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1.
Emerg Microbes Infect ; 11(1): 208-211, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1598042

ABSTRACT

We performed an annotation of 35 mutations in the spike protein of the SARS-CoV-2 Omicron variant. Our analysis of the mutations indicates that Omicron has gained prominent immune evasion and potential for enhanced transmissibility. Previous modeling study has revealed that continued evolution in both immune evasion and enhanced transmissibility by SARS-CoV-2 would compromise vaccines as tools for the pandemic control. To combat the future variants of SARS-CoV-2, the world needs novel antiviral drugs that are effective at curb viral spreading without introducing additional selective pressure towards resistant variants.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/drug therapy , Drug Design/methods , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Antiviral Agents/chemical synthesis , Antiviral Agents/therapeutic use , Binding Sites , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/transmission , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/adverse effects , Humans , Immune Evasion , Mutation , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/immunology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
2.
Signal Transduct Target Ther ; 6(1): 420, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1585885

ABSTRACT

COVID-19 is identified as a zoonotic disease caused by SARS-CoV-2, which also can cross-transmit to many animals but not mice. Genetic modifications of SARS-CoV-2 or mice enable the mice susceptible to viral infection. Although neither is the natural situation, they are currently utilized to establish mouse infection models. Here we report a direct contact transmission of SARS-CoV-2 variant B.1.351 in wild-type mice. The SARS-CoV-2 (B.1.351) replicated efficiently and induced significant pathological changes in lungs and tracheas, accompanied by elevated proinflammatory cytokines in the lungs and sera. Mechanistically, the receptor-binding domain (RBD) of SARS-CoV-2 (B.1.351) spike protein turned to a high binding affinity to mouse angiotensin-converting enzyme 2 (mACE2), allowing the mice highly susceptible to SARS-CoV-2 (B.1.351) infection. Our work suggests that SARS-CoV-2 (B.1.351) expands the host range and therefore increases its transmission route without adapted mutation. As the wild house mice live with human populations quite closely, this possible transmission route could be potentially risky. In addition, because SARS-CoV-2 (B.1.351) is one of the major epidemic strains and the mACE2 in laboratory-used mice is naturally expressed and regulated, the SARS-CoV-2 (B.1.351)/mice could be a much convenient animal model system to study COVID-19 pathogenesis and evaluate antiviral inhibitors and vaccines.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/transmission , Host-Pathogen Interactions/genetics , Receptors, Virus/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , COVID-19/immunology , COVID-19/virology , Cytokines/genetics , Cytokines/immunology , Disease Models, Animal , Gene Expression , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Lung/pathology , Lung/virology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding , Protein Domains , Receptors, Virus/immunology , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Virus Replication
3.
J Cell Mol Med ; 26(1): 25-34, 2022 01.
Article in English | MEDLINE | ID: covidwho-1570773

ABSTRACT

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , COVID-19 Vaccines/chemistry , COVID-19/prevention & control , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/ultrastructure , Serine Endopeptidases/chemistry , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virion/drug effects , Virion/pathogenicity , Virion/ultrastructure
4.
J Clin Invest ; 131(21)2021 11 01.
Article in English | MEDLINE | ID: covidwho-1495789

ABSTRACT

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.


Subject(s)
COVID-19/immunology , COVID-19/virology , Killer Cells, Natural/immunology , Receptors, Natural Killer Cell/immunology , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Animals , Antigens, Differentiation, T-Lymphocyte/immunology , Cell Adhesion Molecules/immunology , Cohort Studies , Cytotoxicity, Immunologic , Female , Heterografts , Host Microbial Interactions/immunology , Humans , Immunophenotyping , In Vitro Techniques , Ligands , Male , Mice , Mice, SCID , Middle Aged , NK Cell Lectin-Like Receptor Subfamily D/immunology , Pandemics , Receptors, Immunologic/immunology , Receptors, Virus/immunology , Viral Load , Young Adult
5.
Nat Commun ; 12(1): 250, 2021 01 11.
Article in English | MEDLINE | ID: covidwho-1387324

