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1.
PLoS Pathog ; 18(7): e1010686, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1951569

ABSTRACT

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.


Subject(s)
Antibodies, Neutralizing , COVID-19 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral , COVID-19/prevention & control , Mice , Mice, Inbred C57BL , Pandemics/prevention & control , Spike Glycoprotein, Coronavirus/immunology
2.
Physiology (Bethesda) ; 36(6): 359-366, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1495701

ABSTRACT

Compared with adults, children are less likely infected with SARS-CoV-2 and are often asymptomatic when infected. However, infection in children can lead to severe disease. The pandemic affects the lives of all children, especially those with lower socioeconomic status. This review highlights the physiological impacts of COVID-19 in early life.


Subject(s)
COVID-19 , Adult , Child , Humans , Infant , Pandemics , SARS-CoV-2
3.
mSystems ; 6(6): e0113621, 2021 Dec 21.
Article in English | MEDLINE | ID: covidwho-1494994

ABSTRACT

Environmental monitoring in public spaces can be used to identify surfaces contaminated by persons with coronavirus disease 2019 (COVID-19) and inform appropriate infection mitigation responses. Research groups have reported detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on surfaces days or weeks after the virus has been deposited, making it difficult to estimate when an infected individual may have shed virus onto a SARS-CoV-2-positive surface, which in turn complicates the process of establishing effective quarantine measures. In this study, we determined that reverse transcription-quantitative PCR (RT-qPCR) detection of viral RNA from heat-inactivated particles experiences minimal decay over 7 days of monitoring on eight out of nine surfaces tested. The properties of the studied surfaces result in RT-qPCR signatures that can be segregated into two material categories, rough and smooth, where smooth surfaces have a lower limit of detection. RT-qPCR signal intensity (average quantification cycle [Cq]) can be correlated with surface viral load using only one linear regression model per material category. The same experiment was performed with untreated viral particles on one surface from each category, with essentially identical results. The stability of RT-qPCR viral signal demonstrates the need to clean monitored surfaces after sampling to establish temporal resolution. Additionally, these findings can be used to minimize the number of materials and time points tested and allow for the use of heat-inactivated viral particles when optimizing environmental monitoring methods. IMPORTANCE Environmental monitoring is an important tool for public health surveillance, particularly in settings with low rates of diagnostic testing. Time between sampling public environments, such as hospitals or schools, and notifying stakeholders of the results should be minimal, allowing decisions to be made toward containing outbreaks of coronavirus disease 2019 (COVID-19). The Safer At School Early Alert program (SASEA) (https://saseasystem.org/), a large-scale environmental monitoring effort in elementary school and child care settings, has processed >13,000 surface samples for SARS-CoV-2, detecting viral signals from 574 samples. However, consecutive detection events necessitated the present study to establish appropriate response practices around persistent viral signals on classroom surfaces. Other research groups and clinical labs developing environmental monitoring methods may need to establish their own correlation between RT-qPCR results and viral load, but this work provides evidence justifying simplified experimental designs, like reduced testing materials and the use of heat-inactivated viral particles.

4.
Antimicrob Agents Chemother ; 65(10): e0115521, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1416580

ABSTRACT

Remdesivir (RDV; GS-5734) is currently the only FDA-approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The drug is approved for use in adults or children 12 years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2-infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed, and several, including molnupiravir and PF-07321332, are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of remdesivir nucleoside (RVn; GS-441524) that are processed to RVn monophosphate, the precursor of the active RVn triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma, and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types, including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells, and Huh7.5 cells. In Syrian hamsters, oral treatment with 1-O-octadecyl-2-O-benzyl-glycero-3-phosphate RVn (ODBG-P-RVn) was well tolerated and achieved therapeutic levels in plasma above the 90% effective concentration (EC90) for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.


Subject(s)
COVID-19 , Prodrugs , Adenosine/analogs & derivatives , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Caco-2 Cells , Cricetinae , Humans , Lipids , SARS-CoV-2
5.
PMC; 2020.
Preprint in English | PMC | ID: ppcovidwho-290515

ABSTRACT

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities. ### Competing Interest Statement J.D.E. is a co-founder of TEGA Therapeutics. J.D.E. and The Regents of the University of California have licensed a University invention to and have an equity interest in TEGA Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. C.A.G and B.E.T are employees of TEGA Therapeutics.

