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1.
Cell Res ; 31(1): 25-36, 2021 01.
Article in English | MEDLINE | ID: covidwho-1387275

ABSTRACT

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 , Epitopes/immunology , SARS-CoV-2/immunology , Single-Chain Antibodies/immunology , Animals , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , COVID-19/immunology , COVID-19/prevention & control , Chlorocebus aethiops , Disease Models, Animal , Humans , Single-Chain Antibodies/pharmacology , Vero Cells
2.
Int J Infect Dis ; 111: 347-353, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1370538

ABSTRACT

AIMS: To explore the contagiousness and new SARS-CoV-2 mutations in pediatric COVID-19. METHODS: This cohort study enrolled all pediatric patients admitted to 8 hospitals in Zhejiang Province of China between 21 January and 29 February 2020, their family members and close-contact classmates. Epidemiological, demographic, clinical and laboratory data were collected. Bioinformatics was used to analyze the features of SARS-CoV-2. Individuals were divided into 3 groups by the first-generation case: Groups 1 (unclear), 2 (adult), and 3 (child). The secondary attack rate (SAR) and R0 were compared among the groups. RESULTS: The infection rate among 211 individuals was 64% (135/211). The SAR in Groups 2 and 3 was 71% (73/103) and 3% (1/30), respectively; the median R0 in Groups 2 and 3 was 2 (range: 1-8) and 0 (range: 0-1), respectively. Compared with adult cases, the SAR and R0 of pediatric cases were significantly lower (p<0.05). We obtained SARS-CoV-2 sequences from the same infant's throat and fecal samples at a two-month interval and found that the new spike protein A958D mutation detected in the stool improved thermostability theoretically. CONCLUSIONS: Children have lower ability to spread SARS-CoV-2. The new A958D mutation is a potential reason for its long residence in the intestine.


Subject(s)
COVID-19 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Adult , COVID-19/virology , Child , China/epidemiology , Cohort Studies , Humans , Incidence , Infant , Mutation , SARS-CoV-2/genetics
3.
Front Immunol ; 12: 708184, 2021.
Article in English | MEDLINE | ID: covidwho-1346403

ABSTRACT

There is a worldwide pandemic of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; yet our understanding remains limited on the characteristic of antibodies, especially for dynamic long-term tracking. Sequential serum samples were collected up to 416 days post onset of symptoms (POS) from 102 patients who were hospitalized with coronavirus disease 2019 (COVID-19). Immunoglobulin (Ig)G, IgM, and IgA levels targeting SARS-CoV-2 spike 1 receptor-binding domain (S1-RBD), spike 2 extracellular domain (S2-ECD), and nucleocapsid protein (N) were quantified as well as neutralizing activity. We were pleasantly surprised to find that the antibody remained detective and effective for more than a year POS. We also found the varied reactions of different antibodies as time passed: N-IgA rose most rapidly in the early stage of infection, while S2-IgG was present at a high level in the long time of observation. This study described the long traceable antibody response of the COVID-19 and offered hints about targets to screen for postinfectious immunity and for vaccination development of SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/immunology , SARS-CoV-2/immunology , Aged , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Female , Follow-Up Studies , Hospitalization , Humans , Immunoglobulin Isotypes/blood , Immunoglobulin Isotypes/immunology , Kinetics , Male , Middle Aged , Models, Theoretical , Phosphoproteins/immunology , Protein Domains/immunology , SARS-CoV-2/isolation & purification , Seroconversion , Spike Glycoprotein, Coronavirus/immunology
4.
BMC Infect Dis ; 21(1): 357, 2021 Apr 16.
Article in English | MEDLINE | ID: covidwho-1315854

