scholarly journals Identifying Epitopes Responsible for Neutralizing Antibody and DC-SIGN Binding on the Spike Glycoprotein of the Severe Acute Respiratory Syndrome Coronavirus

2006 ◽  
Vol 80 (21) ◽  
pp. 10315-10324 ◽  
Author(s):  
Yi-Ping Shih ◽  
Chia-Yen Chen ◽  
Shih-Jen Liu ◽  
Kuan-Hsuan Chen ◽  
Yuan-Ming Lee ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Binding Capacity ◽  
Neutralizing Antibody ◽  
Observation Data ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Capture Assay ◽  
Immature Dendritic Cells ◽  
Glycosylation Sites ◽  
Peptide Display

ABSTRACT The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) uses dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) to facilitate cell entry via cellular receptor-angiotensin-converting enzyme 2. For this project, we used recombinant baculoviruses expressing different lengths of SARS-CoV spike (S) protein in a capture assay to deduce the minimal DC-SIGN binding region. Our results identified the region location between amino acid (aa) residues 324 to 386 of the S protein. We then generated nine monoclonal antibodies (MAbs) against the S protein to map the DC-SIGN-binding domain using capture assays with pseudotyped viruses and observed that MAb SIa5 significantly blocked S protein-DC-SIGN interaction. An enhancement assay using the HKU39849 SARS-CoV strain and human immature dendritic cells confirmed our observation. Data from a pepscan analysis and M13 phage peptide display library system mapped the reactive MAb SIa5 epitope to aa residues 363 to 368 of the S protein. Results from a capture assay testing three pseudotyped viruses with mutated N-linked glycosylation sites of the S protein indicate that only two pseudotyped viruses (N330Q and N357Q, both of which lost glycosylation sites near the SIa5 epitope) had diminished DC-SIGN-binding capacity. We also noted that MAb SIb4 exerted a neutralizing effect against HKU39849; its reactive epitope was mapped to aa residues 435 to 439 of the S protein. We offer the data to facilitate the development of therapeutic agents and preventive vaccines against SARS-CoV infection.

scholarly journals A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies

2005 ◽  
Vol 86 (5) ◽  
pp. 1435-1440 ◽  
Author(s):  
Milosz Faber ◽  
Elaine W. Lamirande ◽  
Anjeanette Roberts ◽  
Amy B. Rice ◽  
Hilary Koprowski ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Protein S ◽  
S Protein ◽  
Cellular Immune Responses ◽  
Glycoprotein G ◽  
Cellular Immune ◽  
Animal Reservoirs ◽  
S Genes

Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.

scholarly journals Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2

2020 ◽  
Author(s):  
Huihui Mou ◽  
Brian D. Quinlan ◽  
Haiyong Peng ◽  
Yan Guo ◽  
Shoujiao Peng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Angiotensin Converting Enzyme ◽  
Converting Enzyme ◽  
Viral Receptor ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Binding Domains ◽  
Protein Receptor ◽  
Horseshoe Bat ◽  
Horseshoe Bats

SUMMARYThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat (R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.

scholarly journals Systemic effects of missense mutations on SARS-CoV-2 spike glycoprotein stability and receptor-binding affinity

2020 ◽  
Author(s):  
Shaolei Teng ◽  
Adebiyi Sobitan ◽  
Raina Rhoades ◽  
Dongxiao Liu ◽  
Qiyi Tang
Keyword(s):  
Receptor Binding ◽  
Saturation Mutagenesis ◽  
Systemic Effects ◽  
Missense Mutations ◽  
Host Cell Membrane ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Glycosylation Sites ◽  
The Stability ◽  
And Function

