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1.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-327732

ABSTRACT

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity. One sentence summary A human monoclonal antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

2.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-310380

ABSTRACT

Control of the ongoing SARS-CoV-2 pandemic is endangered by the emergence of viral variants with increased transmission efficiency, resistance to marketed therapeutic antibodies and reduced sensitivity to vaccine-induced immunity. Here, we screened B cells from COVID-19 donors and identified P5C3, a highly potent and broadly neutralizing monoclonal antibody with picomolar neutralizing activity against all SARS-CoV-2 variants of concern (VOC) identified to date. Structural characterization of P5C3 Fab in complex with the Spike demonstrates a neutralizing activity defined by a large buried surface area, highly overlapping with the receptor-binding domain (RBD) surface necessary for ACE2 interaction. We further demonstrate that P5C3 showed complete prophylactic protection in the SARS-CoV-2 infected hamster challenge model. These results indicate that P5C3 opens exciting perspectives either as a prophylactic agent in immunocompromised individuals with poor response to vaccination or as combination therapy in SARS-CoV-2-infected individuals.Funding: This CARE project has received funding from the Innovative MedicinesInitiative 2 Joint Undertaking (JU) under grant agreement No 101005077. The JU receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA and BILL & MELINDA GATES FOUNDATION, GLOBAL HEALTH DRUG DISCOVERYINSTITUTE, UNIVERSITY OF DUNDEE. Furthermore, funding was also provided through the Lausanne University Hospital, through the Swiss Vaccine Research Institute to G.P., and through the EPFL COVID fund to D.T.Conflict of Interest: None to declare. Ethical Approval: Study design and use of subject samples were approved by the Institutional Review Board of the Lausanne University Hospital and the ‘Commission d’éthique du Canton de Vaud’ (CER-VD).

3.
NPJ Vaccines ; 6(1): 122, 2021 Oct 20.
Article in English | MEDLINE | ID: covidwho-1475297

ABSTRACT

Early in the SARS-CoV-2 pandemic concerns were raised regarding infection of new animal hosts and the effect on viral epidemiology. Infection of other animals could be detrimental by causing clinical disease, allowing further mutations, and bares the risk for the establishment of a non-human reservoir. Cats were the first reported animals susceptible to natural and experimental infection with SARS-CoV-2. Given the concerns these findings raised, and the close contact between humans and cats, we aimed to develop a vaccine candidate that could reduce SARS-CoV-2 infection and in addition to prevent spread among cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus, known to be safe and efficacious in a variety of animal species, could induce neutralizing antibody responses in guinea pigs and cats. The design of the SARS-CoV-2 spike immunogen was critical in developing a strong neutralizing antibody response. Vaccination of cats was able to induce high neutralizing antibody responses, effective also against the SARS-CoV-2 B.1.1.7 variant. Interestingly, in contrast to control animals, the infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after SARS-CoV-2 challenge. Correspondingly, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit the virus. The results show that the RP vaccine induces protective immunity preventing SARS-CoV-2 infection and transmission. These data suggest that this RP vaccine could be a multi-species vaccine useful to prevent infection and spread to and between animals should that approach be required.

4.
Nat Commun ; 12(1): 5621, 2021 09 23.
Article in English | MEDLINE | ID: covidwho-1437680

ABSTRACT

Although serological studies have shown that antibodies against SARS-CoV-2 play an important role in protection against (re)infection, the dynamics of mucosal antibodies during primary infection and their potential impact on viral load and the resolution of disease symptoms remain unclear. During the first pandemic wave, we assessed the longitudinal nasal antibody response in index cases with mild COVID-19 and their household contacts. Nasal and serum antibody responses were analysed for up to nine months. Higher nasal receptor binding domain and spike protein-specific antibody levels at study inclusion were associated with lower viral load. Older age was correlated with more frequent COVID-19 related symptoms. Receptor binding domain and spike protein-specific mucosal antibodies were associated with the resolution of systemic, but not respiratory symptoms. Finally, receptor binding domain and spike protein-specific mucosal antibodies remained elevated up to nine months after symptom onset.


Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , COVID-19/diagnosis , Nasal Mucosa/metabolism , SARS-CoV-2/immunology , Adolescent , Adult , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/statistics & numerical data , Child , Humans , Immunity, Mucosal , Longitudinal Studies , Male , Middle Aged , Nasal Mucosa/immunology , Nasal Mucosa/virology , Severity of Illness Index , Viral Load , Young Adult
5.
Cell Rep ; 37(2): 109814, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1433045

ABSTRACT

Control of the ongoing SARS-CoV-2 pandemic is endangered by the emergence of viral variants with increased transmission efficiency, resistance to marketed therapeutic antibodies, and reduced sensitivity to vaccine-induced immunity. Here, we screen B cells from COVID-19 donors and identify P5C3, a highly potent and broadly neutralizing monoclonal antibody with picomolar neutralizing activity against all SARS-CoV-2 variants of concern (VOCs) identified to date. Structural characterization of P5C3 Fab in complex with the spike demonstrates a neutralizing activity defined by a large buried surface area, highly overlapping with the receptor-binding domain (RBD) surface necessary for ACE2 interaction. We further demonstrate that P5C3 shows complete prophylactic protection in the SARS-CoV-2-infected hamster challenge model. These results indicate that P5C3 opens exciting perspectives either as a prophylactic agent in immunocompromised individuals with poor response to vaccination or as combination therapy in SARS-CoV-2-infected individuals.


Subject(s)
Broadly Neutralizing Antibodies/therapeutic use , COVID-19/drug therapy , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , Cell Line , Cricetinae , Disease Models, Animal , Epitopes/immunology , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Neutralization Tests , Protein Binding/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/ultrastructure , Structure-Activity Relationship , Vaccination
6.
Nat Commun ; 12(1): 4586, 2021 07 28.
Article in English | MEDLINE | ID: covidwho-1387355

ABSTRACT

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoassay/standards , Luminescent Measurements/standards , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/instrumentation , Calibration , GTP-Binding Proteins/chemistry , Genes, Reporter , Humans , Immunoconjugates/chemistry , Limit of Detection , Luciferases/genetics , Luciferases/metabolism , Point-of-Care Testing , SARS-CoV-2/genetics
7.
Nat Commun ; 11(1): 2511, 2020 05 14.
Article in English | MEDLINE | ID: covidwho-1387317

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

8.
J Infect Dis ; 223(12): 2020-2028, 2021 06 15.
Article in English | MEDLINE | ID: covidwho-1246725

ABSTRACT

Effective clinical intervention strategies for coronavirus disease 2019 (COVID-19) are urgently needed. Although several clinical trials have evaluated use of convalescent plasma containing virus-neutralizing antibodies, levels of neutralizing antibodies are usually not assessed and the effectiveness has not been proven. We show that hamsters treated prophylactically with a 1:2560 titer of human convalescent plasma or a 1:5260 titer of monoclonal antibody were protected against weight loss, had a significant reduction of virus replication in the lungs, and showed reduced pneumonia. Interestingly, this protective effect was lost with a titer of 1:320 of convalescent plasma. These data highlight the importance of screening plasma donors for high levels of neutralizing antibodies. Our data show that prophylactic administration of high levels of neutralizing antibody, either monoclonal or from convalescent plasma, prevent severe SARS-CoV-2 pneumonia in a hamster model, and could be used as an alternative or complementary to other antiviral treatments for COVID-19.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , COVID-19/therapy , Lung/pathology , SARS-CoV-2/immunology , Virus Replication/drug effects , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Neutralizing/administration & dosage , COVID-19/immunology , Cricetinae , Disease Models, Animal , Humans , Immunization, Passive , Lung/drug effects , Virus Shedding/drug effects , Weight Loss/drug effects
9.
Clin Transl Immunology ; 10(5): e1285, 2021.
Article in English | MEDLINE | ID: covidwho-1233184

ABSTRACT

OBJECTIVES: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms. METHODS: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling. RESULTS: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay. CONCLUSION: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2.

10.
Nat Struct Mol Biol ; 28(6): 478-486, 2021 06.
Article in English | MEDLINE | ID: covidwho-1226434

ABSTRACT

Three highly pathogenic ß-coronaviruses have crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. To evaluate the possibility of identifying antibodies with broad neutralizing activity, we isolated a monoclonal antibody, termed B6, that cross-reacts with eight ß-coronavirus spike glycoproteins, including all five human-infecting ß-coronaviruses. B6 broadly neutralizes entry of pseudotyped viruses from lineages A and C, but not from lineage B, and the latter includes SARS-CoV and SARS-CoV-2. Cryo-EM, X-ray crystallography and membrane fusion assays reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery. The data indicate that antibody binding sterically interferes with the spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with ß-coronaviruses from three lineages, along with a proof of concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-ß-coronavirus vaccine.


