scholarly journals Isolation of cross-reactive monoclonal antibodies against divergent human coronaviruses that delineate a conserved and vulnerable site on the spike protein

2020 ◽  
Author(s):  
Chunyan Wang ◽  
Rien van Haperen ◽  
Javier Gutiérrez-Álvarez ◽  
Wentao Li ◽  
Nisreen M.A. Okba ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Distinct Species ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Protective Antibodies ◽  
Therapeutic Measures ◽  
Neutralizing Potential ◽  
Immunoglobulin Repertoire ◽  
Human Coronaviruses ◽  
Virion Surface

AbstractThe coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry. As such, it is an attractive target for the development of protective antibodies and vaccines. Here we describe two human monoclonal antibodies, 1.6C7 and 28D9, that display a remarkable cross-reactivity against distinct species from three Betacoronavirus subgenera, capable of binding the spike proteins of SARS-CoV and SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both antibodies, derived from immunized transgenic mice carrying a human immunoglobulin repertoire, blocked MERS-CoV infection in cells, whereas 28D9 also showed weak cross-neutralizing potential against HCoV-OC43, SARS-CoV and SARS-CoV-2 in a neutralization-sensitive virus pseudotyping system, but not against authentic virus. Both cross-reactive monoclonal antibodies were found to target the stem helix in the spike protein S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle, that is antibody-accessible. We demonstrate that administration of these antibodies in mice protects from a lethal MERS-CoV challenge in both prophylactic and/or therapeutic models. Collectively, these antibodies delineate a conserved, immunogenic and vulnerabe site on the spike protein which spurs the development of broad-range diagnostic, preventive and therapeutic measures against coronaviruses.

scholarly journals A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chunyan Wang ◽  
Rien van Haperen ◽  
Javier Gutiérrez-Álvarez ◽  
Wentao Li ◽  
Nisreen M. A. Okba ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cross Reactivity ◽  
Cell Entry ◽  
Spike Protein ◽  
Structural Studies ◽  
Human Monoclonal Antibodies ◽  
Human Coronavirus ◽  
Helical Bundle ◽  
Protective Antibodies ◽  
Virion Surface

AbstractThe 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.

scholarly journals Serological profiles of pan-coronavirus-specific responses in COVID-19 patients using a multiplexed electro-chemiluminescence-based testing platform

2021 ◽  
Author(s):  
Sidhartha Chaudhury ◽  
Jack Hutter ◽  
Jessica S Bolton ◽  
Shilpa Hakre ◽  
Evelyn Mose ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Cross Reactivity ◽  
Multiplex Assay ◽  
Spike Protein ◽  
Contact Tracing ◽  
Epitope Specificity ◽  
Army Hospital ◽  
Protein Receptor ◽  
Healthcare Administrators ◽  
Human Coronaviruses

AbstractSerological assessment of SARS-CoV-2 specific responses are an essential tool for determining the prevalence of past SARS-CoV-2 infections in the population especially when testing occurs after symptoms have developed and limited contact tracing is in place. The goal of our study was to test a new 10-plex electro-chemiluminescence-based assay to measure IgM and IgG responses to the spike proteins from multiple human coronaviruses including SARS-CoV-2, assess the epitope specificity of the SARS-CoV-2 antibody response against full-length spike protein, receptor-binding domain and N-terminal domain of the spike protein, and the nucleocapsid protein. We carried out the assay on samples collected from three sample groups: subjects diagnosed with COVID-19 from the U.S. Army hospital at Camp Humphreys in Pyeongtaek, South Korea; healthcare administrators from the same hospital but with no reported diagnosis of COVID-19; and pre-pandemic samples. We found that the new CoV-specific multiplex assay was highly sensitive allowing plasma samples to be diluted 1:30,000 with a robust signal. The reactivity of IgG responses to SARS-CoV-2 nucleocapsid protein and IgM responses to SARS-CoV-2 spike protein could distinguish COVID-19 samples from non-COVID-19 and pre-pandemic samples. The data from the three sample groups also revealed a unique pattern of cross-reactivity between SARS-CoV-2 and SARS-CoV-1, MERS-CoV, and seasonal coronaviruses HKU1 and OC43. Our findings show that the CoV-2 IgM response is highly specific while the CoV-2 IgG response is more cross-reactive across a range of human CoVs and also showed that IgM and IgG responses show distinct patterns of epitope specificity. In summary, this multiplex assay was able to distinguish samples by COVID-19 status and characterize distinct trends in terms of cross-reactivity and fine-specificity in antibody responses, underscoring its potential value in diagnostic or serosurveillance efforts.

