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1.
Euro Surveill ; 25(28)2020 07.
Article in English | MEDLINE | ID: covidwho-647504

ABSTRACT

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.


Subject(s)
Antibodies, Monoclonal/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , SARS Virus/immunology , Severe Acute Respiratory Syndrome/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Animals , Betacoronavirus/genetics , Blotting, Western , COS Cells , Chlorocebus aethiops , Conserved Sequence , Coronavirus Infections/genetics , Coronavirus Infections/virology , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Genome, Viral , Mice , Pandemics , Peptidyl-Dipeptidase A/immunology , Plasmids , Pneumonia, Viral/genetics , Recombinant Proteins/immunology , SARS Virus/genetics , Sequence Alignment , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/genetics , Transfection , Vero Cells , Virus Integration
2.
Methods Mol Biol ; 2203: 55-65, 2020.
Article in English | MEDLINE | ID: covidwho-761346

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiological agent of MERS, a severe respiratory disease first reported in the Middle East in 2012. Serological assays are used to diagnose MERS-CoV infection and to screen for serum antibodies in seroepidemiological studies. The conventional enzyme-linked immunosorbent assay (ELISA) is the preferred tool for detecting serum antibodies specific for pathogens; however, the utility of conventional ELISA with respect to detection of MERS-CoV antibodies is limited due to the number of false-positives caused by cross-reactivity of serum antibodies with antigens that are conserved among coronaviruses. The competitive ELISA (cELISA) uses a pathogen-specific monoclonal antibody (MAb) that competes with serum antibodies for binding to an antigen; therefore, it is used widely for serological surveillance of many pathogens. In this chapter, I describe detection of serum antibodies using cELISA based on MAbs specific for MERS-CoV.


Subject(s)
Antibodies, Viral/blood , Coronavirus Infections/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Middle East Respiratory Syndrome Coronavirus/immunology , Animals , Antibodies, Monoclonal , Cross Reactions , Enzyme-Linked Immunosorbent Assay/instrumentation , Humans , Mice, Inbred BALB C
3.
mSphere ; 5(5)2020 09 02.
Article in English | MEDLINE | ID: covidwho-742193

ABSTRACT

The novel coronavirus, SARS-coronavirus (CoV)-2 (SARS-CoV-2), has caused over 17 million infections in just a few months, with disease manifestations ranging from largely asymptomatic infection to critically severe disease. The remarkable spread and unpredictable disease outcomes continue to challenge management of this infection. Among the hypotheses to explain the heterogeneity of symptoms is the possibility that exposure to other coronaviruses (CoVs), or overall higher capability to develop immunity against respiratory pathogens, may influence the evolution of immunity to SARS-CoV-2. Thus, we profiled the immune response across multiple coronavirus receptor binding domains (RBDs), respiratory viruses, and SARS-CoV-2, to determine whether heterologous immunity to other CoV-RBDs or other infections influenced the evolution of the SARS-CoV-2 humoral immune response. Overall changes in subclass, isotype, and Fc-receptor binding were profiled broadly across a cohort of 43 individuals against different coronaviruses-RBDs of SARS-CoV-2 and the more common HKU1 and NL63 viruses. We found rapid functional evolution of responses to SARS-CoV-2 over time, along with broad but relatively more time-invariant responses to the more common CoVs. Moreover, there was little evidence of correlation between SARS-CoV-2 responses and HKU1, NL63, and respiratory infection (influenza and respiratory syncytial virus) responses. These findings suggest that common viral infections including common CoV immunity, targeting the receptor binding domain involved in viral infection, do not appear to influence the rapid functional evolution of SARS-CoV-2 immunity, and thus should not impact diagnostics or shape vaccine-induced immunity.IMPORTANCE A critical step to ending the spread of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the ability to detect, diagnose, and understand why some individuals develop mild and others develop severe disease. For example, defining the early evolutionary patterns of humoral immunity to SARS-CoV-2, and whether prevalent coronaviruses or other common infections influence the evolution of immunity, remains poorly understood but could inform diagnostic and vaccine development. Here, we deeply profiled the evolution of SARS-CoV-2 immunity, and how it is influenced by other coinfections. Our data suggest an early and rapid rise in functional humoral immunity in the first 2 weeks of infection across antigen-specific targets, which is negligibly influenced by cross-reactivity to additional common coronaviruses or common respiratory infections. These data suggest that preexisting receptor binding domain-specific immunity does not influence or bias the evolution of immunity to SARS-CoV-2 and should have negligible influence on shaping diagnostic or vaccine-induced immunity.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/immunology , Immunity, Heterologous , Immunity, Humoral , Pneumonia, Viral/immunology , Antibodies, Viral/blood , Biomarkers/blood , Clinical Laboratory Techniques , Coronavirus Infections/blood , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Cross Reactions , Humans , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology
4.
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 , 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 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
5.
Monoclon Antib Immunodiagn Immunother ; 39(4): 107-111, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-696013

