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1.
Nat Commun ; 12(1): 5469, 2021 09 22.
Article in English | MEDLINE | ID: covidwho-1434103

ABSTRACT

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.


Subject(s)
Antibodies, Neutralizing/pharmacology , COVID-19/drug therapy , Single-Domain Antibodies/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Administration, Intranasal , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Cryoelectron Microscopy , Crystallography, X-Ray , Disease Models, Animal , Dose-Response Relationship, Immunologic , Epitopes/chemistry , Epitopes/metabolism , Female , Male , Mesocricetus , Neutralization Tests , SARS-CoV-2/drug effects , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/immunology , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry
3.
Nat Commun ; 12(1): 1260, 2021 02 24.
Article in English | MEDLINE | ID: covidwho-1101645

ABSTRACT

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.


Subject(s)
COVID-19/immunology , COVID-19/virology , Lung/pathology , Lung/virology , Animals , Disease Models, Animal , Female , Immunity, Cellular/physiology , Interferon-gamma/metabolism , Macaca fascicularis , Macaca mulatta , Male , Pandemics , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity
4.
Br J Pharmacol ; 178(3): 626-635, 2021 02.
Article in English | MEDLINE | ID: covidwho-1066635

ABSTRACT

BACKGROUND AND PURPOSE: Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID-19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS-CoV-2, in vitro, is needed. EXPERIMENTAL APPROACH: Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined. KEY RESULTS: All the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 µg·ml-1 , whereas LMWHs were less inhibitory by ~150-fold (IC50 range 3.4-7.8 mg·ml-1 ). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor. CONCLUSION AND IMPLICATIONS: This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS-CoV-2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.


Subject(s)
Heparin/pharmacology , SARS-CoV-2/growth & development , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Chlorocebus aethiops , Heparin/metabolism , Heparin/therapeutic use , Heparin, Low-Molecular-Weight/pharmacology , Protein Binding/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Viral Plaque Assay
6.
Nat Commun ; 11(1): 4198, 2020 08 21.
Article in English | MEDLINE | ID: covidwho-724360

ABSTRACT

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Immunoglobulin A/immunology , Peptidyl-Dipeptidase A/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Chlorocebus aethiops , Cross Reactions , Epitopes , HEK293 Cells , Humans , Immunoglobulin A/metabolism , Immunoglobulin A, Secretory/immunology , Immunoglobulin A, Secretory/metabolism , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Interaction Domains and Motifs , SARS Virus/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
7.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Article in English | MEDLINE | ID: covidwho-653285

ABSTRACT

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral , Receptors, Virus/metabolism , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/metabolism , Antibodies, Viral/ultrastructure , Antibody Affinity , Antigen-Antibody Reactions/immunology , Betacoronavirus/metabolism , Binding, Competitive , COVID-19 , Cryoelectron Microscopy , Crystallography, X-Ray , Epitopes/immunology , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Models, Molecular , Peptide Library , Peptidyl-Dipeptidase A/ultrastructure , Protein Binding , Protein Conformation , Receptors, Virus/ultrastructure , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , SARS-CoV-2 , Sequence Homology, Amino Acid , Single-Domain Antibodies/metabolism , Single-Domain Antibodies/ultrastructure , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure
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