ABSTRACT

BACKGROUND: In 2020, a new coronavirus, SARS-CoV-2, quickly spread worldwide within a few months. Although coronaviruses typically infect the upper or lower respiratory tract, the virus RNA can be detected in plasma. The risk of transmitting coronavirus via transfusion of blood products remains. As more asymptomatic infections are identified in COVID-19 cases, blood safety has become particularly important. Methylene blue (MB) photochemical technology has been proven to inactivate lipid-enveloped viruses with high efficiency and safety. The present study aimed to investigate the SARS-CoV-2 inactivation effects of MB in plasma. METHODS: The SARS-CoV-2 virus strain was isolated from Zhejiang University. The live virus was harvested from cultured VERO-E6 cells, and mixed with MB in plasma. The MB final concentrations were 0, 1, 2, and 4 µM. The "BX-1 AIDS treatment instrument" was used at room temperature, the illumination adjusted to 55,000 ± 0.5 million Lux, and the plasma was irradiated for 0, 2, 5, 10, 20, and 40 mins using light at a single wavelength of 630 nm. Virus load changes were measured using quantitative reverse transcription- PCR. RESULTS: BX-1 could effectively eliminate SARS-CoV-2 within 2 mins in plasma, and the virus titer declined to 4.5 log10 TCID50 (median tissue culture infectious dose)/mL. CONCLUSION: BX-1 is based on MB photochemical technology, which was designed to inactivate HIV-1 virus in plasma. It was proven to be safe and reliable in clinical trials of HIV treatment. In this study, we showed that BX-1 could also be applied to inactivate SARS-CoV-2. During the current outbreak, this technique it has great potential for ensuring the safety of blood transfusions, for plasma transfusion therapy in recovering patients, and for preparing inactivated vaccines.


Subject(s)
Blood Safety , COVID-19/prevention & control , COVID-19/therapy , Methylene Blue/pharmacology , SARS-CoV-2/drug effects , Virus Inactivation , Animals , Blood Transfusion , Chlorocebus aethiops , Humans , Immunization, Passive , Plasma/virology , RNA, Viral , Vero Cells
6.
J Proteome Res ; 20(7): 3463-3474, 2021 07 02.
Article in English | MEDLINE | ID: covidwho-1253876

ABSTRACT

The COVID-19 pandemic has become a worldwide health crisis. So far, most studies have focused on the epidemiology and pathogenesis of this infectious disease. Little attention has been given to the disease sequelae in patients recovering from COVID-19, and nothing is known about the mechanisms underlying these sequelae. Herein, we profiled the serum proteome of a cohort of COVID-19 patients in the disease onset and recovery stages. Based on the close integration of our proteomic analysis with clinical data, we propose that COVID-19 is associated with prolonged disorders in cholesterol metabolism and myocardium, even in the recovery stage. We identify potential biomarkers for these disorders. Moreover, severely affected patients presented more serious disturbances in these pathways. Our findings potentially support clinical decision-making to improve the prognosis and treatment of patients.


Subject(s)
COVID-19 , Proteomics , Cholesterol , Humans , Myocardium , Pandemics , Proteome , SARS-CoV-2
7.
BMC Infect Dis ; 21(1): 357, 2021 Apr 16.
Article in English | MEDLINE | ID: covidwho-1190056

ABSTRACT

BACKGROUND: In 2020, a new coronavirus, SARS-CoV-2, quickly spread worldwide within a few months. Although coronaviruses typically infect the upper or lower respiratory tract, the virus RNA can be detected in plasma. The risk of transmitting coronavirus via transfusion of blood products remains. As more asymptomatic infections are identified in COVID-19 cases, blood safety has become particularly important. Methylene blue (MB) photochemical technology has been proven to inactivate lipid-enveloped viruses with high efficiency and safety. The present study aimed to investigate the SARS-CoV-2 inactivation effects of MB in plasma. METHODS: The SARS-CoV-2 virus strain was isolated from Zhejiang University. The live virus was harvested from cultured VERO-E6 cells, and mixed with MB in plasma. The MB final concentrations were 0, 1, 2, and 4 µM. The "BX-1 AIDS treatment instrument" was used at room temperature, the illumination adjusted to 55,000 ± 0.5 million Lux, and the plasma was irradiated for 0, 2, 5, 10, 20, and 40 mins using light at a single wavelength of 630 nm. Virus load changes were measured using quantitative reverse transcription- PCR. RESULTS: BX-1 could effectively eliminate SARS-CoV-2 within 2 mins in plasma, and the virus titer declined to 4.5 log10 TCID50 (median tissue culture infectious dose)/mL. CONCLUSION: BX-1 is based on MB photochemical technology, which was designed to inactivate HIV-1 virus in plasma. It was proven to be safe and reliable in clinical trials of HIV treatment. In this study, we showed that BX-1 could also be applied to inactivate SARS-CoV-2. During the current outbreak, this technique it has great potential for ensuring the safety of blood transfusions, for plasma transfusion therapy in recovering patients, and for preparing inactivated vaccines.