ABSTRACT

Understanding the mechanism for antibody neutralization of SARS-CoV-2 is critical for the development of effective therapeutics and vaccines. We recently isolated a large number of monoclonal antibodies from SARS-CoV-2 infected individuals. Here we select the top three most potent yet variable neutralizing antibodies for in-depth structural and functional analyses. Crystal structural comparisons reveal differences in the angles of approach to the receptor binding domain (RBD), the size of the buried surface areas, and the key binding residues on the RBD of the viral spike glycoprotein. One antibody, P2C-1F11, most closely mimics binding of receptor ACE2, displays the most potent neutralizing activity in vitro and conferred strong protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice. It also occupies the largest binding surface and demonstrates the highest binding affinity to RBD. More interestingly, P2C-1F11 triggers rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein, with only minimal such effect by the remaining two antibodies. These results offer a structural and functional basis for potent neutralization via disruption of the very first and critical steps for SARS-CoV-2 cell entry.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/immunology , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Disease Models, Animal , Epitopes , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Virus/immunology , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
6.
Elife ; 102021 08 02.
Article in English | MEDLINE | ID: covidwho-1377103

ABSTRACT

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking viral receptor angiotensin-converting enzyme 2 (ACE2). The lead drug candidate possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD ~3000 times more tightly than ACE2 did and inhibited SARS-CoV-2 pseudovirus ~160 times more efficiently than ACE2 did. Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models. Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented an excellent in vivo stability and a high tissue bioavailability. As effective and inexpensive drug candidates, Nanosota-1 may contribute to the battle against COVID-19.


Subject(s)
Antibodies, Viral/immunology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Single-Domain Antibodies/pharmacology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Molecular , Pandemics , Protein Binding , Protein Conformation , Receptors, Virus/immunology , Receptors, Virus/metabolism , Single-Domain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/metabolism
7.
FEBS Lett ; 595(18): 2323-2340, 2021 09.
Article in English | MEDLINE | ID: covidwho-1332924

ABSTRACT

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin-like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS-CoV-2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Bacterial Proteins/chemistry , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/chemistry , DNA-Binding Proteins/chemistry , Ferritins/chemistry , Humans , Immunization , Mice , Nanoparticles , Protein Domains , Protein Multimerization , Spike Glycoprotein, Coronavirus/chemistry , Sulfolobus
8.
mBio ; 12(4): e0100221, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1327614

ABSTRACT

After first emerging in late 2019 in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized, but the supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the full-length spike protein of SARS-CoV-2. In this study, we generated mouse monoclonal antibodies (MAbs) against different epitopes on the RBD and assessed binding and neutralization of authentic SARS-CoV-2. We demonstrate that antibodies with neutralizing activity, but not nonneutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the MAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variant in vitro. IMPORTANCE Cross-neutralization of SARS-CoV-2 variants by RBD-targeting antibodies is still not well understood, and very little is known about the potential protective effect of nonneutralizing antibodies in vivo. Using a panel of mouse monoclonal antibodies, we investigate both of these points.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/immunology , Receptors, Virus/immunology , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Viral/immunology , Antibodies, Viral/pharmacology , COVID-19/drug therapy , Chlorocebus aethiops , Female , Humans , Mice , Mice, Inbred BALB C , Neutralization Tests , Protein Binding , Protein Domains/immunology , SARS-CoV-2/immunology , Vero Cells , Viral Load
9.
J Immunol ; 207(3): 878-887, 2021 08 01.
Article in English | MEDLINE | ID: covidwho-1323338

ABSTRACT

Tools to monitor SARS-CoV-2 transmission and immune responses are needed. We present a neutralization ELISA to determine the levels of Ab-mediated virus neutralization and a preclinical model of focused immunization strategy. The ELISA is strongly correlated with the elaborate plaque reduction neutralization test (ρ = 0.9231, p < 0.0001). The neutralization potency of convalescent sera strongly correlates to IgG titers against SARS-CoV-2 receptor-binding domain (RBD) and spike (ρ = 0.8291 and 0.8297, respectively; p < 0.0001) and to a lesser extent with the IgG titers against protein N (ρ = 0.6471, p < 0.0001). The preclinical vaccine NMRI mice models using RBD and full-length spike Ag as immunogens show a profound Ab neutralization capacity (IC50 = 1.9 × 104 to 2.6 × 104 and 3.9 × 103 to 5.2 × 103, respectively). Using a panel of novel high-affinity murine mAbs, we also show that a majority of the RBD-raised mAbs have inhibitory properties, whereas only a few of the spike-raised mAbs do. The ELISA-based viral neutralization test offers a time- and cost-effective alternative to the plaque reduction neutralization test. The immunization results indicate that vaccine strategies focused only on the RBD region may have advantages compared with the full spike.