ABSTRACT

In this hypothesis, we address the biological/immunological pathway leading to severe disease or death after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The underlying immune response is described with "original antigenic sin" (OAS) whereby previous infections influence the response to future virus encounters. We cite evidence for OAS-induced immunopathology in HIV-1 disease. We hypothesize that similar immune abnormalities can occur after infection with SARS-CoV-2. This hypothesis is supported by recent analysis of the antibodies in infected patients demonstrating serological and B cell abnormalities. The concept of symmetrical clonal regulation developed earlier for the immune network illustrates the pathway suggested by our hypothesis and may be helpful to develop strategies avoiding severe coronavirus disease 2019.


Subject(s)
Antibodies, Viral/immunology , B-Lymphocytes/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Immune Evasion/immunology , Pneumonia, Viral/immunology , Antibodies, Monoclonal/immunology , Coronavirus Infections/pathology , Cross Reactions/immunology , Cytokine Release Syndrome/immunology , HIV/immunology , HIV-1/immunology , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Immunologic Memory/immunology , Pandemics , Pneumonia, Viral/pathology
6.
Braz J Microbiol ; 51(3): 1117-1123, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-695574

ABSTRACT

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 107 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Fluorescent Dyes/economics , Organic Chemicals/economics , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/economics , Adolescent , Adult , Animals , Betacoronavirus/genetics , Child , Chlorocebus aethiops , Coronavirus Infections/economics , Cross Reactions , Humans , Middle Aged , Nasopharynx/virology , Oropharynx/virology , Pandemics/economics , Pneumonia, Viral/economics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Vero Cells , Young Adult
8.
EBioMedicine ; 58: 102890, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-666030

ABSTRACT

BACKGROUND: The novel coronavirus (SARS-CoV-2) shares approximately 80% whole genome sequence identity and 66% spike (S) protein identity with that of SARS-CoV. The cross-neutralization between these viruses is currently not well-defined. METHODS: Here, by using the live SARS-CoV-2 virus infection assay as well as HIV-1 based pseudotyped-virus carrying the spike (S) gene of the SARS-CoV-2 (ppSARS-2) and SARS-CoV (ppSARS), we examined whether infections with SARS-CoV and SARS-CoV-2 can induce cross-neutralizing antibodies. FINDINGS: We confirmed that SARS-CoV-2 infects cells via angiotensin converting enzyme 2 (ACE2), the functional receptor for SARS-CoV, and we also found that the recombinant receptor binding domain (RBD) of the S protein of SARS-CoV effectively inhibits ppSARS-2 entry in Huh7.5 cells. However, convalescent sera from SARS-CoV and SARS-CoV-2 patients showed high neutralizing activity only against the homologous virus, with no or limited cross-neutralization activity against the other pseudotyped virus. Similar results were also observed in vaccination studies in mice. INTERPRETATION: Our study demonstrates that although both SARS-CoV and SARS-CoV-2 use ACE2 as a cellular receptor, the neutralization epitopes are not shared by these two closely-related viruses, highlighting challenges towards developing a universal vaccine against SARS-CoV related viruses. FUNDING: This work was supported by the National Key Research and Development Program of China, the National Major Project for Control and Prevention of Infectious Disease in China, and the One Belt and One Road Major Project for infectious diseases.


