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1.
mBio ; : e0344221, 2022 Jan 25.
Article in English | MEDLINE | ID: covidwho-1649813

ABSTRACT

Recent studies have shown a temporal increase in the neutralizing antibody potency and breadth to SARS-CoV-2 variants in coronavirus disease 2019 (COVID-19) convalescent individuals. Here, we examined longitudinal antibody responses and viral neutralizing capacity to the B.1 lineage virus (Wuhan related), to variants of concern (VOC; Alpha, Beta, Gamma, and Delta), and to a local variant of interest (VOI; Lambda) in volunteers receiving the Sputnik V vaccine in Argentina. Longitudinal serum samples (N = 536) collected from 118 volunteers obtained between January and October 2021 were used. The analysis indicates that while anti-spike IgG levels significantly wane over time, the neutralizing capacity for the Wuhan-related lineages of SARS-CoV-2 and VOC is maintained within 6 months of vaccination. In addition, an improved antibody cross-neutralizing ability for circulating variants of concern (Beta and Gamma) was observed over time postvaccination. The viral variants that displayed higher escape to neutralizing antibodies with respect to the original virus (Beta and Gamma variants) were the ones showing the largest increase in susceptibility to neutralization over time after vaccination. Our observations indicate that serum neutralizing antibodies are maintained for at least 6 months and show a reduction of VOC escape to neutralizing antibodies over time after vaccination. IMPORTANCE Vaccines have been produced in record time for SARS-CoV-2, offering the possibility of halting the global pandemic. However, inequalities in vaccine accessibility in different regions of the world create a need to increase international cooperation. Sputnik V is a recombinant adenovirus-based vaccine that has been widely used in Argentina and other developing countries, but limited information is available about its elicited immune responses. Here, we examined longitudinal antibody levels and viral neutralizing capacity elicited by Sputnik V vaccination. Using a cohort of 118 volunteers, we found that while anti-spike antibodies wane over time, the neutralizing capacity to viral variants of concern and local variants of interest is maintained within 4 months of vaccination. In addition, we observed an increased cross-neutralization activity over time for the Beta and Gamma variants. This study provides valuable information about the immune response generated by a vaccine platform used in many parts of the world.

2.
mBio ; : e0337721, 2022 Jan 18.
Article in English | MEDLINE | ID: covidwho-1637923

ABSTRACT

Pathogenic coronaviruses are a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified small-molecule inhibitors that potently block the replication of severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with a 50% effective concentration of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types, including primary human bronchial epithelial cells, against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens. IMPORTANCE The coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 infection, is an ongoing public health disaster worldwide. Although several vaccines are available as a preventive measure and the FDA approval of an orally bioavailable drug is on the horizon, there remains a need for developing antivirals against SARS-CoV-2 that could work on the early course of infection. By using infectious reporter viruses, we screened small-molecule inhibitors for antiviral activity against SARS-CoV-2. Among the top hits was JIB-04, a compound previously studied for its anticancer activity. Here, we showed that JIB-04 inhibits the replication of SARS-CoV-2 as well as different DNA and RNA viruses. Furthermore, JIB-04 conferred protection in a porcine model of coronavirus infection, although to a lesser extent when given as therapeutic rather than prophylactic doses. Our findings indicate a limited but still promising utility of JIB-04 as an antiviral agent in the combat against COVID-19 and potentially other viral diseases.

3.
Non-conventional in English | MEDLINE, Grey literature | ID: grc-750513

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and hundreds of thousands of deaths. Accordingly, an effective vaccine is of critical importance in mitigating coronavirus induced disease 2019 (COVID-19) and curtailing the pandemic. We developed a replication-competent vesicular stomatitis virus (VSV)-based vaccine by introducing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high titers of antibodies that neutralize SARS-CoV-2 infection and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung indicating protection against pneumonia. Finally, passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals protects naïve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

4.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Article in English | MEDLINE | ID: covidwho-1493345

ABSTRACT

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.


