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1.
Cell Rep ; 39(11): 110954, 2022 Jun 14.
Article in English | MEDLINE | ID: covidwho-1866958

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response.


Subject(s)
COVID-19 , Viral Nonstructural Proteins , Cell Line , Humans , RNA Stability , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
2.
Cell Discov ; 8(1): 16, 2022 Feb 15.
Article in English | MEDLINE | ID: covidwho-1692632

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with angiotensin converting enzyme 2 (ACE2) interface, which enables 2G1 to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar half maximal inhibitory concentration in vitro. In SARS-CoV-2, Beta or Delta variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 is potentially capable of dealing with emerging SARS-CoV-2 variants in the future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.

3.
J Hazard Mater ; 429: 127709, 2022 05 05.
Article in English | MEDLINE | ID: covidwho-1654743

ABSTRACT

Dry heat decontamination has been shown to effectively inactivate viruses without compromising the integrity of delicate personal protective equipment (PPE), allowing safe reuse and helping to alleviate shortages of PPE that have arisen due to COVID-19. Unfortunately, current thermal decontamination guidelines rely on empirical data which are often sparse, limited to a specific virus, and unable to provide fundamental insight into the underlying inactivation reaction. In this work, we experimentally quantified dry heat decontamination of SARS-CoV-2 on disposable masks and validated a model that treats the inactivation reaction as thermal degradation of macromolecules. Furthermore, upon nondimensionalization, all of the experimental data collapse onto a unified curve, revealing that the thermally driven decontamination process exhibits self-similar behavior. Our results show that heating surgical masks to 70 °C for 5 min inactivates over 99.9% of SARS-CoV-2. We also characterized the chemical and physical properties of disposable masks after heat treatment and did not observe degradation. The model presented in this work enables extrapolation of results beyond specific temperatures to provide guidelines for safe PPE decontamination. The modeling framework and self-similar behavior are expected to extend to most viruses-including yet-unencountered novel viruses-while accounting for a range of environmental conditions.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/prevention & control , Decontamination/methods , Equipment Reuse , Hot Temperature , Humans , Personal Protective Equipment
4.
Viruses ; 14(1)2021 12 31.
Article in English | MEDLINE | ID: covidwho-1580398

ABSTRACT

We report the discovery of several highly potent small molecules with low-nM potency against severe acute respiratory syndrome coronavirus (SARS-CoV; lowest half-maximal inhibitory concentration (IC50: 13 nM), SARS-CoV-2 (IC50: 23 nM), and Middle East respiratory syndrome coronavirus (MERS-CoV; IC50: 76 nM) in pseudovirus-based assays with excellent selectivity index (SI) values (>5000), demonstrating potential pan-coronavirus inhibitory activities. Some compounds showed 100% inhibition against the cytopathic effects (CPE; IC100) of an authentic SARS-CoV-2 (US_WA-1/2020) variant at 1.25 µM. The most active inhibitors also potently inhibited variants of concern (VOCs), including the UK (B.1.1.7) and South African (B.1.351) variants and the Delta variant (B.1.617.2) originally identified in India in pseudovirus-based assay. Surface plasmon resonance (SPR) analysis with one potent inhibitor confirmed that it binds to the prefusion SARS-CoV-2 spike protein trimer. These small-molecule inhibitors prevented virus-mediated cell-cell fusion. The absorption, distribution, metabolism, and excretion (ADME) data for one of the most active inhibitors, NBCoV1, demonstrated drug-like properties. An in vivo pharmacokinetics (PK) study of NBCoV1 in rats demonstrated an excellent half-life (t1/2) of 11.3 h, a mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect these lead inhibitors to facilitate the further development of preclinical and clinical candidates.