Subject(s)
Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Betacoronavirus/immunology , Coronavirus Infections/immunology , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Coronavirus Infections/prevention & control , Cross Reactions , Epitopes , Female , HEK293 Cells , Humans , Mice , Protein Binding , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
11.
Sci Adv ; 7(23)2021 06.
Article in English | MEDLINE | ID: covidwho-1219234

ABSTRACT

The emergence of SARS-CoV-2 antibody escape mutations highlights the urgent need for broadly neutralizing therapeutics. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralizing SARS-CoV-2 and SARS-CoV and protecting against the associated respiratory disease in an animal model. Here, we report cryo-EM structures of both trimeric spike ectodomains in complex with the 47D11 Fab. 47D11 binds to the closed receptor-binding domain, distal to the ACE2 binding site. The CDRL3 stabilizes the N343 glycan in an upright conformation, exposing a mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. 47D11 stabilizes a partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies targeting the exposed receptor-binding motif. Together, these results reveal a cross-protective epitope on the SARS-CoV-2 spike and provide a structural roadmap for the development of 47D11 as a prophylactic or postexposure therapy for COVID-19.


Subject(s)
Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , SARS Virus , SARS-CoV-2 , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Humans , SARS Virus/chemistry , SARS Virus/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Structure-Activity Relationship
12.
Emerg Infect Dis ; 27(5): 1362-1370, 2021 05.
Article in English | MEDLINE | ID: covidwho-1202205

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many animal species, including minks, cats, and dogs. To gain insights into SARS-CoV-2 infections in cats and dogs, we developed and validated a set of serologic assays, including ELISA and virus neutralization. Evaluation of samples from animals before they acquired coronavirus disease and samples from cats roaming SARS-CoV-2-positive mink farms confirmed the suitability of these assays for specific antibody detection. Furthermore, our findings exclude SARS-CoV-2 nucleocapsid protein as an antigen for serologic screening of cat and dog samples. We analyzed 500 serum samples from domestic cats and dogs in the Netherlands during April-May 2020. We showed 0.4% of cats and 0.2% of dogs were seropositive. Although seroprevalence in cats and dogs that had unknown SARS-CoV-2 exposure was low during the first coronavirus disease wave, our data stress the need for development of continuous serosurveillance for SARS-CoV-2 in these 2 animal species.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Cats , Dogs , Humans , Mink , Netherlands/epidemiology , Seroepidemiologic Studies
13.
Nat Commun ; 12(1): 1715, 2021 03 17.
Article in English | MEDLINE | ID: covidwho-1139739

ABSTRACT

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.


Subject(s)
Antibodies, Monoclonal, Humanized/immunology , Betacoronavirus/immunology , Epitopes/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/immunology , Betacoronavirus/classification , Camelus , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cross Reactions , Epitopes/chemistry , Epitopes/genetics , Humans , Mice , Protein Conformation , Protein Subunits , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
14.
J Virol ; 95(3)2021 01 13.
Article in English | MEDLINE | ID: covidwho-1028544