Subject(s)
Antibodies, Neutralizing/blood , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Neutralization Tests/methods , Receptors, Virus/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/therapy , COVID-19 Vaccines/immunology , Humans , Immunization , Immunization, Passive , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Mice , Protein Domains/immunology
10.
Mol Biol Rep ; 48(7): 5745-5758, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1321814

ABSTRACT

To date, the latest research results suggest that the novel severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) can enter host cells directly via the gastrointestinal tract by binding to the enterocyte-expressed ACE2 receptor, or indirectly as a result of infection of type II alveolar epithelial cells. At the same time, entry of SARS-CoV-2 through the gastrointestinal tract initiates the activation of innate and adaptive immune responses, the formation of an excessive inflammatory reaction and critical increase in the expression of proinflammatory cytokines, which, subsequently, can presumably increase inflammation and induce intestinal damage in patients suffering from inflammatory bowel disease (IBD). The aims of the present review were to reveal and analyze possible molecular pathways and consequences of the induction of an innate and adaptive immune response during infection with SARS-CoV-2 in patients with IBD. A thorough literature search was carried out by using the keywords: IBD, SARS-CoV-2, COVID-19. Based on the screening, a number of intracellular and extracellular pathways were considered and discussed, which can impact the immune response during SARS-CoV-2 infection in IBD patients. Additionally, the possible consequences of the infection for such patients were estimated. We further hypothesize that any virus, including the new SARS-CoV-2, infecting intestinal tissues and/or entering the host's body through receptors located on intestinal enterocytes may be a trigger for the onset of IBD in individuals with a genetic predisposition and/or the risk of developing IBD associated with other factors.


Subject(s)
Adaptive Immunity , COVID-19/epidemiology , Gastrointestinal Tract , Immunity, Innate , Inflammatory Bowel Diseases , COVID-19/immunology , COVID-19/virology , Gastrointestinal Tract/immunology , Gastrointestinal Tract/pathology , Gastrointestinal Tract/virology , Humans , Inflammatory Bowel Diseases/epidemiology , Inflammatory Bowel Diseases/immunology , Receptors, Virus/immunology , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Virus Internalization
11.
Nat Commun ; 12(1): 4210, 2021 07 09.
Article in English | MEDLINE | ID: covidwho-1303772

ABSTRACT

Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Receptors, Virus/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites/immunology , COVID-19/immunology , Cricetinae , Disease Models, Animal , Epitopes/immunology , Female , Humans , Male , Neutralization Tests , Receptors, Antigen, B-Cell/immunology , Spike Glycoprotein, Coronavirus/immunology
12.
Nat Commun ; 12(1): 4144, 2021 07 06.
Article in English | MEDLINE | ID: covidwho-1298839

ABSTRACT

To investigate the duration of humoral immune response in convalescent coronavirus disease 2019 (COVID-19) patients, we conduct a 12-month longitudinal study through collecting a total of 1,782 plasma samples from 869 convalescent plasma donors in Wuhan, China and test specific antibody responses. The results show that positive rate of IgG antibody against receptor-binding domain of spike protein (RBD-IgG) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the COVID-19 convalescent plasma donors exceeded 70% for 12 months post diagnosis. The level of RBD-IgG decreases with time, with the titer stabilizing at 64.3% of the initial level by the 9th month. Moreover, male plasma donors produce more RBD-IgG than female, and age of the patients positively correlates with the RBD-IgG titer. A strong positive correlation between RBD-IgG and neutralizing antibody titers is also identified. These results facilitate our understanding of SARS-CoV-2-induced immune memory to promote vaccine and therapy development.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Immunoglobulin G/blood , Receptors, Virus/immunology , SARS-CoV-2/immunology , Adult , Animals , Blood Donors , COVID-19/therapy , Cell Line , China , Chlorocebus aethiops , Convalescence , Female , Humans , Immunity, Humoral/immunology , Immunization, Passive , Immunologic Memory/immunology , Longitudinal Studies , Male , Sex Factors , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
13.
Structure ; 29(7): 655-663.e4, 2021 07 01.
Article in English | MEDLINE | ID: covidwho-1263379

ABSTRACT

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively, show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of the spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented an immune response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies, such as 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Receptors, Virus/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Binding Sites , Cloning, Molecular , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Gene Expression , HEK293 Cells , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/genetics , Receptors, Virus/immunology , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
14.
PLoS Biol ; 19(5): e3001209, 2021 05.
Article in English | MEDLINE | ID: covidwho-1219261

