The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies.

A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2-infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection.

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Cross-reactive neutralization of SARS-CoV-2 by serum antibodies from recovered SARS patients and immunized animals

Science Advances ◽

10.1126/sciadv.abc9999 ◽

2020 ◽

Vol 6 (45) ◽

pp. eabc9999 ◽

Cited By ~ 4

Author(s):

Yuanmei Zhu ◽

Danwei Yu ◽

Yang Han ◽

Hongxia Yan ◽

Huihui Chong ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Binding Domain ◽

Full Length ◽

Receptor Binding Domain ◽

Serum Antibodies ◽

Serum Samples ◽

S Protein ◽

Novel Coronavirus

The current coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus genetically close to SARS-CoV. To investigate the effects of previous SARS-CoV infection on the ability to recognize and neutralize SARS-CoV-2, we analyzed 20 convalescent serum samples collected from individuals infected with SARS-CoV during the 2003 SARS outbreak. All patient sera reacted strongly with the S1 subunit and receptor binding domain (RBD) of SARS-CoV; cross-reacted with the S ectodomain, S1, RBD, and S2 proteins of SARS-CoV-2; and neutralized both SARS-CoV and SARS-CoV-2 S protein–driven infections. Analysis of antisera from mice and rabbits immunized with a full-length S and RBD immunogens of SARS-CoV verified cross-reactive neutralization against SARS-CoV-2. A SARS-CoV–derived RBD from palm civets elicited more potent cross-neutralizing responses in immunized animals than the RBD from a human SARS-CoV strain, informing strategies for development of universal vaccines against emerging coronaviruses.

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Recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus S protein elicits neutralizing antibodies: Implication for developing SARS vaccines

Virology ◽

10.1016/j.virol.2006.03.049 ◽

2006 ◽

Vol 353 (1) ◽

pp. 6-16 ◽

Cited By ~ 29

Author(s):

Lanying Du ◽

Yuxian He ◽

Yijia Wang ◽

Haojie Zhang ◽

Selene Ma ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Adeno Associated Virus ◽

S Protein

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Xeno-Nucleic Acid (XNA) 2’-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding

Viruses ◽

10.3390/v13101983 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1983

Author(s):

Irani Alves Ferreira-Bravo ◽

Jeffrey J. DeStefano

Keyword(s):

Nucleic Acid ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Dissociation Constants ◽

High Specificity ◽

Binding Domain ◽

Host Cells ◽

Receptor Binding Domain ◽

S Protein ◽

Equilibrium Dissociation Constants

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2ʹ-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10–20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

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An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines

mBio ◽

10.1128/mbio.00930-21 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Yan Guo ◽

Wenhui He ◽

Huihui Mou ◽

Lizhou Zhang ◽

Jing Chang ◽

...

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Binding Domain ◽

Full Length ◽

Vaccine Antigen ◽

Receptor Binding Domain ◽

Protein Vaccine ◽

Wild Type ◽

S Protein ◽

Glycosylation Sites

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines. IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

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Immunogenicity of SARS-CoV: the Receptor-Binding Domain of S Protein is a Major Target of Neutralizing Antibodies

Advances in Experimental Medicine and Biology - The Nidoviruses ◽

10.1007/978-0-387-33012-9_98 ◽

2006 ◽

pp. 539-542 ◽

Cited By ~ 3

Author(s):

Yuxian He

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

S Protein ◽

Major Target

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An engineered receptor-binding domain improves the immunogenicity of multivalent SARS-CoV-2 vaccines

10.1101/2020.11.18.388934 ◽

2020 ◽

Author(s):

Brian D. Quinlan ◽

Wenhui He ◽

Huihui Mou ◽

Lizhou Zhang ◽

Yan Guo ◽

...

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Viral Entry ◽

Binding Domain ◽

Vaccine Antigen ◽

Receptor Binding Domain ◽

Protein Vaccine ◽

Wild Type ◽

S Protein ◽

H Pylori

ABSTRACTThe SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD expresses inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) expresses markedly more efficiently, and generates a more potent neutralizing responses as a DNA vaccine antigen, than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers such as an H. pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.

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