scholarly journals Mechanisms of Zoonotic Severe Acute Respiratory Syndrome Coronavirus Host Range Expansion in Human Airway Epithelium

2007 ◽  
Vol 82 (5) ◽  
pp. 2274-2285 ◽  
Author(s):  
Timothy Sheahan ◽  
Barry Rockx ◽  
Eric Donaldson ◽  
Amy Sims ◽  
Raymond Pickles ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Infectious Clone ◽  
Human Monoclonal Antibody ◽  
Protein S ◽  
Vero Cells ◽  
Mouse Lung ◽  
Airway Epithelial ◽  
Human Airway

ABSTRACT In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and caused over 8,000 human cases of infection and more than 700 deaths worldwide. Zoonotic SARS-CoV likely evolved to infect humans by a series of transmission events between humans and animals for sale in China. Using synthetic biology, we engineered the spike protein (S) from a civet strain, SZ16, into our epidemic strain infectious clone, creating the chimeric virus icSZ16-S, which was infectious but yielded progeny viruses incapable of propagating in vitro. After introducing a K479N mutation within the S receptor binding domain (RBD) of SZ16, the recombinant virus (icSZ16-S K479N) replicated in Vero cells but was severely debilitated in growth. The in vitro evolution of icSZ16-S K479N on human airway epithelial (HAE) cells produced two viruses (icSZ16-S K479N D8 and D22) with enhanced growth on HAE cells and on delayed brain tumor cells expressing the SARS-CoV receptor, human angiotensin I converting enzyme 2 (hACE2). The icSZ16-S K479N D8 and D22 virus RBDs contained mutations in ACE2 contact residues, Y442F and L472F, that remodeled S interactions with hACE2. Further, these viruses were neutralized by a human monoclonal antibody (MAb), S230.15, but the parent icSZ16-S K479N strain was eight times more resistant than the mutants. These data suggest that the human adaptation of zoonotic SARS-CoV strains may select for some variants that are highly susceptible to select MAbs that bind to RBDs. The epidemic, icSZ16-S K479N, and icSZ16-S K479N D22 viruses replicate similarly in the BALB/c mouse lung, highlighting the potential use of these zoonotic spike SARS-CoVs to assess vaccine or serotherapy efficacy in vivo.

scholarly journals Escape from Human Monoclonal Antibody Neutralization Affects In Vitro and In Vivo Fitness of Severe Acute Respiratory Syndrome Coronavirus

2010 ◽  
Vol 201 (6) ◽  
pp. 946-955 ◽  
Author(s):  
Barry Rockx ◽  
Eric Donaldson ◽  
Matthew Frieman ◽  
Timothy Sheahan ◽  
Davide Corti ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Severe Acute Respiratory Syndrome ◽  
Human Monoclonal Antibody ◽  
Antibody Neutralization

scholarly journals The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

2009 ◽  
Vol 84 (2) ◽  
pp. 1097-1109 ◽  
Author(s):  
Eric C. Freundt ◽  
Li Yu ◽  
Cynthia S. Goldsmith ◽  
Sarah Welsh ◽  
Aaron Cheng ◽  
...  
Keyword(s):  
Cell Death ◽  
Severe Acute Respiratory Syndrome ◽  
Vero Cells ◽  
Small Gtpase ◽  
Open Reading Frames ◽  
Accessory Proteins ◽  
Reading Frame ◽  
Golgi Fragmentation

ABSTRACT The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

scholarly journals Incorporation of Adeno-Associated Virus in a Calcium Phosphate Coprecipitate Improves Gene Transfer to Airway Epithelia In Vitro and In Vivo

2000 ◽  
Vol 74 (1) ◽  
pp. 535-540 ◽  
Author(s):  
Robert W. Walters ◽  
Dongsheng Duan ◽  
John F. Engelhardt ◽  
Michael J. Welsh
Keyword(s):  
Calcium Phosphate ◽  
Gene Transfer ◽  
Mouse Lung ◽  
Apical Surface ◽  
Airway Epithelia ◽  
Adeno Associated Virus ◽  
Human Airway

ABSTRACT Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaPicoprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo.

scholarly journals Novel proteinase-activated receptor-2 (PAR2) antagonist C391 blocks Alternaria-induced human airway epithelial signaling and asthma indicators in murine models

2021 ◽  
Author(s):  
Candy Rivas ◽  
Michael Yee ◽  
Kenneth Addison ◽  
Marissa Lovett ◽  
Kasturi Pal ◽  
...  
Keyword(s):  
Cell Line ◽  
Mapk Signaling ◽  
Pharmacological Intervention ◽  
Airway Epithelial ◽  
Murine Models ◽  
Mucus Cell ◽  
Hek 293 ◽  
Human Airway

Background and Purpose: Despite availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in pre-clinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 induces intracellular Ca2+, mitogen activated protein kinase (MAPK) and -arrestin signaling the airway, leading to both inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signaling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 reduction with C391 would attenuate allergen-induced asthma indicators in murine models. Experimental Approach: We evaluated the ability for C391 to alter Alternaria alternata-induced PAR2 signaling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata-induced asthma indicators in vivo in two murine strains. Key Results: C391 blocked A. alternata-induced, PAR2-dependent Ca2+ and MAPK signaling in 16HBE14o- cells, as well as -arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute asthma murine models. Conclusions and Implications: To our knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs.

scholarly journals Extracellular Vesicle-Mediated siRNA Delivery, Protein Delivery, and CFTR Complementation in Well-Differentiated Human Airway Epithelial Cells

