzyme-linked immunosorbent assay (ELISA) using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 RT-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 to the two different viral proteins showed a moderate correlation. Antibodies to N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.6% 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 cautions 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 Holden, Nikolas S. Hopkins, Alexandria Harris, Jerusha J. Kumpati, Michael A. Whitt, J. Jack Lee, Luis Ostrosky-Zeichner, Ramesha Papanna, Valerie LeBleu, James 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 to those observed in critically ill patients with 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-1b, IL-1RA, IL-6, IL-8, IL-18, and TNFα) between patients with moderate COVID-19, severe COVID-19, and ARDS or sepsis. Results: 15 hospitalized COVID-19 patients, 9 of whom were critically ill, were compared to critically ill patients with ARDS (n = 12) or sepsis (n = 16). There were no statistically significant differences in baseline levels of IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNFα between patients with COVID-19 and critically ill controls with ARDS or sepsis. Conclusions: 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: A.J.R.: Stanford ICU Biobank NHLBI K23 HL125663. C.A.B.: Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases #1016687; NIH/NIAID U19AI057229-16 (PI MM Davis); Stanford Maternal Child Health Research Institute; Chan Zuckerberg Biohub.
Jennifer G. Wilson, Laura J. Simpson, Anne-Maud Ferreira, Arjun Rustagi, Jonasel A. Roque, Adijat Asuni, Thanmayi Ranganath, Philip M. Grant, Aruna K. Subramanian, Yael Rosenberg-Hasson, Holden Maecker, Susan Holmes, Joseph E. Levitt, Catherine Blish, Angela J. Rogers
COVID-19-associated morbidity and mortality have been attributed to a pathologic host response. Two divergent hypotheses have been proposed: a hyper-inflammatory ‘cytokine-storm’-mediated injury versus failure of host protective immunity resulting 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 COVID-19, 51 septic, 18 critically-ill non-septic (CINS), and 27 healthy controls 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% to 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 greater than 1,000 pg/mL that were not associated with elevations in other canonical pro-inflammatory cytokines. Collectively, these findings support the hypothesis that COVID-19 suppresses host functional adaptive and innate immunity. Importantly, Interleukin-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. Moldawer, Richard S. Hotchkiss
Reprogramming of host metabolism supports viral pathogenesis by fueling viral proliferation, by providing, for example, free amino acids and fatty acids as building blocks. To investigate metabolic effects of SARS-COV-2 infection, we evaluated serum metabolites of COVID-19 patients (n = 33; diagnosed by nucleic acid testing), as compared to COVID-19-negative controls (n = 16). 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). 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.
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
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, cytokine and chemokines in blood of hospitalised COVID-19 patients from serial sampling and follow up over four 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 disease (18 patients) were enrolled with 18 healthy volunteers. We measured 34 immune mediators, cytokines and chemokines in peripheral blood every 4-7 days over one month per patient using a bio-plex 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-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-gamma were only significantly elevated in the late stage of severe COVID-19 illness. TNF- alpha and GM-CSF showed no significant differences between severe and mild cases. Conclusion Together our data suggest 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, IL-10 in blood individually and in combination might be useful prognostic bio-markers 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 J. McMichael, Ronghua Jin, Julian C. Knight, Tao Dong, Yonghong Zhang
BACKGROUND. Severe acute respiratory coronavirus 2 (SARS-CoV-2) caused coronavirus disease 2019 (COVID-19) has become a pandemic. This study addressed the clinical and immunopathological characteristics of severe COVID-19. METHODS. Sixty-nine COVID-19 patients were classified into as severe and non-severe groups to analyze their clinical and laboratory characteristics. A panel of blood cytokines was quantified over time. Biopsy specimens from two deceased cases were obtained for immunopathological, ultrastructural, and in situ hybridization examinations. RESULTS. Circulating cytokines, including IL8, IL6, TNFα, IP10, MCP1, and RANTES, were significantly elevated in severe COVID-19 patients. Dynamic IL6 and IL8 were associated with disease progression. SARS-CoV-2 was demonstrated to infect type II, 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 SARS-CoV-2 caused cytopathy and immunopathologic damage. Strategies that encourage pulmonary recruitment and overactivation of inflammatory cells by suppressing cytokine storm might improve the outcomes of severe COVID-19 patients.
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 more than 3.1 million cases and 224,000 deaths as of May 1, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (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 immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (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. 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 in 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 5,630 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% (1,952/5,630). Male (P = 0.025) and older age (P = 2.525 × 10–39) were two significant risk factors of SARS-CoV-2 infection. People were generally susceptible, and most cases concentrated in people of 30- to 69-years-old. Besides, we investigated the association between diagnosis rate and the number of testing in 501 subjects. Results revealed a 1.27-fold improvement (35.5%/27.9%) of diagnosis rate from testing once to twice (P = 5.847 × 10–9), and a 1.43-fold improvement (39.9%/27.9%) from testing once to three times (P = 7.797 × 10–14). More than three testing times 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 age are two significant risk factors. Increasing the number 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 for the first time, and to perform three testing times is better if available.
Na Shen, Yaowu Zhu, Xiong Wang, Jing Peng, Weiyong Liu, Feng Wang, Yanjun Lu, Liming Cheng, Ziyong Sun
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