Subject(s)
Benzothiazoles/pharmacology , COVID-19/drug therapy , Oligopeptides/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/genetics , Animals , Benzamidines/chemistry , Benzothiazoles/pharmacokinetics , COVID-19/genetics , COVID-19/virology , Cell Line , Drug Design , Epithelial Cells/drug effects , Epithelial Cells/virology , Esters/chemistry , Guanidines/chemistry , Humans , Lung/drug effects , Lung/virology , Mice , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Oligopeptides/pharmacokinetics , SARS-CoV-2/pathogenicity , Serine Endopeptidases/drug effects , Serine Endopeptidases/ultrastructure , Small Molecule Libraries/pharmacology , Substrate Specificity/drug effects , Virus Internalization/drug effects
5.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Article in English | MEDLINE | ID: covidwho-1470027

ABSTRACT

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , COVID-19/immunology , COVID-19/prevention & control , Pandemics , SARS-CoV-2/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Antigen-Presenting Cells/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/epidemiology , COVID-19 Vaccines/administration & dosage , Camelids, New World/immunology , Female , Histocompatibility Antigens Class II/immunology , Humans , Immunity, Cellular , Immunity, Humoral , Immunization, Secondary , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Pandemics/prevention & control , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/genetics , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
6.
Cell Host Microbe ; 29(1): 44-57.e9, 2021 01 13.
Article in English | MEDLINE | ID: covidwho-1385265

ABSTRACT

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.


Subject(s)
SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Binding Sites , Epitopes/immunology , Gene Library , High-Throughput Nucleotide Sequencing , Humans , Protein Domains , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Spike Glycoprotein, Coronavirus/chemistry
7.
Cell Rep ; 36(2): 109364, 2021 07 13.
Article in English | MEDLINE | ID: covidwho-1283971

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.


Subject(s)
COVID-19/immunology , COVID-19/metabolism , Receptors, Virus , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Substitution , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Cell Cycle , Cell Line, Tumor , Chlorocebus aethiops , Gene Expression Profiling , Heparitin Sulfate/metabolism , Humans , Interferon Type I/metabolism , Interferon-Induced Helicase, IFIH1/metabolism , Models, Biological , Protein Binding , Protein Domains , Proteomics , Receptors, Virus/metabolism , SARS-CoV-2 , Serine Endopeptidases/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Internalization , Virus Replication
8.
Cell ; 184(9): 2332-2347.e16, 2021 04 29.
Article in English | MEDLINE | ID: covidwho-1135276

ABSTRACT

The SARS-CoV-2 spike (S) glycoprotein contains an immunodominant receptor-binding domain (RBD) targeted by most neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite (designated site i) recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge, albeit selecting escape mutants in some animals. Indeed, several SARS-CoV-2 variants, including the B.1.1.7, B.1.351, and P.1 lineages, harbor frequent mutations within the NTD supersite, suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs for protective immunity and vaccine design.


Subject(s)
Antigens, Viral/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/virology , Cricetinae , Epitope Mapping , Genetic Variation , Models, Molecular , Mutation/genetics , Neutralization Tests , Protein Domains , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/ultrastructure
9.
Cell Host Microbe ; 29(3): 477-488.e4, 2021 03 10.
Article in English | MEDLINE | ID: covidwho-1053270

ABSTRACT

Neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics. However, viral escape mutants could compromise efficacy. To define immune-selected mutations in the S protein, we exposed a VSV-eGFP-SARS-CoV-2-S chimeric virus, in which the VSV glycoprotein is replaced with the S protein, to 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) and generated 50 different escape mutants. Each mAb had a unique resistance profile, although many shared residues within an epitope of the RBD. Some variants (e.g., S477N) were resistant to neutralization by multiple mAbs, whereas others (e.g., E484K) escaped neutralization by convalescent sera. Additionally, sequential selection identified mutants that escape neutralization by antibody cocktails. Comparing these antibody-mediated mutations with sequence variation in circulating SARS-CoV-2 revealed substitutions that may attenuate neutralizing immune responses in some humans and thus warrant further investigation.


Subject(s)
Antibodies, Monoclonal/blood , Antibodies, Viral/blood , Mutation , Neutralization Tests/methods , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , COVID-19/virology , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Female , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Binding , Receptors, Virus/metabolism , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
10.
Proc Natl Acad Sci U S A ; 117(50): 32105-32113, 2020 12 15.
Article in English | MEDLINE | ID: covidwho-947594

ABSTRACT

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.


Subject(s)
COVID-19/drug therapy , Endosomes/genetics , Hydroxycholesterols/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Antiviral Agents/pharmacology , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Endosomes/metabolism , Humans , Interferons/metabolism , Membrane Fusion/drug effects , SARS Virus/drug effects , SARS Virus/metabolism , SARS Virus/pathogenicity , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization/drug effects , Virus Replication/drug effects
11.
bioRxiv ; 2021 Jun 04.
Article in English | MEDLINE | ID: covidwho-808504

ABSTRACT

Pathogenic coronaviruses represent a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified several small-molecule inhibitors that potently block the replication of the newly emerged severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with an EC50 of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens.