scholarly journals Systematic evaluation of SARS-CoV-2 spike protein derived peptides for diagnosis of COVID-19 patients

2020 ◽  
Author(s):  
Yang Li ◽  
Danyun Lai ◽  
Qing Lei ◽  
Zhaowei Xu ◽  
Hongyan Hou ◽  
...  
Keyword(s):  
Serological Test ◽  
Protein S ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Systematic Evaluation ◽  
Peptide Microarray ◽  
Serological Tests ◽  
S Protein ◽  
Asymptomatic Patients ◽  
Human Coronaviruses

Serological test plays an essential role in monitoring and combating COVID-19 pandemic. Recombinant spike protein (S protein), especially S1 protein is one of the major reagents for serological tests. However, the high cost in production of S protein, and the possible cross-reactivity with other human coronaviruses poses unneglectable challenges. Taking advantage of a peptide microarray of full spike protein coverage, we analyzed 2,434 sera from 858 COVID-19 patients, sera from 63 asymptomatic patients and 610 controls collected from multiple clinical centers. Based on the results of the peptide microarray, we identified several S protein derived 12-mer peptides that have high diagnosis performance. Particularly, for monitoring IgG response, one peptide (aa 1148-1159 or S2-78) has a comparable sensitivity (95.5%, 95% CI 93.7-96.9%) and specificity (96.7%, 95% CI 94.8-98.0%) to that of S1 protein for detection of both COVID-19 patients and asymptomatic infections. Furthermore, the performance of S2-78 IgG for diagnosis was successfully validated by ELISA with an independent sample cohort. By combining S2-78/ S1 with other peptides, a two-step strategy was proposed to ensure both the sensitivity and specificity of S protein based serological assay. The peptide/s identified in this study could be applied independently or in combination with S1 protein for accurate, affordable, and accessible COVID-19 diagnosis.

scholarly journals Cross-Reactivity, Epitope Mapping, and Potency of Monoclonal Antibodies to Class 5 Fimbrial Tip Adhesins of Enterotoxigenic Escherichia coli

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Yang Liu ◽  
Sakina Shahabudin ◽  
Sami Farid ◽  
Lanfong H. Lee ◽  
Annette L. McVeigh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Sequence Similarity ◽  
Bacterial Pathogen ◽  
Cross Reactivity ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Functional Activities ◽  
Colonization Factor ◽  
Protective Antibodies

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is a leading diarrheagenic bacterial pathogen among travelers and children in resource-limited regions. Adherence to host intestinal cells mediated by ETEC fimbriae is believed to be a critical first step in ETEC pathogenesis. These fimbriae are categorized into related classes based on sequence similarity, with members of the class 5 fimbrial family being the best characterized. The eight related members of the ETEC class 5 fimbrial family are subdivided into three subclasses (5a, 5b, and 5c) that share similar structural arrangements, including a fimbrial tip adhesin. However, sequence variability among the class 5 adhesins may hinder the generation of cross-protective antibodies. To better understand functional epitopes of the class 5 adhesins and their ability to induce intraclass antibody responses, we produced 28 antiadhesin monoclonal antibodies (MAbs) to representative adhesins CfaE, CsbD, and CotD, respectively. We determined the MAb cross-reactivities, localized the epitopes, and measured functional activities as potency in inhibition of hemagglutination induced by class 5 fimbria-bearing ETEC. The MAbs’ reactivities to a panel of class 5 adhesins in enzyme-linked immunosorbent assays (ELISAs) revealed several reactivity patterns, including individual adhesin specificity, intrasubclass specificity, intersubclass specificity, and class-wide cross-reactivity, suggesting that some conserved epitopes, including two conserved arginines, are shared by the class 5 adhesins. However, the cross-reactive MAbs had functional activities limited to strains expressing colonization factor antigen I (CFA/I), coli surface antigen 17 (CS17), or CS1, suggesting that the breadth of functional activities of the MAbs was more restricted than the repertoire of cross-reactivities measured by ELISA. The results imply that multivalent adhesin-based ETEC vaccines or prophylactics need more than one active component to reach broad protection.

