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

2021 ◽  
Vol 118 (19) ◽  
pp. e2100425118
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

Binding 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 to 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 Cryo-EM Structures of the N501Y SARS-CoV-2 Spike Protein in Complex with ACE2 and Two Potent Neutralizing Antibodies

2021 ◽  
Author(s):  
Xing Zhu ◽  
Dhiraj Mannar ◽  
Shanti S. Srivastava ◽  
Alison M. Berezuk ◽  
Jean-Philippe Demers ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Structural Changes ◽  
Neutralizing Antibody ◽  
Antibody Fragments ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Binding Interface ◽  
Short Summary

AbstractThe recently reported “UK variant” of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments.Short summaryThe N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.

scholarly journals Development of a Rapid Point-Of-Care Test that Measures Neutralizing Antibodies to SARS-CoV-2

2020 ◽  
Author(s):  
Douglas F. Lake ◽  
Alexa J. Roeder ◽  
Erin Kaleta ◽  
Paniz Jasbi ◽  
Sivakumar Periasamy ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Point Of Care ◽  
Neutralizing Antibody ◽  
Antibody Test ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Plasma Therapy ◽  
Angiotensin Converting Enzyme 2 ◽  
Point Of Care Test ◽  
Protein Receptor

As increasing numbers of people recover from and are vaccinated against COVID-19, tests are needed to measure levels of protective, neutralizing antibodies longitudinally to help determine duration of immunity. We developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies in plasma, serum or whole blood. The LFA is based on the principle that neutralizing antibodies inhibit binding of the spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2). The test classifies high levels of neutralizing antibodies in sera that were titered using authentic SARS-CoV-2 and pseudotype neutralization assays with an accuracy of 98%. Sera obtained from patients with seasonal coronavirus did not prevent RBD from binding to ACE2. As a demonstration for convalescent plasma therapy, we measured conversion of non-immune plasma into strongly neutralizing plasma. This is the first report of a neutralizing antibody test that is rapid, highly portable and relatively inexpensive that might be useful in assessing COVID-19 vaccine immunity.

scholarly journals Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies

PLoS Biology ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. e3001237
Author(s):  
Xing Zhu ◽  
Dhiraj Mannar ◽  
Shanti S. Srivastava ◽  
Alison M. Berezuk ◽  
Jean-Philippe Demers ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Neutralizing Antibodies ◽  
Structural Changes ◽  
Neutralizing Antibody ◽  
Antibody Fragments ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Structural Explanation ◽  
Binding Interface ◽  
Cryo Electron Microscopy

The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.

scholarly journals Long-term persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific and neutralizing antibodies in recovered COVID-19 patients

2021 ◽  
Author(s):  
Jira Chansaenroj ◽  
Ritthideach Yorsaeng ◽  
Nasamon Wanlapakorn ◽  
Chintana Chirathaworn ◽  
Natthinee Sudhinaraset ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Symptom Onset ◽  
Neutralizing Antibodies ◽  
Serum Antibody ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Spike Protein ◽  
Immunological Study ◽  
Igg Antibody ◽  
Vaccine Schedule

Abstract Understanding antibody responses after natural severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can guide the coronavirus disease 2019 (COVID-19) vaccine schedule. This study aimed to assess the dynamics of SARS-CoV-2 antibodies, including anti-spike protein 1 (S1) immunoglobulin (Ig)G, anti-receptor-binding domain (RBD) total Ig, anti-S1 IgA, and neutralizing antibody against wild-type SARS-CoV-2 in a cohort of patients who were previously infected with SARS-CoV-2. Between March and May 2020, 531 individuals with virologically confirmed cases of SARS-CoV-2 infection were enrolled in our immunological study. The neutralizing titers against SARS-CoV-2 were detected in 95.2%, 86.7%, 85.0%, and 85.4% of recovered COVID-19 patients at 3, 6, 9, and 12 months after symptom onset, respectively. The seropositivity rate of anti-S1 IgG, anti-RBD total Ig, anti-S1 IgA, and neutralizing titers remained at 68.6%, 89.6%, 77.1%, and 85.4%, respectively, at 12 months after symptom onset. The half-life of neutralizing titers was estimated at 100.7 days (95% confidence interval = 44.5 – 327.4 days, R2 = 0.106). These results support that the decline in serum antibody levels over time depends on the symptom severity, and the individuals with high IgG antibody titers experienced a significantly longer persistence of SARS-CoV-2-specific antibody responses than those with lower titers.

