BACKGROUND. While most children experience mild COVID-19, high-risk children with underlying conditions may develop severe disease, requiring interventions. Kinetics of antibodies transferred via COVID-19 convalescent plasma early in disease, have not been characterized. METHODS. In this study (NCT04377672), high-risk children were prospectively enrolled to receive high-titer COVID-19 convalescent plasma (>1:320 anti-spike IgG; Euroimmun). Passive transfer of antibodies and endogenous antibody production were serially evaluated for up to 2 months after transfusion. Commercial and research ELISA assays, virus neutralization assays, high-throughput phage-display assay utilizing a coronavirus epitope library and pharmacokinetic analyses were performed. RESULTS. Fourteen high-risk children (median age 7.5 years) received high-titer COVID-19 convalescent plasma, nine children within five days (range 2-7) of symptom onset and five children within 4 days (range 3-5) after exposure to SARS-CoV-2. There were no serious adverse events related to transfusion. Antibodies to SARS-CoV-2 were transferred from the donor to the recipient, but antibody titers declined by 14-21 days with a 15.1-day t½ for spike protein IgG. Donor plasma had significant neutralization capacity which was transferred to the recipient. However, as early as 30 minutes post-transfusion, recipient plasma had low neutralization capacity. CONCLUSIONS. Convalescent plasma transfused to high-risk children appears to be safe with expected antibody kinetics, regardless of weight or age. However, current use of convalescent plasma in high-risk children achieves low neutralizing capacity.
Oren Gordon, Mary Katherine Brosnan, Steve Yoon, Dawoon Jung, Kirsten Littlefield, Abhinaya Ganesan, Christopher A. Caputo, Maggie Li, William R. Morgenlander, Stephanie N. Henson, Alvaro A. Ordonez, Patricia De Jesus, Elizabeth W. Tucker, Nadine Peart Akindele, Zexu Ma, Jo Wilson, Camilo A. Ruiz-Bedoya, M. Elizabeth M. Younger, Evan M. Bloch, Shmuel Shoham, David Sullivan, Aaron A.R. Tobian, Kenneth R. Cooke, Ben Larman, Jogarao V.S. Gobburu, Arturo Casadevall, Andrew Pekosz, Howard M. Lederman, Sabra L. Klein, Sanjay K. Jain
Sangivamycin is a nucleoside analog that is well-tolerated by humans and broadly active against phylogenetically distinct viruses, including arenaviruses, filoviruses, and orthopoxviruses. Here, we show that sangivamycin is a potent antiviral against multiple variants of replicative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with half-maximal inhibitory concentration (IC50) in the nanomolar range in several cell types. Sangivamycin suppressed SARS-CoV-2 replication with greater efficacy than remdesivir (another broad-spectrum nucleoside analog). When we investigated sangivamycin’s potential for clinical administration, pharmacokinetic, ADME (absorption, distribution, metabolism, and excretion), and toxicity properties were found to be favorable. When tested in combination with remdesivir, efficacy was additive rather than competitive against SARS-CoV-2. The proven safety in humans, long half-life, potent antiviral activity (compared to remdesivir), and combinatorial potential suggest that sangivamycin is likely to be efficacious alone or in combination therapy to suppress viremia in patients. Sangivamycin may also have the ability to help combat drug-resistant or vaccine-escaping SARS-CoV-2 variants since it is antivirally active against several tested variants. Our results support the pursuit of sangivamycin for further preclinical and clinical development as a potential coronavirus disease 2019 (COVID-19) therapeutic.
Ryan P. Bennett, Elena N. Postnikova, Brett P. Eaton, Yingyun Cai, Shuiqing Yu, Charles O. Smith, Janie Liang, Huanying Zhou, Gregory A. Kocher, Michael J. Murphy, Harold C. Smith, Jens H. Kuhn
Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected broncho-alveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated influenza patients, as a non-COVID-19 viral pneumonia cohort. Compared to influenza, BAL fluids of COVID-19 patients contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1β, IL-1RA, IL-17A, TNF-α and G-CSF, the chemokines CCL7, CXCL1, CXCL8, CXCL11 and CXCL12α, and the protease inhibitors elafin, secretory leukocyte protease inhibitor (SLPI) and tissue inhibitor of metalloproteinases 1 (TIMP-1). In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotics treatment for bacterial co-infections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes and NK cells were detected in COVID-19 patients whereas concentrations tended to decrease in influenza patients, highlighting the persistent immunological response to co-infections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.