scholarly journals Systematic evaluation of IgG responses to SARS-CoV-2 spike protein-derived peptides for monitoring COVID-19 patients

2021 ◽  
Author(s):  
Yang Li ◽  
Dan-yun Lai ◽  
Qing Lei ◽  
Zhao-wei Xu ◽  
Feng Wang ◽  
...  
Keyword(s):  
Diagnostic Performance ◽  
Protein Production ◽  
Protein S ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Systematic Evaluation ◽  
Serological Tests ◽  
S Protein ◽  
Asymptomatic Patients ◽  
Human Coronaviruses

AbstractSerological tests play an essential role in monitoring and combating the COVID-19 pandemic. Recombinant spike protein (S protein), especially the S1 protein, is one of the major reagents used for serological tests. However, the high cost of S protein production and possible cross-reactivity with other human coronaviruses pose unavoidable challenges. By taking advantage of a peptide microarray with full spike protein coverage, we analyzed 2,434 sera from 858 COVID-19 patients, 63 asymptomatic patients and 610 controls collected from multiple clinical centers. Based on the results, we identified several S protein-derived 12-mer peptides that have high diagnostic performance. In particular, for monitoring the IgG response, one peptide (aa 1148–1159 or S2–78) exhibited a sensitivity (95.5%, 95% CI 93.7–96.9%) and specificity (96.7%, 95% CI 94.8–98.0%) comparable to those of the S1 protein for the detection of both symptomatic and asymptomatic COVID-19 cases. Furthermore, the diagnostic performance of the S2–78 (aa 1148–1159) IgG was successfully validated by ELISA in an independent sample cohort. A panel of four peptides, S1–93 (aa 553–564), S1–97 (aa 577–588), S1–101 (aa 601–612) and S1–105 (aa 625–636), that likely will avoid potential cross-reactivity with sera from patients infected by other coronaviruses was constructed. The peptides identified in this study may be applied independently or in combination with the S1 protein for accurate, affordable, and accessible COVID-19 diagnosis.

scholarly journals Phage Display as a Strategy to Obtain Anti-flavivirus Monoclonal Antibodies

2020 ◽  
Author(s):  
Isaura Beatriz Borges Silva ◽  
Renato Kaylan Alves de Oliveira França ◽  
Jacyelly Medeiros Silva ◽  
Andrea Queiroz Maranhão ◽  
Carlos Roberto Prudencio
Keyword(s):  
Monoclonal Antibodies ◽  
Phage Display ◽  
Diagnostic Tests ◽  
Severe Disease ◽  
Cross Reactivity ◽  
Negative Consequences ◽  
Human Mabs ◽  
Neutralizing Potential ◽  
Serological Biomarkers ◽  
Great Complexity

Arbovirus of the Flaviviridae family represents an issue worldwide, particularly because it can lead to serious illness and death in some countries. There is still a great complexity in obtaining effective therapies and specific and sensitive diagnostic tests, due to the high antigenic similarity between them. This similarity may account for antibodies cross reactivity which has positive and negative consequences for the course of infectious diseases. Among dengue virus (DENV) serotype infections, the cross-reactivity can increase virus replication and the risk of a severe disease by a mechanism known as an antibody-dependent enhancement (ADE). The search for serological biomarkers through monoclonal antibodies (MAbs) that identify unique viral regions can assist in the differential detection, whereas the development of recombinant antibodies with a neutralizing potential can lead to the establishment of efficacious treatments. The Phage Display methodology emerged as one of the main alternatives for the selection of human MAbs with high affinity for a specific target. Therefore, this technology can be a faster alternative for the development of specific diagnostic platforms and efficient and safe treatments for flavivirus infections. In this context, we propose for this chapter a discussion about Phage Display as a strategy to obtain MAbs for DENV and other flaviviruses.