12.
Cell Host Microbe ; 28(3): 465-474.e4, 2020 09 09.
Article in English | MEDLINE | ID: covidwho-710174

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections, and an effective vaccine is critical to mitigate coronavirus-induced disease 2019 (COVID-19). Previously, we developed a replication-competent vesicular stomatitis virus (VSV) expressing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Here, we show that vaccination with VSV-eGFP-SARS-CoV-2 generates neutralizing immune responses and protects mice from SARS-CoV-2. Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high antibody titers that neutralize SARS-CoV-2 and target the receptor binding domain that engages human angiotensin-converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice that expressed human ACE2 and were immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung, indicating protection against pneumonia. Passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals also protects naive mice from SARS-CoV-2 challenge. These data support development of VSV-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.


Subject(s)
Betacoronavirus , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Vesicular stomatitis Indiana virus/genetics , Viral Vaccines/genetics , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Chlorocebus aethiops , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Disease Models, Animal , Genetic Vectors , Green Fluorescent Proteins/genetics , Host Microbial Interactions/immunology , Humans , Lung/immunology , Lung/pathology , Lung/virology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Receptors, Virus/genetics , SARS-CoV-2 , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/pharmacology , Vero Cells , Vesicular stomatitis Indiana virus/immunology , Viral Vaccines/immunology , Viral Vaccines/pharmacology
13.
Proc Natl Acad Sci U S A ; 117(34): 20803-20813, 2020 08 25.
Article in English | MEDLINE | ID: covidwho-695945

ABSTRACT

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.


Subject(s)
Betacoronavirus/drug effects , Ebolavirus/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases , Triazines/pharmacology , Virus Internalization/drug effects , Animals , Betacoronavirus/physiology , COVID-19 , Cells, Cultured , Coronavirus Infections , Ebolavirus/physiology , Gene Editing , Humans , Hydrazones , Pandemics , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Pneumonia, Viral , Pyrimidines , SARS-CoV-2 , Viral Envelope Proteins/genetics
14.
bioRxiv ; 2020 Jul 10.
Article in English | MEDLINE | ID: covidwho-663086

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and hundreds of thousands of deaths. Accordingly, an effective vaccine is of critical importance in mitigating coronavirus induced disease 2019 (COVID-19) and curtailing the pandemic. We developed a replication-competent vesicular stomatitis virus (VSV)-based vaccine by introducing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high titers of antibodies that neutralize SARS-CoV-2 infection and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung indicating protection against pneumonia. Finally, passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals protects naïve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

15.
Nat Med ; 26(9): 1422-1427, 2020 09.
Article in English | MEDLINE | ID: covidwho-640071

ABSTRACT

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/isolation & purification , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/virology , Humans , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology
16.
Cell Host Microbe ; 28(3): 475-485.e5, 2020 09 09.
Article in English | MEDLINE | ID: covidwho-626409

ABSTRACT

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious molecular clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput-imaging-based neutralization assay at biosafety level 2. We also developed a focus-reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc soluble decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Additionally, replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/immunology , Coronavirus Infections/therapy , Peptidyl-Dipeptidase A/immunology , Pneumonia, Viral/therapy , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/genetics , Betacoronavirus/physiology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Green Fluorescent Proteins/genetics , Host Microbial Interactions/immunology , Humans , Immunization, Passive , Neutralization Tests , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/immunology , Virus Internalization , Virus Replication
17.
Sci Immunol ; 5(47)2020 05 13.
Article in English | MEDLINE | ID: covidwho-260039

ABSTRACT

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19 patients. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.


Subject(s)
Betacoronavirus/physiology , Enterocytes/virology , Membrane Proteins/metabolism , Serine Endopeptidases/metabolism , Virus Internalization , Angiotensin-Converting Enzyme 2 , Animals , Cell Line , Duodenum/cytology , Enterocytes/pathology , Humans , Mice , Organoids/virology , Peptidyl-Dipeptidase A/metabolism , Rotavirus/physiology , SARS-CoV-2 , Vesiculovirus/genetics
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