ABSTRACT

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/virology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/immunology , COVID-19/epidemiology , COVID-19/immunology , COVID-19/metabolism , Epitopes/immunology , Humans , Mice , Mice, Transgenic , Neutralization Tests , Pandemics , Protein Binding , Protein Domains , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/immunology
15.
Commun Biol ; 4(1): 500, 2021 04 23.
Article in English | MEDLINE | ID: covidwho-1213942

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic, which has resulted in more than two million deaths at 2021 February . There is currently no approved therapeutics for treating COVID-19. The SARS-CoV-2 Spike protein is considered a key therapeutic target by many researchers. Here we describe the identification of several monoclonal antibodies that target SARS-CoV-2 Spike protein. One human antibody, CA521FALA, demonstrated neutralization potential by immunizing human antibody transgenic mice. CA521FALA showed potent SARS-CoV-2-specific neutralization activity against SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 infection in vitro. CA521FALA also demonstrated having a long half-life of 9.5 days in mice and 9.3 days in rhesus monkeys. CA521FALA inhibited SARS-CoV-2 infection in SARS-CoV-2 susceptible mice at a therapeutic setting with virus titer of the lung reduced by 4.5 logs. Structural analysis by cryo-EM revealed that CA521FALA recognizes an epitope overlapping with angiotensin converting enzyme 2 (ACE2)-binding sites in SARS-CoV-2 RBD in the Spike protein. CA521FALA blocks the interaction by binding all three RBDs of one SARS-CoV-2 spike trimer simultaneously. These results demonstrate the importance for antibody-based therapeutic interventions against COVID-19 and identifies CA521FALA a promising antibody that reacts with SARS-CoV-2 Spike protein to strongly neutralize its activity.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19/epidemiology , COVID-19/virology , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Pandemics , Protein Binding/drug effects , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism
16.
Cell Rep ; 35(6): 109109, 2021 05 11.
Article in English | MEDLINE | ID: covidwho-1201425

ABSTRACT

It is unclear whether individuals with enormous diversity in B cell receptor repertoires are consistently able to mount effective antibody responses against SARS-CoV-2. We analyzed antibody responses in a cohort of 55 convalescent patients and isolated 54 potent neutralizing monoclonal antibodies (mAbs). While most of the mAbs target the angiotensin-converting enzyme 2 (ACE2) binding surface on the receptor binding domain (RBD) of SARS-CoV-2 spike protein, mAb 47D1 binds only to one side of the receptor binding surface on the RBD. Neutralization by 47D1 is achieved independent of interfering RBD-ACE2 binding. A crystal structure of the mAb-RBD complex shows that the IF motif at the tip of 47D1 CDR H2 interacts with a hydrophobic pocket in the RBD. Diverse immunoglobulin gene usage and convergent epitope targeting characterize neutralizing antibody responses to SARS-CoV-2, suggesting that vaccines that effectively present the receptor binding site on the RBD will likely elicit neutralizing antibody responses in a large fraction of the population.


Subject(s)
Antibodies, Neutralizing/genetics , COVID-19/genetics , Immunoglobulins/genetics , Adult , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/immunology , COVID-19/immunology , COVID-19/therapy , Epitopes/genetics , Epitopes/immunology , Female , Genes, Immunoglobulin/genetics , Genetic Variation/genetics , Humans , Immunization, Passive/methods , Immunoglobulins/immunology , Male , Middle Aged , Peptidyl-Dipeptidase A/metabolism , Protein Binding/immunology , Protein Domains/genetics , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity
17.
Infect Genet Evol ; 91: 104832, 2021 07.
Article in English | MEDLINE | ID: covidwho-1164210

