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1.
PLoS Pathog ; 17(12): e1010175, 2021 12.
Article in English | MEDLINE | ID: covidwho-1592244

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.


Subject(s)
Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Protein Domains , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites , Binding Sites, Antibody , COVID-19/prevention & control , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Immunoglobulin Fc Fragments/therapeutic use , Immunoglobulin G/therapeutic use , Mice, Transgenic , Neutralization Tests , Protein Binding , Recombinant Fusion Proteins/therapeutic use , SARS-CoV-2/drug effects , Vero Cells
2.
J Immunol Methods ; 495: 113082, 2021 08.
Article in English | MEDLINE | ID: covidwho-1243047

ABSTRACT

The development of new diagnostic assays become a priority for managing COVID-19. To this aim, we presented here an in-house ELISA based on the production of two major recombinant and high-quality antigens from SARS-CoV-2. Full-length N and S-RBD fragment proteins fused to mouse IgG2a-Fc were produced in the CHO cell line. Secreted recombinant proteins were easily purified with standard Protein A chromatography and were used in an in-house ELISA to detect anti-N and anti-RBD IgGs in the plasma of COVID-19 RTPCR-positive patients. High reactivity against recombinant antigens was readily detected in all positive plasma samples, whereas no recognition was observed with control healthy subject's plasmas. Remarkably, unpurified recombinant N protein obtained from cell culture supernatant was also suitable for the monitoring by ELISA of IgG levels in positive patients. This work provides an early prospection for low price but high-quality serological kit development.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Recombinant Proteins/metabolism , SARS-CoV-2/physiology , Animals , Antibodies, Viral/blood , CHO Cells , COVID-19 Serological Testing/economics , Costs and Cost Analysis , Cricetulus , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Recombinant Proteins/genetics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology
3.
Cell Rep Med ; 2(6): 100313, 2021 06 15.
Article in English | MEDLINE | ID: covidwho-1240648

ABSTRACT

The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis-and in some cases trogocytosis-but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies.


Subject(s)
Antibodies, Monoclonal/immunology , Epitopes/immunology , Immunoglobulin Fc Fragments/metabolism , Spike Glycoprotein, Coronavirus/immunology , Animals , Antigen-Antibody Reactions , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , COVID-19/pathology , COVID-19/virology , Cell Line , Cross Reactions/immunology , Epitope Mapping , Female , Humans , Immunoglobulin Fc Fragments/immunology , Mice , Mice, Inbred BALB C , Phagocytosis , Protein Subunits/immunology , SARS Virus/immunology , SARS Virus/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
4.
Cell Rep Med ; 2(6): 100296, 2021 06 15.
Article in English | MEDLINE | ID: covidwho-1225428

ABSTRACT

The capacity of antibodies to engage with immune cells via the Fc region is important in preventing and controlling many infectious diseases. The evolution of such antibodies during convalescence from coronavirus disease 2019 (COVID-19) is largely unknown. We develop assays to measure Fc-dependent antibody functions against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-expressing cells in serial samples from subjects primarily with mild-moderate COVID-19 up to 149 days post-infection. We find that S-specific antibodies capable of engaging Fcγ receptors decay over time, with S-specific antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP) activity within plasma declining accordingly. Although there is significant decay in ADCC and ADP activity, they remain readily detectable in almost all subjects at the last time point studied (94%) in contrast with neutralization activity (70%). Although it remains unclear the degree to which Fc effector functions contribute to protection against SARS-CoV-2 re-infection, our results indicate that antibodies with Fc effector functions persist longer than neutralizing antibodies.


Subject(s)
Antibodies, Viral/metabolism , COVID-19/immunology , Immunoglobulin Fc Fragments/metabolism , Antibodies, Viral/blood , Antibody-Dependent Cell Cytotoxicity/immunology , COVID-19/pathology , COVID-19/virology , Cell Line, Tumor , Dimerization , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Kinetics , Neutralization Tests , Phagocytosis , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Severity of Illness Index , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
5.
Proteins ; 89(9): 1065-1078, 2021 09.
Article in English | MEDLINE | ID: covidwho-1222689