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 351 ◽  
Author(s):  
Brajesh K. Singh ◽  
Ashley L. Cooney ◽  
Sateesh Krishnamurthy ◽  
Patrick L. Sinn
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Target Cells ◽  
Airway Epithelial ◽  
Long Distance ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelial Cells

Extracellular vesicles (EVs) are a class of naturally occurring secreted cellular bodies that are involved in long distance cell-to-cell communication. Proteins, lipids, mRNA, and miRNA can be packaged into these vesicles and released from the cell. This information is then delivered to target cells. Since EVs are naturally adapted molecular messengers, they have emerged as an innovative, inexpensive, and robust method to deliver therapeutic cargo in vitro and in vivo. Well-differentiated primary cultures of human airway epithelial cells (HAE) are refractory to standard transfection techniques. Indeed, common strategies used to overexpress or knockdown gene expression in immortalized cell lines simply have no detectable effect in HAE. Here we use EVs to efficiently deliver siRNA or protein to HAE. Furthermore, EVs can deliver CFTR protein to cystic fibrosis donor cells and functionally correct the Cl− channel defect in vitro. EV-mediated delivery of siRNA or proteins to HAE provides a powerful genetic tool in a model system that closely recapitulates the in vivo airways.

scholarly journals Evaluation of Human Monoclonal Antibody 80R for Immunoprophylaxis of Severe Acute Respiratory Syndrome by an Animal Study, Epitope Mapping, and Analysis of Spike Variants

2005 ◽  
Vol 79 (10) ◽  
pp. 5900-5906 ◽  
Author(s):  
Jianhua Sui ◽  
Wenhui Li ◽  
Anjeanette Roberts ◽  
Leslie J. Matthews ◽  
Akikazu Murakami ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Human Monoclonal Antibody ◽  
Neutralizing Antibody ◽  
Index Patient ◽  
Binding Domain ◽  
S Protein

ABSTRACT In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding domain. Amino acids critical for 80R binding were identified. In addition, the effects of various 80R-binding domain amino acid substitutions which occur in SARS-like-CoV from civet cats, and which evolved during the 2002/2003 outbreak and in a 2003/2004 Guangdong index patient, were analyzed. The results demonstrated that the vast majority of SARS-CoVs are sensitive to 80R. We propose that by establishing the susceptibility and resistance profiles of newly emerging SARS-CoVs through early S1 genotyping of the core 180-amino-acid neutralizing epitope of 80R, an effective immunoprophylaxis strategy with 80R should be possible in an outbreak setting. Our study also cautions that for any prophylaxis strategy based on neutralizing antibody responses, whether by passive or active immunization, a genotyping monitor will be necessary for effective use.

scholarly journals Does the ΔF508-CFTR mutation induce a proinflammatory response in human airway epithelial cells?

2012 ◽  
Vol 303 (6) ◽  
pp. L509-L518 ◽  
Author(s):  
Thomas H. Hampton ◽  
Alicia E. Ballok ◽  
Jennifer M. Bomberger ◽  
Melanie R. Rutkowski ◽  
Roxanna Barnaby ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Inflammatory Response ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Chemokine Production ◽  
Human Airway ◽  
Δf508 Cftr ◽  
Human Airway Epithelial Cells

In the clinical setting, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene enhance the inflammatory response in the lung to Pseudomonas aeruginosa ( P. aeruginosa ) infection. However, studies on human airway epithelial cells in vitro have produced conflicting results regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa, and there are no comprehensive studies evaluating the effect of P. aeruginosa on the inflammatory response in airway epithelial cells with the ΔF508/ΔF508 genotype and their matched CF cell line rescued with wild-type (wt)-CFTR. CFBE41o- cells (ΔF508/ΔF508) and CFBE41o- cells complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this pair of cells. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2 and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o-ΔF508/ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo . Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.

scholarly journals Hydroxychloroquine as a Chemoprophylactic Agent for COVID-19: A Clinico-Pharmacological Review

2020 ◽  
Vol 11 ◽  
Author(s):  
Mudit Agarwal ◽  
Piyush Ranjan ◽  
Upendra Baitha ◽  
Ankit Mittal
Keyword(s):  
Clinical Trials ◽  
Viral Entry ◽  
Vero Cells ◽  
Inhibitory Effect ◽  
Airway Epithelial Cells ◽  
Point Of View ◽  
High Risk Population ◽  
Airway Epithelial

Hydroxychloroquine has gained much attention as one of the candidate drugs that can be repurposed as a prophylactic agent against SARS-CoV-2, the agent responsible for the COVID-19 pandemic. Due to high transmissibility and presence of asymptomatic carriers and presymptomatic transmission, there is need for a chemoprophylactic agent to protect the high-risk population. In this review, we dissect the currently available evidence on hydroxychloroquine prophylaxis from a clinical and pharmacological point of view. In vitro studies on Vero cells show that hydroxychloroquine effectively inhibits SARS-CoV-2 by affecting viral entry and viral transport via endolysosomes. However, this efficacy has failed to replicate in in vivo animal models as well as in most clinical observational studies and clinical trials assessing pre-exposure prophylaxis and postexposure prophylaxis in healthcare workers. An analysis of the pharmacology of HCQ in COVID-19 reveals certain possible reasons for this failure—a pharmacokinetic failure due to failure to achieve adequate drug concentration at the target site and attenuation of its inhibitory effect due to the presence of TMPRSS2 in airway epithelial cells. Currently, many clinical trials on HCQ prophylaxis in HCW are ongoing; these factors should be taken into account. Using higher doses of HCQ for prophylaxis is likely to be associated with increased safety concerns; thus, it may be worthwhile to focus on other possible interventions.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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