scholarly journals Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Chunhua Li ◽  
Wentao Li ◽  
Eduardo Lucio de Esesarte ◽  
Hongbo Guo ◽  
Paul van den Elzen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Porcine Epidemic Diarrhea Virus ◽  
Neutralizing Antibodies ◽  
Cell Attachment ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs, resulting in significant economic losses to the swine industry worldwide. Current vaccination approaches against this emerging coronavirus are only partially effective, though natural infection protects pigs against reinfection and provides lactogenic immunity to suckling piglets. The viral spike (S) glycoprotein, responsible for receptor binding and cell entry, is the major target for neutralizing antibodies. However, knowledge of antibody epitopes, their nature and location in the spike structure, and the mechanisms by which the antibodies interfere with infection is scarce. Here we describe the generation and characterization of 10 neutralizing and nonneutralizing mouse monoclonal antibodies raised against the S1 receptor binding subunit of the S protein. By expression of different S1 protein fragments, six antibody epitope classes distributed over the five structural domains of the S1 subunit were identified. Characterization of antibodies for cross-reactivity and cross-neutralization revealed antigenic differences among PEDV strains. The epitopes of potent neutralizing antibodies segregated into two epitope classes and mapped within the N-terminal sialic acid binding domain and in the more C-terminal receptor binding domain. Antibody neutralization escape mutants displayed single amino acid substitutions that impaired antibody binding and neutralization and defined the locations of the epitopes. Our observations picture the antibody epitope landscape of the PEDV S1 subunit and reveal that its cell attachment domains are key targets of neutralizing antibodies. IMPORTANCE Porcine epidemic diarrhea virus (PEDV), an emerging porcine coronavirus, causes an economically important enteric disease in pigs. Effective PEDV vaccines for disease control are currently lacking. The spike (S) glycoprotein on the virion surface is the key player in virus cell entry and, therefore, the main target of neutralizing antibodies. To understand the antigenic landscape of the PEDV spike protein, we developed monoclonal antibodies against the spike protein's S1 receptor binding region and characterized their epitopes, neutralizing activity, and cross-reactivity toward multiple PEDV strains. Epitopes of antibodies segregated into six epitope classes dispersed over the multidomain S1 structure. Monoclonal antibodies revealed antigenic variability in B-cell epitopes between PEDV strains. The epitopes of neutralizing antibodies mapped to two distinct domains in S1 that are involved in binding to carbohydrate and proteinaceous cell surface molecules, respectively, indicating the importance of these cell attachment sites on the PEDV spike protein in eliciting a protective humoral immune response.

scholarly journals Serological profiles of pan-coronavirus-specific responses in COVID-19 patients using a multiplexed electro-chemiluminescence-based testing platform

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252628
Author(s):  
Sidhartha Chaudhury ◽  
Jack Hutter ◽  
Jessica S. Bolton ◽  
Shilpa Hakre ◽  
Evelyn Mose ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Cross Reactivity ◽  
Multiplex Assay ◽  
Spike Protein ◽  
Contact Tracing ◽  
Epitope Specificity ◽  
Army Hospital ◽  
Protein Receptor ◽  
Healthcare Administrators ◽  
Human Coronaviruses

Serological assessment of SARS-CoV-2 specific responses are an essential tool for determining the prevalence of past SARS-CoV-2 infections in the population especially when testing occurs after symptoms have developed and limited contact tracing is in place. The goal of our study was to test a new 10-plex electro-chemiluminescence-based assay to measure IgM and IgG responses to the spike proteins from multiple human coronaviruses including SARS-CoV-2, assess the epitope specificity of the SARS-CoV-2 antibody response against full-length spike protein, receptor-binding domain and N-terminal domain of the spike protein, and the nucleocapsid protein. We carried out the assay on samples collected from three sample groups: subjects diagnosed with COVID-19 from the U.S. Army hospital at Camp Humphreys in Pyeongtaek, South Korea; healthcare administrators from the same hospital but with no reported diagnosis of COVID-19; and pre-pandemic samples. We found that the new CoV-specific multiplex assay was highly sensitive allowing plasma samples to be diluted 1:30,000 with a robust signal. The reactivity of IgG responses to SARS-CoV-2 nucleocapsid protein and IgM responses to SARS-CoV-2 spike protein could distinguish COVID-19 samples from non-COVID-19 and pre-pandemic samples. The data from the three sample groups also revealed a unique pattern of cross-reactivity between SARS-CoV-2 and SARS-CoV-1, MERS-CoV, and seasonal coronaviruses HKU1 and OC43. Our findings show that the CoV-2 IgM response is highly specific while the CoV-2 IgG response is more cross-reactive across a range of human CoVs and also showed that IgM and IgG responses show distinct patterns of epitope specificity. In summary, this multiplex assay was able to distinguish samples by COVID-19 status and characterize distinct trends in terms of cross-reactivity and fine-specificity in antibody responses, underscoring its potential value in diagnostic or serosurveillance efforts.