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses to the spike (S) protein monomer, S protein native trimeric form, or the nucleocapsid (N) proteins were evaluated in cohorts of individuals with acute infection (n = 93) and in individuals enrolled in a postinfection seroprevalence population study (n = 578) in Switzerland. Commercial assays specific for the S1 monomer, for the N protein, or within a newly developed Luminex assay using the S protein trimer were found to be equally sensitive in antibody detection in the acute-infection-phase samples. Interestingly, compared to anti-S antibody responses, those against the N protein appear to wane in the postinfection cohort. Seroprevalence in a "positive patient contacts" group (n = 177) was underestimated by N protein assays by 10.9 to 32.2%, while the "randomly selected" general population group (n = 311) was reduced by up to 45% relative to the S protein assays. The overall reduction in seroprevalence targeting only anti-N antibodies for the total cohort ranged from 9.4 to 31%. Of note, the use of the S protein in its native trimer form was significantly more sensitive compared to monomeric S proteins. These results indicate that the assessment of anti-S IgG antibody responses against the native trimeric S protein should be implemented to estimate SARS-CoV-2 infections in population-based seroprevalence studies.IMPORTANCE In the present study, we have determined SARS-CoV-2-specific antibody responses in sera of acute and postinfection phase subjects. Our results indicate that antibody responses against viral S and N proteins were equally sensitive in the acute phase of infection, but that responses against N appear to wane in the postinfection phase where those against the S protein persist over time. The most sensitive serological assay in both acute and postinfection phases used the native S protein trimer as the binding antigen, which has significantly greater conformational epitopes for antibody binding compared to the S1 monomer protein used in other assays. We believe these results are extremely important in order to generate correct estimates of SARS-CoV-2 infections in the general population. Furthermore, the assessment of antibody responses against the trimeric S protein will be critical to evaluate the durability of the antibody response and for the characterization of a vaccine-induced antibody response.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , COVID-19/blood , COVID-19/epidemiology , Female , Humans , Immunoassay , Immunoglobulin A/blood , Immunoglobulin G/blood , Male , Phosphoproteins/immunology , Protein Multimerization , Sensitivity and Specificity , Seroepidemiologic Studies , Spike Glycoprotein, Coronavirus/chemistry , Switzerland/epidemiology , Time Factors
15.
Sci Rep ; 10(1): 21447, 2020 12 08.
Article in English | MEDLINE | ID: covidwho-969123

ABSTRACT

Currently, infections with SARS-Coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, are responsible for substantial morbidity and mortality worldwide. Older adults subjects > 60 years of age account for > 95% of the over one million fatal cases reported to date. It is unclear why in this age group SARS-CoV-2 infection causes more severe disease than in young adults. We hypothesized that differences in SARS-CoV-2 cross-reactive cellular immunity induced after infection with human coronaviruses (HCoVs), like OC43 and NL63, were at the basis of the differential mortality (and morbidity) observed after SARS-CoV-2 infection, because a small proportion of HCoV-specific T cells cross-react with SARS-CoV-2. Our data demonstrate that pre-existing T cell immunity induced by circulating human alpha- and beta-HCoVs is present in young adult individuals, but virtually absent in older adult subjects. Consequently, the frequency of cross-reactive T cells directed to the novel pandemic SARS-CoV-2 was minimal in most older adults. To the best of our knowledge, this is the first time that the presence of cross-reactive T cells to SARS-CoV-2 is compared in young and older adults. Our findings provide at least a partial explanation for the more severe clinical outcome of SARS-CoV-2 infection observed in the elderly. Moreover, this information could help to design efficacious vaccines for this age group, aiming at the induction of cell-mediated immunity.


Subject(s)
Antibodies, Viral/immunology , Coronavirus NL63, Human/immunology , Coronavirus OC43, Human/immunology , SARS-CoV-2/immunology , T-Lymphocytes/immunology , Adult , Aged , COVID-19/immunology , COVID-19/pathology , Cross Reactions/immunology , Humans , Immunity, Cellular/immunology , Middle Aged , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology , Young Adult
16.
J Immunol ; 205(12): 3491-3499, 2020 12 15.
Article in English | MEDLINE | ID: covidwho-895432

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections often cause only mild disease that may evoke relatively low Ab titers compared with patients admitted to hospitals. Generally, total Ab bridging assays combine good sensitivity with high specificity. Therefore, we developed sensitive total Ab bridging assays for detection of SARS-CoV-2 Abs to the receptor-binding domain (RBD) and nucleocapsid protein in addition to conventional isotype-specific assays. Ab kinetics was assessed in PCR-confirmed, hospitalized coronavirus disease 2019 (COVID-19) patients (n = 41) and three populations of patients with COVID-19 symptoms not requiring hospital admission: PCR-confirmed convalescent plasmapheresis donors (n = 182), PCR-confirmed hospital care workers (n = 47), and a group of longitudinally sampled symptomatic individuals highly suspect of COVID-19 (n = 14). In nonhospitalized patients, the Ab response to RBD is weaker but follows similar kinetics, as has been observed in hospitalized patients. Across populations, the RBD bridging assay identified most patients correctly as seropositive. In 11/14 of the COVID-19-suspect cases, seroconversion in the RBD bridging assay could be demonstrated before day 12; nucleocapsid protein Abs emerged less consistently. Furthermore, we demonstrated the feasibility of finger-prick sampling for Ab detection against SARS-CoV-2 using these assays. In conclusion, the developed bridging assays reliably detect SARS-CoV-2 Abs in hospitalized and nonhospitalized patients and are therefore well suited to conduct seroprevalence studies.


