scholarly journals Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaewta Rattanapisit ◽  
Balamurugan Shanmugaraj ◽  
Suwimon Manopwisedjaroen ◽  
Priyo Budi Purwono ◽  
Konlavat Siriwattananon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Receptor Binding ◽  
Nicotiana Benthamiana ◽  
Specific Binding ◽  
Expression System ◽  
Disease Diagnosis ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Global Public Health

Abstract Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 μg/g and 130 μg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.

scholarly journals A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core

2020 ◽  
Author(s):  
Juliette Fedry ◽  
Daniel L. Hurdiss ◽  
Chunyan Wang ◽  
Wentao Li ◽  
Gonzalo Obal ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Receptor Binding ◽  
Human Monoclonal Antibody ◽  
Binding Domain ◽  
Structural Basis ◽  
Binding Motif ◽  
Receptor Binding Domain ◽  
Hamster Model ◽  
Health Crisis

AbstractSARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

scholarly journals Functional analysis of the receptor binding domain of SARS coronavirus S1 region and its monoclonal antibody

2014 ◽  
Vol 36 (3) ◽  
pp. 387-397 ◽  
Author(s):  
Hyun Kim ◽  
Yeongjin Hong ◽  
Keigo Shibayama ◽  
Yasuhiko Suzuki ◽  
Nobutaka Wakamiya ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Functional Analysis ◽  
Receptor Binding ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Sars Coronavirus

Discovery of potential inhibitors of the receptor-binding domain (RBD) of pandemic disease-causing SARS-CoV-2 Spike Glycoprotein from Triphala through molecular docking

2021 ◽  
Vol 01 ◽  
Author(s):  
Sharuk L. Khan ◽  
Falak A. Siddiqui ◽  
Mohd Sayeed Shaikh ◽  
Nitin V. Nema ◽  
Aijaz A. Shaikh
Keyword(s):  
Molecular Docking ◽  
Hydrogen Bonds ◽  
Receptor Binding ◽  
Chemical Constituents ◽  
Antioxidant Properties ◽  
Binding Domain ◽  
Quality Data ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein

Background: COVID-19 (SARS-CoV-2 infection) has affected almost every region of the world. Presently, there is no defined line of treatment available for it. Triphala is already proven to have a safe biological window and well known for its antioxidant and immunomodulatory properties. Objective: Present work has been carried out to study Triphala's effectiveness for the treatment of COVID-19. Methods: The Receptor-binding domain (RBD) of SARS-CoV-2 Spike Glycoprotein responsible for the invasion into the host cell, which leads to further infection. The molecular docking (MD) was performed to explore the binding affinities (kcal/mol) of Triphala's chemical constituents and compared them with the existing drugs under investigation for the treatment of COVID-19 epidemiology. Results: Chebulinic acid binding affinity -8.5 kcal/mol with the formation of 10 hydrogen bonds. Almost all the major chemical constituents have formed two or more hydrogen bonds with RBD of SARS-CoV-2 Spike Glycoprotein. Conclusion: The present study showed that Triphala might perform vital roles in the treatment of COVID-19 and expand its usefulness to physicians to treat this illness. There is a need to complete the in-vitro, in-vivo biological testing of Triphala on SARS-CoV-2 disease to create more quality data. The binding mode of Chebulinic acid in the allosteric cavity allows a better understanding of RBD of SARS-CoV-2 Spike Glycoprotein target and provides insight for the design of new inhibitors. Triphala is already proven to have a safe biological window, which indicates we can skip the pre-clinical trials. Apart from this, Triphala is well known for its antioxidant properties, which ultimately improves the immunity of the COVID-19 patient.

scholarly journals Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro

2020 ◽  
Vol 11 ◽  
Author(s):  
Balamurugan Shanmugaraj ◽  
Kaewta Rattanapisit ◽  
Suwimon Manopwisedjaroen ◽  
Arunee Thitithanyanont ◽  
Waranyoo Phoolcharoen
Keyword(s):  
Monoclonal Antibodies ◽  
Nicotiana Benthamiana ◽  
Specific Binding ◽  
Expression System ◽  
Protein A ◽  
Single Step ◽  
Affinity Column ◽  
Plant Expression ◽  
Plant Expression System