Seppe Cambier, Mieke Metzemaekers, Ana C. Carvalho, Amber Nooyens, Cato Jacobs, Lore Vanderbeke, Bert Malengier-Devlies, Mieke Gouwy, Elisabeth Heylen, Philippe Meersseman, Greet Hermans, Els Wauters, Alexander Wilmer, Contagious Consortium, Dominique Schols, Patrick Matthys, Ghislain Opdenakker, Rafael Elias Marques, Joost Wauters, Jennifer Vandooren, Paul Proost
A substantial proportion of patients who have recovered from coronavirus disease-2019 (COVID-19) experience COVID-19-related symptoms, even months after hospital discharge. We extensively immunologically characterized patients who recovered from COVID-19. In these patients, T cells were exhausted, with increased PD-1+ T cells, as compared to healthy controls. Plasma levels of IL-1ß, IL-1RA and IL-8, among others, were also increased in patients who recovered from COVID-19. This altered immunophenotype was mirrored by a reduced ex vivo T cell response to both nonspecific and specific stimulation, revealing a dysfunctional status of T cells, including a poor response to SARS-CoV-2 antigens. Altered levels of plasma soluble PD-L1 as well as of PD1 promoter methylation and PD1-targeting miR-15-5p in CD8+ T cells were also observed, suggesting abnormal function of the PD-1/PD-L1 immune checkpoint axis. Notably, ex vivo blockade of PD-1 nearly normalized the aforementioned immunophenotype and restored T cell function, reverting the observed post-COVID-19 immune abnormalities; indeed, we also noted an increased T cell-mediated response to SARS-CoV-2 peptides. Finally, in a neutralization assay, PD-1 blockade did not alter the ability of T cells to neutralize SARS-CoV-2 spike pseudotyped lentivirus infection. Immune checkpoint blockade ameliorates post-COVID-19 immune abnormalities and stimulates an anti-SARS-CoV-2 immune response.
Cristian Loretelli, Ahmed Abdelsalam, Francesca D'Addio, Moufida Ben Nasr, Emma Assi, Vera Usuelli, Anna Maestroni, Andy Joe Seelam, Elio Ippolito, Stefania Di Maggio, Lara Loreggian, Dejan Radovanovic, Claudia Vanetti, Jun Yang, Basset El Essawy, Antonio Rossi, Ida Pastore, Laura Montefusco, Maria Elena Lunati, Andrea M. Bolla, Mara Biasin, Spinello Antinori, Pierachille Santus, Agostino Riva, Gianvincenzo Zuccotti, Massimo Galli, Stefano Rusconi, Paolo Fiorina
Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules amongst diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflect proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants Alpha, Beta, Gamma, Kappa, and Delta exhibit comparable levels of replication in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.
Louisa Helms, Silvia Marchiano, Ian B. Stanaway, Tien-Ying Hsiang, Benjamin A. Juliar, Shally Saini, Yan Ting Zhao, Akshita Khanna, Rajasree Menon, Fadhl Alakwaa, Carmen Mikacenic, Eric D. Morrell, Mark M. Wurfel, Matthias Kretzler, Jennifer L. Harder, Charles E. Murry, Jonathan Himmelfarb, Hannele Ruohola-Baker, Pavan K. Bhatraju, Michael Gale, Jr., Benjamin S. Freedman
COVID-19 is caused by SARS-CoV-2 (SC2) and is more prevalent and severe in elderly and patients with comorbid diseases (CM). Because chitinase 3-like-1 (CHI3L1) is induced during aging and CM, the relationships between CHI3L1 and SC2 were investigated. Here, we demonstrate that CHI3L1 is a potent stimulator of the SC2 receptor angiotensin converting enzyme 2 (ACE2) and viral spike protein priming proteases (SPP), that ACE2 and SPP are induced during aging, and that anti-CHI3L1, kasugamycin, and inhibitors of phosphorylation abrogate these ACE2- and SPP-inductive events. Human studies also demonstrate that the levels of circulating CHI3L1 are increased in the elderly and patients with CM, where they correlate with COVID-19 severity. These studies demonstrate that CHI3L1 is a potent stimulator of ACE2 and SPP, that this induction is a major mechanism contributing to the effects of aging during SC2 infection, and that CHI3L1 co-opts the CHI3L1 axis to augment SC2 infection. CHI3L1 plays a critical role in the pathogenesis of and is an attractive therapeutic target in COVID-19.