Abstract The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the binding to the permissive cells. The receptor-binding domain (RBD) of SARS-CoV-2 S protein directly interacts with the human angiotensin-converting enzyme 2 (ACE2) on the host cell membrane. In this study, we used computational saturation mutagenesis approaches, including structure-based energy calculations and sequence-based pathogenicity predictions, to quantify the systemic effects of missense mutations on SARS-CoV-2 S protein structure and function. A total of 18 354 mutations in S protein were analyzed, and we discovered that most of these mutations could destabilize the entire S protein and its RBD. Specifically, residues G431 and S514 in SARS-CoV-2 RBD are important for S protein stability. We analyzed 384 experimentally verified S missense variations and revealed that the dominant pandemic form, D614G, can stabilize the entire S protein. Moreover, many mutations in N-linked glycosylation sites can increase the stability of the S protein. In addition, we investigated 3705 mutations in SARS-CoV-2 RBD and 11 324 mutations in human ACE2 and found that SARS-CoV-2 neighbor residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD–ACE2 interaction. The findings comprehensively provide potential target sites in the development of drugs and vaccines against COVID-19.

scholarly journals Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry

2020 ◽  
Author(s):  
Seiya Ozono ◽  
Yanzhao Zhang ◽  
Hirotaka Ode ◽  
Toong Seng Tan ◽  
Kazuo Imai ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Angiotensin Converting Enzyme ◽  
Causative Agent ◽  
Cell Entry ◽  
Viral Infectivity ◽  
Converting Enzyme ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Naturally Occurring ◽  
Spike Proteins

AbstractThe causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the angiotensin-converting enzyme-2 (ACE2) receptor and is cleaved by transmembrane protease serine 2 (TMPRSS2). However, whether S mutations affect SARS-CoV-2 infectivity remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural observations. Nevertheless, the D614G mutant remains susceptible to neutralization by antisera against prototypic viruses. Taken together, these data indicate that the D614G mutation enhances viral infectivity while maintaining neutralization susceptibility.

scholarly journals Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2

2008 ◽  
Vol 89 (11) ◽  
pp. 2741-2745 ◽  
Author(s):  
Alison C. Mathewson ◽  
Alexandra Bishop ◽  
Yongxiu Yao ◽  
Fred Kemp ◽  
Junyuan Ren ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Angiotensin Converting Enzyme ◽  
Converting Enzyme ◽  
Severe Respiratory Distress ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Lower Affinity ◽  
Common Receptor ◽  
Affinity Interaction ◽  
Receptor Binding Protein

Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.

scholarly journals Amino Acid Substitutions in the S2 Region Enhance Severe Acute Respiratory Syndrome Coronavirus Infectivity in Rat Angiotensin-Converting Enzyme 2-Expressing Cells

2007 ◽  
Vol 81 (19) ◽  
pp. 10831-10834 ◽  
Author(s):  
Shuetsu Fukushi ◽  
Tetsuya Mizutani ◽  
Kouji Sakai ◽  
Masayuki Saijo ◽  
Fumihiro Taguchi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Angiotensin Converting Enzyme ◽  
Host Species ◽  
Molecular Basis ◽  
Amino Acid Substitutions ◽  
Converting Enzyme ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Virus Adaptation

ABSTRACT To clarify the molecular basis of severe acute respiratory syndrome coronavirus (SARS-CoV) adaptation to different host species, we serially passaged SARS-CoV in rat angiotensin-converting enzyme 2 (ACE2)-expressing cells. After 15 passages, the virus (Rat-P15) came to replicate effectively in rat ACE2-expressing cells. Two amino acid substitutions in the S2 region were found on the Rat-P15 S gene. Analyses of the infectivity of the pseudotype-bearing S protein indicated that the two substitutions in the S2 region, especially the S950F substitution, were responsible for efficient infection. Therefore, virus adaptation to different host species can be induced by amino acid substitutions in the S2 region.

scholarly journals Evaluation of Human Monoclonal Antibody 80R for Immunoprophylaxis of Severe Acute Respiratory Syndrome by an Animal Study, Epitope Mapping, and Analysis of Spike Variants

2005 ◽  
Vol 79 (10) ◽  
pp. 5900-5906 ◽  
Author(s):  
Jianhua Sui ◽  
Wenhui Li ◽  
Anjeanette Roberts ◽  
Leslie J. Matthews ◽  
Akikazu Murakami ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Human Monoclonal Antibody ◽  
Neutralizing Antibody ◽  
Index Patient ◽  
Binding Domain ◽  
S Protein