6.
Sci Adv ; 7(34)2021 08.
Article in English | MEDLINE | ID: covidwho-1365115

ABSTRACT

Novel coronavirus disease 2019 (COVID-19) severity is highly variable, with pediatric patients typically experiencing less severe infection than adults and especially the elderly. The basis for this difference is unclear. We find that mRNA and protein expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, increases with advancing age in distal lung epithelial cells. However, in humans, ACE2 expression exhibits high levels of intra- and interindividual heterogeneity. Further, cells infected with SARS-CoV-2 experience endoplasmic reticulum stress, triggering an unfolded protein response and caspase-mediated apoptosis, a natural host defense system that halts virion production. Apoptosis of infected cells can be selectively induced by treatment with apoptosis-modulating BH3 mimetic drugs. Notably, epithelial cells within young lungs and airways are more primed to undergo apoptosis than those in adults, which may naturally hinder virion production and support milder COVID-19 severity.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Apoptosis/genetics , COVID-19/genetics , Gene Expression Profiling/methods , Age Factors , Aged , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Chlorocebus aethiops , Female , Humans , Infant , Lung/cytology , Lung/metabolism , Lung/virology , Male , Mice, Inbred C57BL , Middle Aged , SARS-CoV-2/physiology , Severity of Illness Index , Vero Cells , Virus Internalization
7.
EMBO J ; 40(5): e107651, 2021 03 01.
Article in English | MEDLINE | ID: covidwho-1082516

ABSTRACT

Defining the pulmonary cell types infected by SARS-CoV-2 and finding ways to prevent subsequent tissue damage are key goals for controlling COVID-19. Recent work establishing a human lung organoid-derived air-liquid interface model permissive to SARS-CoV-2 infection identifies alveolar type II cells as the primary cell type infected, reports an infection-induced interferon response and demonstrates the effectiveness of interferon lambda 1 treatment in dampening lung infection.


Subject(s)
Alveolar Epithelial Cells/metabolism , COVID-19/metabolism , Models, Biological , Organoids/metabolism , SARS-CoV-2/physiology , Virus Replication , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , COVID-19/drug therapy , COVID-19/pathology , Humans , Organoids/pathology , Organoids/virology
8.
Curr Protoc Stem Cell Biol ; 54(1): e118, 2020 09.
Article in English | MEDLINE | ID: covidwho-635380

ABSTRACT

The normal development of the pulmonary system is critical to transitioning from placental-dependent fetal life to alveolar-dependent newborn life. Human lung development and disease have been difficult to study due to the lack of an in vitro model system containing cells from the large airways and distal alveolus. This article describes a system that allows human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to differentiate and form three-dimensional (3D) structures that emulate the development, cytoarchitecture, and function of the lung ("organoids"), containing epithelial and mesenchymal cell populations, and including the production of surfactant and presence of ciliated cells. The organoids can also be invested with mesoderm derivatives, differentiated from the same human pluripotent stem cells, such as alveolar macrophages and vasculature. Such lung organoids may be used to study the impact of environmental modifiers and perturbagens (toxins, microbial or viral pathogens, alterations in microbiome) or the efficacy and safety of drugs, biologics, and gene transfer. © 2020 Wiley Periodicals LLC. Basic Protocol: hESC/hiPSC dissection, definitive endoderm formation, and lung progenitor cell induction.


Subject(s)
Coronavirus Infections/pathology , Lung/cytology , Organoids/cytology , Pneumonia, Viral/pathology , Respiratory Tract Infections/pathology , Betacoronavirus , COVID-19 , Cell Culture Techniques , Cell Differentiation , Coronavirus Infections/therapy , Endoderm/cytology , Human Embryonic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Lung/growth & development , Lung/physiology , Models, Biological , Pandemics , Patient-Specific Modeling , Pneumonia, Viral/therapy , Respiratory Tract Infections/therapy , SARS-CoV-2 , Time-Lapse Imaging
9.
Cell ; 183(4): 1043-1057.e15, 2020 11 12.
Article in English | MEDLINE | ID: covidwho-756808

ABSTRACT

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.


Subject(s)
Betacoronavirus/physiology , Heparitin Sulfate/metabolism , Peptidyl-Dipeptidase A/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Betacoronavirus/isolation & purification , Binding Sites , COVID-19 , Cell Line , Coronavirus Infections/pathology , Coronavirus Infections/virology , Heparin/chemistry , Heparin/metabolism , Heparitin Sulfate/chemistry , Humans , Kidney/metabolism , Lung/metabolism , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Binding , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
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