ABSTRACT

MicroRNAs are gene expression regulators, associated with several human pathologies, including the ones caused by virus infections. Although their role in infection diseases is not completely known, they can exert double functions in the infected cell, by mediating the virus infection and/or regulating the immunity-related gene targets through complex networks of virus-host cell interactions. In this systematic review, the Pubmed, EMBASE, Scopus, Lilacs, Scielo, and EBSCO databases were searched for research articles published until October 22nd, 2020 that focused on describing the role, function, and/or association of miRNAs in SARS-CoV-2 human infection and COVID-19. Following the PRISMA 2009 protocol, 29 original research articles were selected. Most of the studies reported miRNA data based on the genome sequencing of SARS-CoV-2 isolates and computational prediction analysis. The latter predicted, by at least one independent study, 1266 host miRNAs to target the viral genome. Thirteen miRNAs were identified by four independent studies to target SARS-CoV-2 specific genes, suggested to act by interfering with their cleavage and/or translation process. The studies selected also reported on viral and host miRNAs that targeted host genes, on the expression levels of miRNAs in biological specimens of COVID-19 patients, and on the impact of viral genome mutations on miRNA function. Also, miRNAs that regulate the expression levels of the ACE2 and TMPRSS2 proteins, which are critical for the virus entrance in the host cells, were reported. In conclusion, despite the limited number of studies identified, based on the search terms and eligibility criteria applied, this systematic review provides evidence on the impact of miRNAs on SARS-CoV-2 infection and COVID-19. Although most of the reported viral/host miRNAs interactions were based on in silico prediction analysis, they demonstrate the relevance of the viral/host miRNA interaction for viral activity and host responses. In addition, the identified studies highlight the potential use of miRNAs as therapeutic targets against COVID-19, and other viral human diseases (This review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) database (#CRD42020199290).


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Genome, Viral , MicroRNAs/genetics , SARS-CoV-2/genetics , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/immunology , COVID-19/immunology , COVID-19/pathology , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , MicroRNAs/classification , MicroRNAs/immunology , Mutation , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/immunology , SARS-CoV-2/immunology , Serine Endopeptidases/immunology , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology
18.
Int J Mol Sci ; 22(5)2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-1150749

ABSTRACT

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Serological Testing/standards , COVID-19/blood , COVID-19/immunology , Neutralization Tests/methods , Neutralization Tests/standards , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cross Reactions , Flow Cytometry/methods , Fluorescence , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Microspheres , Receptors, Virus/chemistry , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
19.
Rev Med Virol ; 31(6): e2227, 2021 11.
Article in English | MEDLINE | ID: covidwho-1148855

ABSTRACT

Severe acute respiratory syndrome related coronavirus-2 (SARS-CoV-2) is the cause of Covid-19 which was classified as a global pandemic in March 2020. The increasing global health and economic burden of SARS-CoV-2 has necessitated urgent investigations into the pathogenesis of disease and development of therapeutic and vaccination regimens. Human trials of vaccine and antiviral candidates have been undertaken, but basic pathogenetic studies are still required to inform these trials. Gaps in understanding of cellular infection by, and immunity to, SARS-CoV-2 mean additional models are required to assist in improved design of these therapeutics. Human organoids are three-dimensional models that contain multiple cell types and mimic human organs in ex vivo culture conditions. The SARS-CoV-2 virus has been implicated in causing not only respiratory injury but also injury to other organs such as the brain, liver and kidneys. Consequently, a variety of different organoid models have been employed to investigate the pathogenic mechanisms of disease due to SARS-CoV-2. Data on these models have not been systematically assembled. In this review, we highlight key findings from studies that have utilised different human organoid types to investigate the expression of SARS-CoV-2 receptors, permissiveness, immune response, dysregulation of cellular functions, and potential antiviral therapeutics.


Subject(s)
Host-Pathogen Interactions/immunology , Models, Biological , Organoids/immunology , Receptors, Virus/antagonists & inhibitors , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Antiviral Agents/pharmacology , Brain/drug effects , Brain/immunology , Brain/virology , COVID-19/drug therapy , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Cell Culture Techniques , Colon/drug effects , Colon/immunology , Colon/virology , Cytokines/genetics , Cytokines/immunology , Host-Pathogen Interactions/drug effects , Humans , Liver/drug effects , Liver/immunology , Liver/virology , Lung/drug effects , Lung/immunology , Lung/virology , Organoids/drug effects , Organoids/virology , Receptors, Virus/genetics , Receptors, Virus/immunology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Serine Endopeptidases/genetics , Serine Endopeptidases/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
20.
Int J Mol Sci ; 22(5)2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-1134167

ABSTRACT

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Serological Testing/standards , COVID-19/blood , COVID-19/immunology , Neutralization Tests/methods , Neutralization Tests/standards , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cross Reactions , Flow Cytometry/methods , Fluorescence , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Microspheres , Receptors, Virus/chemistry , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
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