Subject(s)
Antiviral Agents/pharmacology , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Biological Availability , Cell Line , Cell Survival/drug effects , Coronavirus/classification , Coronavirus/drug effects , HIV Fusion Inhibitors/chemistry , HIV Fusion Inhibitors/pharmacokinetics , HIV Fusion Inhibitors/pharmacology , Humans , Protein Binding , Rats , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacokinetics , Small Molecule Libraries/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors
5.
Sci Adv ; 7(37): eabh1547, 2021 Sep 10.
Article in English | MEDLINE | ID: covidwho-1405213

ABSTRACT

A "universal" platform that can rapidly generate multiplex vaccine candidates is critically needed to control pandemics. Using the severe acute respiratory syndrome coronavirus 2 as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates was engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers was found to be the most potent vaccine candidate in animal models. Without any adjuvant, this vaccine stimulated robust immune responses, both T helper cell 1 (TH1) and TH2 immunoglobulin G subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new nanovaccine design framework might allow the rapid deployment of effective adjuvant-free phage-based vaccines against any emerging pathogen in the future.

6.
PLoS Pathog ; 17(9): e1009897, 2021 09.
Article in English | MEDLINE | ID: covidwho-1398941

ABSTRACT

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Immunogenicity, Vaccine , Nanoparticles/therapeutic use , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Disease Models, Animal , Drug Design , Female , HEK293 Cells , Humans , Lung/virology , Mice , Mice, Inbred BALB C , Protein Domains/immunology
7.
mBio ; 11(6)2020 12 11.
Article in English | MEDLINE | ID: covidwho-1388458

ABSTRACT

SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is an ∼30-amino-acid (aa)-long helix. Here, we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 to 94% helicity). In contrast, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus-based single-cycle assay in HT1080/ACE2 cells and human lung cell line A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (50% inhibitory concentration [IC50]: 1.9 to 4.1 µM) and A549/ACE2 (IC50: 2.2 to 2.8 µM) cells. The linear peptide NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient, preventing the complete formation of cytopathic effects (CPEs) at an IC100 of 17.2 µM. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 µM. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T 1/2) of >289 min.IMPORTANCE SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. Therefore, there is an urgent need to develop novel therapeutics. Structural studies revealed critical interactions between the binding site helix of the ACE2 receptor and SARS-CoV-2 receptor-binding domain (RBD). Therefore, targeting the entry pathway of SARS-CoV-2 is ideal for both prevention and treatment as it blocks the first step of the viral life cycle. We report the design of four double-stapled peptides, three of which showed potent antiviral activity in HT1080/ACE2 cells and human lung carcinoma cells, A549/ACE2. Most significantly, the active stapled peptides with antiviral activity against SARS-CoV-2 showed high α-helicity (60 to 94%). The most active stapled peptide, NYBSP-4, showed substantial resistance to degradation by proteolytic enzymes in human plasma. The lead stapled peptides are expected to pave the way for further optimization of a clinical candidate.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Peptides/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Attachment/drug effects , A549 Cells , Animals , Binding Sites , Chlorocebus aethiops , Humans , Inhibitory Concentration 50 , Peptides/chemical synthesis , Protein Binding , Vero Cells
8.
J Med Chem ; 64(15): 11267-11287, 2021 08 12.
Article in English | MEDLINE | ID: covidwho-1319012

ABSTRACT

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.


Subject(s)
Aldehydes/chemistry , COVID-19/drug therapy , Chagas Disease/drug therapy , Cysteine Proteinase Inhibitors/therapeutic use , SARS-CoV-2/enzymology , Trypanosoma cruzi/enzymology , Aldehydes/metabolism , Aldehydes/pharmacology , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cysteine Endopeptidases/metabolism , Cysteine Proteases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Drug Design , Humans , Kinetics , Models, Molecular , Molecular Structure , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/metabolism , SARS-CoV-2/drug effects , Structure-Activity Relationship , Trypanosoma cruzi/drug effects
10.
Methods Mol Biol ; 2099: 99-106, 2020.
Article in English | MEDLINE | ID: covidwho-1292548

ABSTRACT

Since the emergence of the Middle East respiratory syndrome-coronavirus (MERS-CoV) in 2012, more than 2280 confirmed human infections and 800 associated deaths had been reported to the World Health Organization. MERS-CoV is a single-stranded RNA virus that belongs to the Coronaviridae family. MERS-CoV infection leads to a variety of clinical outcomes in humans ranging from asymptomatic and mild infection to severe acute lung injury and multi-organ failure and death. To study the pathogenesis of MERS-CoV infection and development of medical countermeasures (MCMs) for MERS, a number of genetically modified mouse models have been developed, including various versions of transgenic mice expressing the human DPP4 viral receptor. Tracking and quantifying viral infection, among others, in permissive hosts is a key endpoint for studying MERS pathogenesis and evaluating the efficacy of selected MCMs developed for MERS. In addition to quantifying infectious progeny virus which requires high-containment biosafety level (BSL)-3 laboratory, here we outlined an established real-time quantitative RT-PCR (RT-qPCR)-based procedure to unequivocally quantify MERS-CoV-specific RNAs within the lungs of infected human DPP4 (hDPP4, transgenic (hDPP4 Tg) mice under a standard BSL-2 laboratory.