The ongoing coronavirus disease 2019 (COVID-19) outbreak caused by novel zoonotic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially reported in Wuhan city, Hubei Province of China, in late December 2019. The rapid global spread of the virus calls for the urgent development of vaccines or therapeutics for human applications to combat the coronavirus infection. Monoclonal antibodies (mAbs) have been utilized as effective therapeutics for treating various infectious diseases. In the present study, we evaluated the feasibility of plant expression system for the rapid production of recently identified therapeutically suitable human anti-SARS-CoV-2 mAbs B38 and H4. Transient co-expression of heavy-chain and light-chain sequences of both the antibodies by using plant expression geminiviral vector resulted in rapid accumulation of assembled mAbs in Nicotiana benthamiana leaves within 4 days post-infiltration. Furthermore, both the mAbs were purified from the plant crude extracts with single-step protein A affinity column chromatography. The expression level of mAb B38 and H4 was estimated to be 4 and 35 μg/g leaf fresh weight, respectively. Both plant-produced mAbs demonstrated specific binding to receptor binding domain (RBD) of SARS-CoV-2 and exhibited efficient virus neutralization activity in vitro. To the best of our knowledge, this is the first report of functional anti-SARS-CoV-2 mAbs produced in plants, which demonstrates the ability of using a plant expression system as a suitable platform for the production of effective, safe, and affordable SARS-CoV-2 mAbs to fight against the spread of this highly infectious pathogen.

scholarly journals Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 using a competitive phage biopanning strategy

2020 ◽  
Vol 3 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Xin Zeng ◽  
Lingfang Li ◽  
Jing Lin ◽  
Xinlei Li ◽  
Bin Liu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Magnetic Beads ◽  
Human Monoclonal Antibody ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Antibody Library ◽  
Synthetic Antibody ◽  
Blocking Antibodies

Abstract The infection of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200 000 deaths, but no vaccine or therapeutic monoclonal antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor-binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 can bind to the free RBD-His in the supernatant and be subsequently separated by the nickel-nitrilotriacetic acid magnetic beads. rRBD-15 from the competitive biopanning of our synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 and potently inhibit SARS-CoV-2 pseudovirus infection with IC50 values of 12 nM. Nevertheless, rRBD-16 from the standard biopanning can only bind to RBD in vitro, but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

scholarly journals A synthetic bispecific antibody capable of neutralizing SARS-CoV-2 Delta and Omicron

2022 ◽  
Author(s):  
Tom Z Yuan ◽  
Carolina Lucas ◽  
Valter S Monteiro ◽  
Akiko Iwasaki ◽  
Marisa L Yang ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Receptor Binding ◽  
Bispecific Antibody ◽  
Immune Escape ◽  
Binding Domain ◽  
Bispecific Antibodies ◽  
Receptor Binding Domain ◽  
Promising Strategy

Bispecific antibodies have emerged as a promising strategy for curtailing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape. This brief report highlights RBT-0813 (also known as TB493-04), a synthetic, humanized, receptor-binding domain (RBD)-targeted bispecific antibody that retains picomolar affinity to the Spike (S) trimers of all major variants of concern and neutralizes both SARS-CoV-2 Delta and Omicron in vitro.

scholarly journals Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies

2020 ◽  
Author(s):  
Christopher O. Barnes ◽  
Claudia A. Jette ◽  
Morgan E. Abernathy ◽  
Kim-Marie A. Dam ◽  
Shannon R. Esswein ◽  
...  
Keyword(s):  
Immune Responses ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Health Crisis ◽  
Naturally Occurring ◽  
Insight Into

AbstractThe COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike1–5 show therapeutic promise and are being evaluated clincally6–8. To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs5 in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to “up” RBDs, (2) ACE2-blocking hNAbs that bind both “up” and “down” RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize “up” and “down” RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only “up” RBDs9. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent “down” RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.

scholarly journals Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants

2021 ◽  
Author(s):  
Lingshu Wang ◽  
Tongqing Zhou ◽  
Yi Zhang ◽  
Eun Sung Yang ◽  
Chaim A. Schramm ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Therapeutic Antibodies ◽  
Resistance Development ◽  
Neutralizing Activity ◽  
Escape Mutants

AbstractThe emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 μg/mL; IC80 < 0.0006 to 0.0251 μg/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.One Sentence SummaryUltrapotent antibodies from convalescent donors neutralize and mitigate resistance of SARS-CoV-2 variants of concern.

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