Subject(s)
Antibodies, Viral/immunology , Betacoronavirus/immunology , Cross Reactions , SARS Virus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibody Specificity , Betacoronavirus/genetics , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Female , Humans , Mice , Mice, Inbred BALB C , SARS Virus/genetics , Sequence Homology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
10.
Nature ; 584(7821): 443-449, 2020 08.
Article in English | MEDLINE | ID: covidwho-647154

ABSTRACT

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/immunology , Pneumonia, Viral/prevention & control , Animals , Antibodies, Monoclonal/immunology , Betacoronavirus/chemistry , Binding, Competitive , Cell Line , Cross Reactions , Disease Models, Animal , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Female , Humans , Macaca mulatta , Male , Mice , Middle Aged , Neutralization Tests , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pre-Exposure Prophylaxis , SARS Virus/chemistry , SARS Virus/immunology , Severe Acute Respiratory Syndrome/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
11.
Rheumatol Int ; 40(10): 1539-1554, 2020 10.
Article in English | MEDLINE | ID: covidwho-646938

ABSTRACT

The coronavirus disease-2019 (COVID-19) pandemic is likely to pose new challenges to the rheumatology community in the near and distant future. Some of the challenges, like the severity of COVID-19 among patients on immunosuppressive agents, are predictable and are being evaluated with great care and effort across the globe. A few others, such as atypical manifestations of COVID-19 mimicking rheumatic musculoskeletal diseases (RMDs) are being reported. Like in many other viral infections, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can potentially lead to an array of rheumatological and autoimmune manifestations by molecular mimicry (cross-reacting epitope between the virus and the host), bystander killing (virus-specific CD8 + T cells migrating to the target tissues and exerting cytotoxicity), epitope spreading, viral persistence (polyclonal activation due to the constant presence of viral antigens driving immune-mediated injury) and formation of neutrophil extracellular traps. In addition, the myriad of antiviral drugs presently being tried in the treatment of COVID-19 can result in several rheumatic musculoskeletal adverse effects. In this review, we have addressed the possible spectrum and mechanisms of various autoimmune and rheumatic musculoskeletal manifestations that can be precipitated by COVID-19 infection, its therapy, and the preventive strategies to contain the infection.


Subject(s)
Autoimmune Diseases/physiopathology , Coronavirus Infections/physiopathology , Musculoskeletal Diseases/physiopathology , Pneumonia, Viral/physiopathology , Rheumatic Diseases/physiopathology , Antibodies, Antinuclear/immunology , Antibodies, Antiphospholipid/immunology , Antiviral Agents/adverse effects , Arthralgia/etiology , Arthralgia/immunology , Arthralgia/physiopathology , Autoimmune Diseases/etiology , Autoimmune Diseases/immunology , Betacoronavirus , Blood Coagulation Disorders/etiology , Blood Coagulation Disorders/immunology , Blood Coagulation Disorders/physiopathology , Coronavirus Infections/complications , Coronavirus Infections/drug therapy , Coronavirus Infections/immunology , Cross Reactions/immunology , Extracellular Traps/immunology , Fibrin Fibrinogen Degradation Products , Guillain-Barre Syndrome/etiology , Guillain-Barre Syndrome/immunology , Guillain-Barre Syndrome/physiopathology , Humans , Lupus Coagulation Inhibitor/immunology , Molecular Mimicry , Mucocutaneous Lymph Node Syndrome/etiology , Mucocutaneous Lymph Node Syndrome/immunology , Mucocutaneous Lymph Node Syndrome/physiopathology , Muscle Weakness/etiology , Muscle Weakness/immunology , Muscle Weakness/physiopathology , Musculoskeletal Diseases/etiology , Musculoskeletal Diseases/immunology , Myalgia/etiology , Myalgia/immunology , Myalgia/physiopathology , Myocarditis/etiology , Myocarditis/immunology , Myocarditis/physiopathology , Pandemics , Pneumonia, Viral/complications , Pneumonia, Viral/drug therapy , Pneumonia, Viral/immunology , Rheumatic Diseases/etiology , Rheumatic Diseases/immunology
12.
Am J Clin Pathol ; 154(4): 459-465, 2020 09 08.
Article in English | MEDLINE | ID: covidwho-646825