Suchitra Kamle, Bing Ma, Chuan Hua He, Bedia Akosman, Yang Zhou, Chang-Min Lee, Wafik S. El-Deiry, Kelsey Huntington, Olin Liang, Jason T. Machan, Min-Jong Kang, Hyeon Jun Shin, Emiko Mizoguchi, Chun Geun Lee, Jack A. Elias
Severe respiratory coronavirus 2 (SARS-CoV-2) promotes an imbalanced host response which underlies the development and severity of COVID-19. Infections with viruses are known to modulate transposable elements (TEs) which can exert downstream effects by modulating host gene expression, innate immune sensing, or activities encoded by their protein products. We investigated the impact of SARS-CoV-2 infection on TE expression using RNA-seq data from cell lines and from primary patient samples. Using a bioinformatic tool, Telescope, we showed that SARS-CoV-2 infection led to up- or down-regulation of TE transcripts, a subset of which differed from cells infected with SARS, MERS, RSV, HPIV3 or IAV. Differential expression of key retroelements specifically identified distinct virus families such as coronaviridae, with unique retroelement expression subdividing viral species. Analysis of ChIP-seq data shows that TEs differentially expressed in SARS-CoV-2 infection are enriched for binding sites for TFs involved in immune responses and for pioneer transcription factors. In COVID-19 patient samples, there was a significant TE overexpression in bronchoalveolar lavage fluid and downregulation in peripheral blood mononuclear cells. Thus, while the host gene transcriptome is altered by infection with SARS-CoV-2, the retrotranscriptome may contain the most distinctive features of the cellular response to SARS-CoV-2 infection.
Jez L. Marston, Matthew Greenig, Manvendra Singh, Matthew L. Bendall, Rodrigo R.R. Duarte, Cédric Feschotte, Luis P. Iñiguez, Douglas F. Nixon
Understanding the presence and durability of antibodies against SARS-CoV-2 in the airways is required to provide insights on the ability of individuals to neutralize the virus locally and prevent viral spread. Here, we longitudinally assessed both systemic and airway immune responses upon SARS-CoV-2 infection in a clinically well-characterized cohort of 147 infected individuals representing the full spectrum of COVID-19 severity; from asymptomatic infection to fatal disease. In addition, we evaluated how SARS-CoV-2 vaccination influenced the antibody responses in a subset of these individuals during convalescence as compared to naïve individuals. Not only systemic but also airway antibody responses correlated with the degree of COVID-19 disease severity. However, while systemic IgG levels were durable for up to 8 months, airway IgG and IgA had declined significantly within 3 months. After vaccination, there was an increase in both systemic and airway antibodies, in particular IgG, often exceeding the levels found during acute disease. In contrast, naïve individuals showed low airway antibodies after vaccination. In the former COVID-19 patients, airway antibody levels were significantly elevated after the boost vaccination, highlighting the importance of prime and boost vaccination also for previously infected individuals to obtain optimal mucosal protection.
Alberto Cagigi, Meng Yu, Björn Österberg, Julia Svensson, Sara Falck-Jones, Sindhu Vangeti, Eric Åhlberg, Lida Azizmohammadi, Anna Warnqvist, Ryan Falck-Jones, Pia C. Gubisch, Mert Ödemis, Farangies Ghafoor, Mona Eisele, Klara Lenart, Max Bell, Niclas Johansson, Jan Albert, Jörgen Sälde, Deleah D. Pettie, Michael P. Murphy, Lauren Carter, Neil P. King, Sebastian Ols, Johan Normark, Clas Ahlm, Mattias N. Forsell, Anna Färnert, Karin Loré, Anna Smed-Sörensen
Influenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different. With the hypothesis that upper airway immune and epithelial cells responses are also distinct, we performed single-cell RNA-sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples. Nasal wash cells are enriched for macrophages and neutrophils for both influenza and COVID-19 compared to healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells are enriched in COVID-19 samples. A global decrease in interferon (IFN)-associated transcripts in neutrophils, macrophages, and epithelial cells is apparent in COVID-19 compared to influenza. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial immune sensing. Cell-to-cell interaction analyses reveal a decrease in epithelial interactions in COVID-19 and highlight differences in macrophage-macrophage interactions for COVID-19 and influenza. Our study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.
Kevin M. Gao, Alan G. Derr, Zhiru Guo, Kerstin Nundel, Ann Marshak-Rothstein, Robert W. Finberg, Jennifer P. Wang
Endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. Constitutively, the endothelial surface is anticoagulant, a property maintained at least in part via signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and alterations in the Tie2-angiopoietin axis. Primary human endothelial cells treated with plasma from patients with severe COVID-19 upregulated expression of thromboinflammatory genes, inhibited expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. Lung autopsies from COVID-19 patients demonstrated a prothrombotic endothelial signature. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity and highest levels were associated with worse survival. These data highlight the disruption of Tie2-angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Our findings provide rationale for current trials of Tie2-activating therapy with AKB-9778 in COVID-19.
Alec A. Schmaier, Gabriel M. Pajares Hurtado, Zachary J. Manickas-Hill, Kelsey D. Sack, Siyu M. Chen, Victoria Bhambhani, Juweria Quadir, Anjali K. Nath, Ai-ris Y. Collier, Debby Ngo, Dan H. Barouch, Nathan I. Shapiro, Robert E. Gerszten, Xu Yu, Kevin G. Peters, Robert Flaumenhaft, Samir M. Parikh
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