Subject(s)
Antibodies, Viral/immunology , Antibody Formation , COVID-19/immunology , Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Adult , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing , COVID-19 Serological Testing , Convalescence , Female , Humans , Immunologic Tests , Male , Middle Aged
17.
Proc Natl Acad Sci U S A ; 117(41): 25759-25770, 2020 10 13.
Article in English | MEDLINE | ID: covidwho-807358

ABSTRACT

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.


Subject(s)
Coronavirus/physiology , Hemagglutinins, Viral/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Fusion Proteins/genetics , Virion/metabolism , Animals , Biological Evolution , Cell Line , Coronavirus/genetics , Coronavirus/metabolism , Coronavirus Infections/virology , Coronavirus OC43, Human/genetics , Coronavirus OC43, Human/metabolism , Coronavirus OC43, Human/physiology , Coronavirus, Bovine/genetics , Coronavirus, Bovine/metabolism , Coronavirus, Bovine/physiology , Hemagglutinins, Viral/chemistry , Hemagglutinins, Viral/metabolism , Humans , Lectins/genetics , Lectins/metabolism , Mice , Mutation , Protein Binding , Protein Domains , Receptors, Virus/metabolism , Selection, Genetic , Sialic Acids/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/metabolism , Virion/genetics , Virus Attachment , Virus Release
18.
Emerg Microbes Infect ; 9(1): 1965-1973, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-725731

ABSTRACT

Serology is a crucial part of the public health response to the ongoing SARS-CoV-2 pandemic. Here, we describe the development, validation and clinical evaluation of a protein micro-array as a quantitative multiplex immunoassay that can identify S and N-directed SARS-CoV-2 IgG antibodies with high specificity and sensitivity and distinguish them from all currently circulating human coronaviruses. The method specificity was 100% for SARS-CoV-2 S1 and 96% for N antigen based on extensive syndromic (n=230 cases) and population panel (n=94) testing that also confirmed the high prevalence of seasonal human coronaviruses. To assess its potential role for both SARS-CoV-2 patient diagnostics and population studies, we evaluated a large heterogeneous COVID-19 cohort (n=330) and found an overall sensitivity of 89% (≥ 21 days post onset symptoms (dps)), ranging from 86% to 96% depending on severity of disease. For a subset of these patients longitudinal samples were provided up to 56 dps. Mild cases showed absent or delayed, and lower SARS-CoV-2 antibody responses. Overall, we present the development and extensive clinical validation of a multiplex coronavirus serological assay for syndromic testing, to answer research questions regarding to antibody responses, to support SARS-CoV-2 diagnostics and to evaluate epidemiological developments efficiently and with high-throughput.


Subject(s)
Antibodies, Viral/blood , Betacoronavirus/immunology , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Nucleocapsid Proteins/blood , Pneumonia, Viral/diagnosis , Spike Glycoprotein, Coronavirus/blood , Aged , Antigens, Viral/blood , Antigens, Viral/immunology , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/standards , Coronavirus Infections/immunology , Coronavirus Infections/mortality , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Female , Humans , Longitudinal Studies , Male , Middle Aged , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Neutralization Tests , Nucleocapsid Proteins/immunology , Pandemics , Phosphoproteins , Pneumonia, Viral/immunology , Pneumonia, Viral/mortality , Pneumonia, Viral/virology , Protein Array Analysis , SARS Virus/immunology , SARS Virus/pathogenicity , SARS-CoV-2 , Sensitivity and Specificity , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology
19.
Emerg Infect Dis ; 26(7):1478-1488, 2020.
Article in English | MEDLINE | ID: covidwho-655091

ABSTRACT

A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein-specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays;the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.

20.
Emerg Microbes Infect ; 9(1): 1080-1091, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-429885

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a "plug-and-display" SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as SARS-CoV-2.


Subject(s)
Antibody Formation , Antigen Presentation , Coronavirus Infections/immunology , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Affinity , Binding Sites , Coronavirus Infections/prevention & control , Enzyme-Linked Immunosorbent Assay , Female , Genetic Vectors , HEK293 Cells , Humans , Immunogenicity, Vaccine , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/physiology , Neutralization Tests , Protein Binding , Protein Domains , Rabbits , Spike Glycoprotein, Coronavirus/biosynthesis , Virus Replication
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