ABSTRACT

OBJECTIVES: Initial reports indicate adequate performance of some serology-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assays. However, additional studies are required to facilitate interpretation of results, including how antibody levels impact immunity and disease course. METHODS: A total of 967 subjects were tested for IgG antibodies reactive to SARS-CoV-2, including 172 suspected cases of SARS-CoV-2, 656 plasma samples from healthy donors, 49 sera from patients with rheumatic disease, and 90 specimens from individuals positive for polymerase chain reaction (PCR)-based respiratory viral panel. A subgroup of SARS-CoV-2 PCR-positive cases was tested for IgM antibodies by proteome array method. RESULTS: All specificity and cross-reactivity specimens were negative for SARS-CoV-2 IgG antibodies (0/795, 0%). Positive agreement of IgG with PCR was 83% of samples confirmed to be more than 14 days from symptom onset, with less than 100% sensitivity attributable to a case with severe immunosuppression. Virus-specific IgM was positive in a higher proportion of cases less than 3 days from symptom onset. No association was observed between mild and severe disease course with respect to IgG and IgM levels. CONCLUSIONS: The studied SARS-CoV-2 IgG assay had 100% specificity and no adverse cross-reactivity. Measures of IgG and IgM antibodies did not predict disease severity in our patient population.


Subject(s)
Antibodies, Viral/blood , Betacoronavirus/immunology , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Immunoglobulin G/blood , Pneumonia, Viral/diagnosis , Pneumonia, Viral/immunology , Severity of Illness Index , Antibody Formation , Biomarkers/blood , Case-Control Studies , Coronavirus Infections/blood , Cross Reactions , Cross-Sectional Studies , Humans , Immunoglobulin M/blood , Pandemics , Pneumonia, Viral/blood , Sensitivity and Specificity
14.
Front Immunol ; 11: 1120, 2020.
Article in English | MEDLINE | ID: covidwho-615480

ABSTRACT

Human coronavirus (HCoV) is one of the most common causes of respiratory tract infections throughout the world. Two phenomena observed so far in the development of the SARS-CoV-2 pandemic deserve further attention. First, the relative absence of clinical signs of infections in children, second, the early appearance of IgG in certain patients. From the point of view of immune system physiology, such an early rise of specific IgG is expected in secondary immune responses when memory to a cross-reactive antigen is present, usually from an earlier infection with a coronavirus. It is actually typical for the immune system to respond, to what it already knows, a phenomenon that has been observed in many infections with closely related viruses and has been termed "original antigenic sin." The question then arises whether such cross-reactive antibodies are protective or not against the new virus. The worst scenario would be when such cross-reactive memory antibodies to related coronaviruses would not only be non-protective but even enhance infection and the clinical course. Such a phenomenon of antibody dependent enhancement (ADE) has already been described in several viral infections. Thus, the development of IgG against SARS-CoV-2 in the course of COVID-19 might not be a simple sign of viral clearance and developing protection against the virus. On the contrary, due to cross-reaction to related coronavirus strains from earlier infections, in certain patients IgG might enhance clinical progression due to ADE. The patient's viral history of coronavirus infection might be crucial to the development of the current infection with SARS-CoV-2. Furthermore, it poses a note of caution when treating COVID-19 patients with convalescent sera.


Subject(s)
Antibodies, Viral/immunology , Antibody-Dependent Enhancement/immunology , Betacoronavirus/immunology , Cross Protection/immunology , Cross Reactions/immunology , Antibodies, Neutralizing/immunology , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Humans , Immunoglobulin G/immunology , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Spike Glycoprotein, Coronavirus/immunology
15.
Cell ; 182(4): 828-842.e16, 2020 08 20.
Article in English | MEDLINE | ID: covidwho-612079

ABSTRACT

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.


Subject(s)
Antibodies, Neutralizing/chemistry , Betacoronavirus/chemistry , Coronavirus Infections/immunology , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/chemistry , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , Betacoronavirus/immunology , Coronavirus Infections/blood , Coronavirus Infections/therapy , Cross Reactions , Cryoelectron Microscopy , Epitope Mapping , Epitopes , Humans , Immunization, Passive , Immunoglobulin Fab Fragments/blood , Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin Fab Fragments/ultrastructure , Immunoglobulin G/blood , Immunoglobulin G/isolation & purification , Immunoglobulin G/ultrastructure , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/immunology , Models, Molecular , Pandemics , Pneumonia, Viral/blood , SARS Virus/chemistry , SARS Virus/immunology , Spike Glycoprotein, Coronavirus/immunology
16.
Anal Chem ; 92(14): 9895-9900, 2020 07 21.
Article in English | MEDLINE | ID: covidwho-606379

ABSTRACT

The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The Kd values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, respectively. Simulated interaction modeling, along with competitive experiments, suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.