ABSTRACT

SARS coronavirus 2 is neutralized by proteins that block receptor-binding sites on spikes that project from the viral envelope. In particular, substantial research investment has advanced monoclonal antibody therapies to the clinic where they have shown partial efficacy in reducing viral burden and hospitalization. An alternative is to use the host entry receptor, angiotensin-converting enzyme 2 (ACE2), as a soluble decoy that broadly blocks SARS-associated coronaviruses with limited potential for viral escape. Here, we summarize efforts to engineer higher affinity variants of soluble ACE2 that rival the potency of affinity-matured antibodies. Strategies have also been used to increase the valency of ACE2 decoys for avid spike interactions and to improve pharmacokinetics via IgG fusions. Finally, the intrinsic catalytic activity of ACE2 for the turnover of the vasoconstrictor angiotensin II may directly address COVID-19 symptoms and protect against lung and cardiovascular injury, conferring dual mechanisms of action unachievable by monoclonal antibodies. Soluble ACE2 derivatives therefore have the potential to be next generation therapeutics for addressing the immediate needs of the current pandemic and possible future outbreaks.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Molecular Mimicry , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/metabolism , Mutation , Nanoparticles/chemistry , Nanoparticles/metabolism , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/chemistry
6.
Cell Rep ; 33(12): 108528, 2020 12 22.
Article in English | MEDLINE | ID: covidwho-978234

ABSTRACT

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of ß coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Immunoglobulin Fc Fragments/metabolism , Microbodies/metabolism , SARS-CoV-2/drug effects , Amino Acid Sequence , Animals , COVID-19/prevention & control , COVID-19/virology , Disease Models, Animal , Disulfides/metabolism , Female , HEK293 Cells , Humans , Male , Mice, Transgenic , Protein Domains , Protein Multimerization , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virion/metabolism , Virus Internalization/drug effects
7.
Sci Rep ; 10(1): 17806, 2020 10 20.
Article in English | MEDLINE | ID: covidwho-882927

ABSTRACT

SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.


Subject(s)
Betacoronavirus/metabolism , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Antigen-Antibody Reactions , Betacoronavirus/isolation & purification , Binding Sites , COVID-19 , Cell Line , Computer-Aided Design , Coronavirus Infections/pathology , Coronavirus Infections/virology , Epitopes/chemistry , Epitopes/immunology , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Molecular Docking Simulation , Mutagenesis, Site-Directed , Neutralization Tests , Pandemics , Peptide Library , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Stability , SARS-CoV-2 , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
8.
Front Immunol ; 11: 1660, 2020.
Article in English | MEDLINE | ID: covidwho-697898

ABSTRACT

The current outbreak of viral pneumonia, caused by novel coronavirus SARS-CoV-2, is the focus of worldwide attention. The WHO declared the COVID-19 outbreak a pandemic event on Mar 12, 2020, and the number of confirmed cases is still on the rise worldwide. While most infected individuals only experience mild symptoms or may even be asymptomatic, some patients rapidly progress to severe acute respiratory failure with substantial mortality, making it imperative to develop an efficient treatment for severe SARS-CoV-2 pneumonia alongside supportive care. So far, the optimal treatment strategy for severe COVID-19 remains unknown. Intravenous immunoglobulin (IVIg) is a blood product pooled from healthy donors with high concentrations of immunoglobulin G (IgG) and has been used in patients with autoimmune and inflammatory diseases for more than 30 years. In this review, we aim to highlight the known mechanisms of immunomodulatory effects of high-dose IVIg therapy, the immunopathological hypothesis of viral pneumonia, and the clinical evidence of IVIg therapy in viral pneumonia. We then make cautious therapeutic inferences about high-dose IVIg therapy in treating severe COVID-19. These inferences may provide relevant and useful insights in order to aid treatment for COVID-19.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Betacoronavirus/immunology , Coronavirus Infections/drug therapy , Coronavirus Infections/immunology , Immunoglobulin G/therapeutic use , Immunoglobulins, Intravenous/therapeutic use , Immunologic Factors/therapeutic use , Pneumonia, Viral/drug therapy , Pneumonia, Viral/immunology , Adult , Animals , Antibodies, Neutralizing/administration & dosage , Antibody-Dependent Enhancement , COVID-19 , Child , Coronavirus Infections/virology , Cytokines/metabolism , Humans , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/administration & dosage , Immunoglobulins, Intravenous/administration & dosage , Immunologic Factors/administration & dosage , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2
9.
Nat Commun ; 11(1): 2070, 2020 04 24.
Article in English | MEDLINE | ID: covidwho-116533

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Subject(s)
Betacoronavirus/drug effects , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Neutralization Tests , Peptidyl-Dipeptidase A/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/metabolism , Binding, Competitive/drug effects , Cross Reactions , Drug Design , Humans , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin Fc Fragments/pharmacology , Immunoglobulin G/metabolism , Immunoglobulin G/pharmacology , In Vitro Techniques , Inhibitory Concentration 50 , Membrane Fusion/drug effects , Mice , Mice, Inbred BALB C , Mutation , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/pharmacokinetics , Peptidyl-Dipeptidase A/pharmacology , Protein Domains/genetics , Protein Stability , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/chemistry , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacokinetics , SARS Virus/drug effects , SARS Virus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
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