Subject(s)
Blood Safety , COVID-19/prevention & control , COVID-19/therapy , Methylene Blue/pharmacology , SARS-CoV-2/drug effects , Virus Inactivation , Animals , Blood Transfusion , Chlorocebus aethiops , Humans , Immunization, Passive , Plasma/virology , RNA, Viral , Vero Cells
8.
Cell Res ; 31(1): 25-36, 2021 01.
Article in English | MEDLINE | ID: covidwho-952976

ABSTRACT

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 , Epitopes/immunology , SARS-CoV-2/immunology , Single-Chain Antibodies/immunology , Animals , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , COVID-19/immunology , COVID-19/prevention & control , Chlorocebus aethiops , Disease Models, Animal , Humans , Single-Chain Antibodies/pharmacology , Vero Cells
9.
Cell Discov ; 6(1): 76, 2020 Oct 29.
Article in English | MEDLINE | ID: covidwho-904771

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally with more than 33 million patients diagnosed, taking more than a million lives. Abundant mutations were observed but the functional consequences of these mutations are largely unknown. We report the mutation spectrum, replication dynamics, and infectivity of 11 patient-derived viral isolates in diverse cell lines, including the human lung cancer cell line Calu-3. We observed 46 mutations, including 9 different mutations in the spike gene. Importantly, these viral isolates show significant and consistent variations in replication dynamics and infectivity in tested cell lines, up to a 1500-fold difference in viral titers at 24 h after infecting Calu-3 cells. Moreover, we show that the variations in viral titers among viral isolates are positively correlated with blood clotting function but inversely correlated with the amount of red blood cell and hemoglobin in patients. Therefore, we provide direct evidence that naturally occurring mutations in SARS-CoV-2 can substantially change its replication dynamics and infectivity in diverse human cell lines, with clinical implications in vivo.

10.
Cell ; 183(3): 730-738.e13, 2020 10 29.
Article in English | MEDLINE | ID: covidwho-746087

ABSTRACT

SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryoelectron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were determined to average resolutions of 8.7-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diameter lumen.


Subject(s)
Betacoronavirus/physiology , Betacoronavirus/ultrastructure , Virus Assembly , Animals , Chlorocebus aethiops , Cryoelectron Microscopy , Humans , Mass Spectrometry , Models, Molecular , Protein Conformation , SARS-CoV-2 , Vero Cells , Viral Proteins/chemistry , Viral Proteins/ultrastructure , Virus Cultivation
11.
Emerg Microbes Infect ; 9(1): 1474-1488, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-599992

ABSTRACT

The mutations in the SARS-CoV-2 virus genome during COVID-19 dissemination are unclear. In 788 COVID-19 patients from Zhejiang province, we observed decreased rate of severe/critical cases compared with patients in Wuhan. For mechanisms exploration, we isolated one strain of SARS-CoV-2 (ZJ01) from a mild COVID-19 patient. Thirty-five specific gene mutations were identified. Phylogenetic and relative synonymous codon usage analysis suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 global virus strains based on the base (C or T) at positions 8824 and 28247 while ZJ01 has T at both sites. The prediction of the Furin cleavage site (FCS) and sequence alignment indicated that the FCS may be an important site of coronavirus evolution. ZJ01 mutations identified near the FCS (F1-2) caused changes in the structure and electrostatic distribution of the S surface protein, further affecting the binding capacity of Furin. Single-cell sequencing and ACE2-Furin co-expression results confirmed that the Furin expression was especially higher in glands, liver, kidneys, and colon. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 caused mild flu-like symptoms, further showing its potential in differentiating into mild COVID-19 subtypes.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Furin/metabolism , Pneumonia, Viral/virology , Adult , Betacoronavirus/genetics , COVID-19 , China/epidemiology , Codon , Coronavirus Infections/complications , Coronavirus Infections/epidemiology , Disease Progression , Evolution, Molecular , Female , Humans , Male , Middle Aged , Mutation , Pandemics , Phylogeny , Pneumonia, Viral/complications , Pneumonia, Viral/epidemiology , Retrospective Studies , SARS-CoV-2 , Sequence Analysis, RNA
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