scholarly journals Epitope profiling reveals binding signatures of SARS-CoV-2 immune response and cross-reactivity with endemic HCoVs

2020 ◽  
Author(s):  
Caitlin I. Stoddard ◽  
Jared Galloway ◽  
Helen Y. Chu ◽  
Mackenzie M. Shipley ◽  
Hannah L. Itell ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune Responses ◽  
Binding Sites ◽  
Phage Display Library ◽  
Cross Reactivity ◽  
Antibody Responses ◽  
Spike Protein ◽  
Major Goal ◽  
Significant Homology ◽  
Human Coronaviruses

AbstractA major goal of current SARS-CoV-2 vaccine efforts is to elicit antibody responses that confer protection. Mapping the epitope targets of the SARS-CoV-2 antibody response is critical for innovative vaccine design, diagnostics, and development of therapeutics. Here, we developed a phage display library to map antibody binding sites at high resolution within the complete viral proteomes of all human-infecting coronaviruses in patients with mild or moderate/severe COVID-19. The dominant immune responses to SARS-CoV-2 were targeted to regions spanning the Spike protein, Nucleocapsid, and ORF1ab. Some epitopes were identified in the majority of samples while others were rare, and we found variation in the number of epitopes targeted by different individuals. We also identified a set of cross-reactive sequences that were bound by antibodies in SARS-CoV-2 unexposed individuals. Finally, we uncovered a subset of enriched epitopes from commonly circulating human coronaviruses with significant homology to highly reactive SARS-CoV-2 sequences.

scholarly journals Thrombotic Thrombocytopenia after COVID-19 Vaccination: In Search of the Underlying Mechanism

Vaccines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 559
Author(s):  
Piotr Rzymski ◽  
Bartłomiej Perek ◽  
Robert Flisiak
Keyword(s):  
Direct Interaction ◽  
Underlying Mechanism ◽  
The United States ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Vaccine Hesitancy ◽  
Future Research ◽  
Platelet Factor ◽  
Precautionary Measures ◽  
Adenoviral Proteins

The rollout of COVID-19 vaccines brings hope for successful pandemic mitigation and getting the transmission of SARS-CoV-2 under control. The vaccines authorized in Europe displayed a good safety profile in the clinical trials. However, during their post-authorization use, unusual thrombotic events associated with thrombocytopenia have rarely been reported for vector vaccines. This led to the temporary suspension of the AZD1222 vaccine (Oxford/AstraZeneca) in various European countries and the Ad26.COV2 vaccine (Janssen/Johnson&Johnson) in the United States, with regulatory bodies launching investigations into potential causal associations. The thromboembolic reactions were also rarely reported after mRNA vaccines. The exact cause of these adverse effects remains to be elucidated. The present paper outlines the hypotheses on the mechanisms behind the very rare thrombotic thrombocytopenia reported after the COVID-19 vaccination, along with currently existing evidence and future research prospects. The following are discussed: (i) the role of antibodies against platelet factor 4 (PF4), (ii) the direct interaction between adenoviral vector and platelets, (iii) the cross-reactivity of antibodies against SARS-CoV-2 spike protein with PF4, (iv) cross-reactivity of anti-adenovirus antibodies and PF4, (v) interaction between spike protein and platelets, (vi) the platelet expression of spike protein and subsequent immune response, and (vii) the platelet expression of other adenoviral proteins and subsequent reactions. It is also plausible that thrombotic thrombocytopenia after the COVID-19 vaccine is multifactorial. The elucidation of the causes of these adverse events is pivotal in taking precautionary measures and managing vaccine hesitancy. It needs to be stressed, however, that the reported cases are currently sporadic and that the benefits of COVID-19 vaccines vastly outweigh their potential risks.

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