scholarly journals Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology

2014 ◽  
Vol 89 (6) ◽  
pp. 2995-3007 ◽  
Author(s):  
Yoshikazu Honda-Okubo ◽  
Dale Barnard ◽  
Chun Hao Ong ◽  
Bi-Hung Peng ◽  
Chien-Te Kent Tseng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralizing Antibodies ◽  
Case Fatality ◽  
Neutralizing Antibody ◽  
The Elderly ◽  
Spike Protein ◽  
Interleukin 4 ◽  
Unique Problem ◽  
Antibody Titers ◽  
Ifn Γ

ABSTRACTAlthough the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses.IMPORTANCECoronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.

scholarly journals Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

2016 ◽  
Vol 113 (45) ◽  
pp. 12768-12773 ◽  
Author(s):  
Leopold Kong ◽  
David E. Lee ◽  
Rameshwar U. Kadam ◽  
Tong Liu ◽  
Erick Giang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Vaccine Design ◽  
Neutralizing Antibody ◽  
Md Simulations ◽  
Receptor Binding Site ◽  
Structural Investigations ◽  
Thermophilic Organisms

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world’s population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen–deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

scholarly journals Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2

2020 ◽  
Author(s):  
James Brett Case ◽  
Paul W. Rothlauf ◽  
Rita E. Chen ◽  
Zhuoming Liu ◽  
Haiyan Zhao ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Spike Protein ◽  
Glycoprotein Gene ◽  
Level 3 ◽  
Focus Reduction ◽  
High Throughput Imaging ◽  
Biosafety Level

ABSTRACTAntibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune γ-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.

scholarly journals Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations

2021 ◽  
Author(s):  
Vincent Dussupt ◽  
Rajeshwer S. Sankhala ◽  
Letzibeth Mendez-Rivera ◽  
Samantha M. Townsley ◽  
Fabian Schmidt ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Low Dose ◽  
Viral Escape ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Viral Inactivation ◽  
Angiotensin Converting Enzyme 2 ◽  
Therapeutic Monoclonal Antibodies ◽  
Potent Protection

AbstractPrevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

scholarly journals Neutralizing antibody responses to an HIV envelope glycan hole are not easily broadened

2019 ◽  
Author(s):  
Yuhe R. Yang ◽  
Laura E. McCoy ◽  
Marit J. van Gils ◽  
Raiees Andrabi ◽  
Hannah L. Turner ◽  
...  
Keyword(s):  
High Resolution ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Tier 2 ◽  
High Resolution Data ◽  
Subtype B ◽  
Glycosylation Sites ◽  
Antibody Complex ◽  
Subtype A ◽  
Hiv Envelope

ABSTRACTExtensive studies with subtype A BG505-derived HIV envelope glycoprotein (Env) SOSIP immunogens have revealed that the dominant autologous neutralizing site in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. BG505 and B41 SOSIP immunogens were combined to test whether immunization in rabbits could induce broader Tier 2 neutralizing responses to the common glycan hole shared between BG505 and B41. Here we isolated autologous neutralizing antibodies (nAbs) that were induced by immunization with B41 SOSIP alone, as well as B41 and BG505 co-immunization, and describe their structure in complex with the B41 SOSIP trimer. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both immunogens were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the highly conserved N241 glycan (>97% conserved), which is present in B41. Single particle cryo-electron microscopy (cryoEM) studies confirmed that B41 and BG505-specific nAbs bind to overlapping glycan hole epitopes. In an attempt to broaden the reactivity of a B41-specific nAb, mutations in the BG505 glycan hole epitope guided by our high-resolution data only recovered partial binding. Overall, designing prime-boost immunogens to increase the breath of nAb responses directed at glycan holes epitopes remains challenging even when the typically immunodominant glycan holes despite overlap with different Envs.IMPORTANCEA glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer immunized rabbits. Our high-resolution cryoEM studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that eliciting cross-reactive responses to this region would likely require hybrid immunogens that bridge between BG505 and B41.

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