Subject(s)
Coronavirus Infections/virology , Middle East Respiratory Syndrome Coronavirus/genetics , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/metabolism , Disease Models, Animal , Humans , Lung/virology , Mice , Mice, Transgenic , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Real-Time Polymerase Chain Reaction , Receptors, Virus/genetics , Receptors, Virus/metabolism
11.
PLoS One ; 16(6): e0253487, 2021.
Article in English | MEDLINE | ID: covidwho-1280634

ABSTRACT

Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , COVID-19/therapy , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Neutralizing/metabolism , COVID-19/immunology , COVID-19/virology , Chemokines/blood , Chemokines/genetics , Chlorocebus aethiops , Convalescence , Cricetinae , Cytokines/blood , Cytokines/genetics , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Humans , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Macaca mulatta , Male , Mice, Transgenic , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Load
12.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Article in English | MEDLINE | ID: covidwho-1087557

ABSTRACT

Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mp ro). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC50) value below 100 µM in inhibiting Mp ro, and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting Mp ro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti-SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC50) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.


Subject(s)
Antiviral Agents/pharmacology , Bepridil/pharmacology , Drug Discovery , SARS-CoV-2/drug effects , A549 Cells , Animals , Chlorocebus aethiops , Humans , Molecular Docking Simulation , Molecular Structure , Small Molecule Libraries , Vero Cells
13.
ChemMedChem ; 16(6): 942-948, 2021 03 18.
Article in English | MEDLINE | ID: covidwho-959133

ABSTRACT

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549/ACE2 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Alanine/analogs & derivatives , Alanine/metabolism , Alanine/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Catalytic Domain , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Cysteine/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/metabolism , Humans , Microbial Sensitivity Tests , Protein Binding , Pyrrolidinones/chemical synthesis , Pyrrolidinones/metabolism , Pyrrolidinones/pharmacology , SARS-CoV-2/enzymology , Vero Cells
14.
bioRxiv ; 2020 Jul 05.
Article in English | MEDLINE | ID: covidwho-900735

ABSTRACT

We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast- engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ~ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.

15.
Proc Natl Acad Sci U S A ; 117(47): 29832-29838, 2020 11 24.
Article in English | MEDLINE | ID: covidwho-900111

ABSTRACT

Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Vaccines/immunology , COVID-19/therapy , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antibody-Dependent Cell Cytotoxicity , COVID-19 Serological Testing/standards , COVID-19 Vaccines/standards , Chlorocebus aethiops , Cricetinae , Female , Humans , Immunization, Passive/methods , Immunization, Passive/standards , Immunogenicity, Vaccine , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
16.
Nat Commun ; 11(1): 5214, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-872699

ABSTRACT

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , High-Throughput Screening Assays/methods , Neutralization Tests/methods , Pneumonia, Viral/diagnosis , A549 Cells , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/genetics , Betacoronavirus/immunology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/virology , Humans , Luciferases/genetics , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , SARS-CoV-2 , Vero Cells , Virus Internalization/drug effects , Virus Replication/drug effects
17.
Vaccine ; 38(47): 7533-7541, 2020 11 03.
Article in English | MEDLINE | ID: covidwho-779734

ABSTRACT

We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high levels of neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ~30% mortality in mice immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation with Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Subunit/immunology , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , Aluminum Hydroxide/administration & dosage , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/immunology , Female , Humans , Mice , Mice, Inbred BALB C , Protein Domains/immunology , Recombinant Proteins/immunology , SARS-CoV-2 , Severe Acute Respiratory Syndrome/prevention & control , Spike Glycoprotein, Coronavirus/genetics , Viral Load/immunology
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