Subject(s)
Aptamers, Nucleotide/analysis , Betacoronavirus/chemistry , Spike Glycoprotein, Coronavirus/analysis , Algorithms , Animals , Betacoronavirus/genetics , Coronavirus Infections , Cross Reactions , DNA, Viral/chemistry , Humans , Machine Learning , Mice , Molecular Docking Simulation , Mutation , Pandemics , Peptidyl-Dipeptidase A/drug effects , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral , Sensitivity and Specificity
17.
J Clin Virol ; 129: 104510, 2020 08.
Article in English | MEDLINE | ID: covidwho-601933

ABSTRACT

The emergence of a new coronavirus in Wuhan China has triggered a global need for accurate diagnostic assays. Initially, mostly laboratory developed molecular tests were available but shortly thereafter different commercial assays started to appear and are still increasing in number. Although independent performance evaluations are ongoing, available data is still scarce. Here we provide a direct comparison of key performance characteristics of 13 commercial RT-PCR assays. Thirteen RT-PCR assays were selected based on the criteria that they can be used following generic RNA extraction protocols, on common PCR platforms and availability. Using a 10-fold and 2-fold dilution series of a quantified SARS-CoV-2 cell-cultured virus stock, performance was assessed compared to our in house validated assay. Specificity was tested by using RNA extracted from cultured common human coronaviruses. All RT-PCR kits included in this study exhibited PCR efficiencies > 90%, except for the Sentinel Diagnostics B E-gene RUO assay (80%). Analytical sensitivity varied between 3.3 RNA copies to 330 RNA copies. Only one assay cross reacted with another human coronavirus (MERS). This study provides a technical baseline of 13 different commercial PCR assays for SARS-CoV-2 detection that can be used by laboratories interested in purchasing any of these for further full clinical validation.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Cross Reactions , Humans , Pandemics , RNA, Viral/genetics , Sensitivity and Specificity
18.
Science ; 369(6504): 731-736, 2020 08 07.
Article in English | MEDLINE | ID: covidwho-599033

ABSTRACT

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Broadly Neutralizing Antibodies/immunology , SARS Virus/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibody Affinity , B-Lymphocyte Subsets/immunology , Binding Sites , Cross Reactions , Epitopes , Female , Humans , Immunologic Memory , Male , Middle Aged , Neutralization Tests , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Protein Domains , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Severe Acute Respiratory Syndrome/immunology , Somatic Hypermutation, Immunoglobulin , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Young Adult
19.
Emerg Microbes Infect ; 9(1): 1497-1505, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-595010

ABSTRACT

In response to the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, multiple diagnostic tests are required for acute disease diagnosis, contact tracing, monitoring asymptomatic infection rates and assessing herd immunity. While PCR remains the frontline test of choice in the acute diagnostic setting, serological tests are urgently needed. Unlike PCR tests which are highly specific, cross-reactivity is a major challenge for COVID-19 antibody tests considering there are six other coronaviruses known to infect humans. SARS-CoV is genetically related to SARS-CoV-2 sharing approximately 80% sequence identity and both belong to the species SARS related coronavirus in the genus Betacoronavirus of family Coronaviridae. We developed and compared the performance of four different serological tests to comprehensively assess the cross-reactivity between COVID-19 and SARS patient sera. There is significant cross-reactivity when N protein of either virus is used. The S1 or RBD regions from the spike (S) protein offers better specificity. Amongst the different platforms, capture ELISA performed best. We found that SARS survivors all have significant levels of antibodies remaining in their blood 17 years after infection. Anti-N antibodies waned more than anti-RBD antibodies, and the latter is known to play a more important role in providing protective immunity.


Subject(s)
Antibodies, Viral/immunology , Betacoronavirus/immunology , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , SARS Virus/immunology , Serologic Tests/methods , Severe Acute Respiratory Syndrome/diagnosis , Antibodies, Viral/blood , Betacoronavirus/isolation & purification , Cross Reactions , Diagnosis, Differential , Enzyme-Linked Immunosorbent Assay/methods , HEK293 Cells , Humans , Immunoprecipitation , Nucleocapsid Proteins/immunology , Pandemics , Protein Domains/immunology , SARS Virus/isolation & purification , Spike Glycoprotein, Coronavirus/immunology
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