ABSTRACT In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding domain. Amino acids critical for 80R binding were identified. In addition, the effects of various 80R-binding domain amino acid substitutions which occur in SARS-like-CoV from civet cats, and which evolved during the 2002/2003 outbreak and in a 2003/2004 Guangdong index patient, were analyzed. The results demonstrated that the vast majority of SARS-CoVs are sensitive to 80R. We propose that by establishing the susceptibility and resistance profiles of newly emerging SARS-CoVs through early S1 genotyping of the core 180-amino-acid neutralizing epitope of 80R, an effective immunoprophylaxis strategy with 80R should be possible in an outbreak setting. Our study also cautions that for any prophylaxis strategy based on neutralizing antibody responses, whether by passive or active immunization, a genotyping monitor will be necessary for effective use.

scholarly journals Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike

2020 ◽  
Author(s):  
Ahmad Reza Mehdipour ◽  
Gerhard Hummer
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Md Simulations ◽  
Spike Protein ◽  
Dual Nature ◽  
Angiotensin Converting Enzyme 2 ◽  
Fusion Inhibitors ◽  
Binding Interface ◽  
Glycosylation Sites ◽  
Cryptic Epitope

AbstractBinding of the spike protein of SARS-CoV-2 to the human angiotensin converting enzyme 2 (ACE2) receptor triggers translocation of the virus into cells. Both the ACE2 receptor and the spike protein are heavily glycosylated, including at sites near their binding interface. We built fully glycosylated models of the ACE2 receptor bound to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Using atomistic molecular dynamics (MD) simulations, we found that the glycosylation of the human ACE2 receptor contributes substantially to the binding of the virus. Interestingly, the glycans at two glycosylation sites, N90 and N322, have opposite effects on spike protein binding. The glycan at the N90 site partly covers the binding interface of the spike RBD. Therefore, this glycan can interfere with the binding of the spike protein and protect against docking of the virus to the cell. By contrast, the glycan at the N322 site interacts tightly with the RBD of the ACE2-bound spike protein and strengthens the complex. Remarkably, the N322 glycan binds into a conserved region of the spike protein identified previously as a cryptic epitope for a neutralizing antibody. By mapping the glycan binding sites, our MD simulations aid in the targeted development of neutralizing antibodies and SARS-CoV-2 fusion inhibitors.

scholarly journals Genetic variations in the human severe acute respiratory syndrome coronavirus receptor ACE2 and serine protease TMPRSS2

2020 ◽  
pp. jclinpath-2020-206867
Author(s):  
Kohei Fujikura ◽  
Kazuma Uesaka
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Genetic Variations ◽  
Single Nucleotide ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Coding Regions ◽  
Recent Emergence ◽  
Contact Residues ◽  
Host Cell Surface ◽  
Host Genetic

AimsThe recent emergence of novel, pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health emergency. The coronaviral entry requires the spike (S)-protein for attachment to the host cell surface, and employs human angiotensin-converting enzyme 2 (hACE2) for entry and transmembrane protease serine 2 (TMPRSS2) for S-protein priming. Although coronaviruses undergo evolution by mutating themselves, it is also essential to know the host genetic factors. Here, we describe the single nucleotide variations (SNVs) in human ACE2 and TMPRSS2.MethodsThe genetic variants derived from five population-sequencing projects were classified by variant type, allele frequency (AF), ethnic group and estimated pathogenicity. The SNVs in SARS-CoV-2/hACE2 contact residues were investigated. The genetic variability was normalised using non-linear regression and the total number of SNVs was estimated by the derived formulas.ResultsWe detected 349 and 551 SNVs in ACE2 and TMPRSS2, respectively, in a total of 156 513 individuals. The vast majority (>97%) of the SNVs were very rare (AF <0.1%) and population-specific, and were computationally estimated to be more frequently deleterious than the SNVs with high AF. These SNVs were distributed throughout the coding regions; some ACE2 variants were located in the SARS-CoV-2/hACE2 contact residues, with a hemizygous state occurring in males. Using regression analysis, the total numbers of genetic variations in ACE2 and TMPRSS2 were 1.1×103 and 1.5×103, respectively, for a population of one million people.ConclusionThe majority of SNVs in ACE2 and TMPRSS2 are rare, population-specific and deleterious, and a multitude of very rare SNVs may explain different susceptibility to SARS-CoV-2.

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