scholarly journals A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Guangyu Zhao ◽  
Lei He ◽  
Shihui Sun ◽  
Hongjie Qiu ◽  
Wanbo Tai ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Broad Spectrum ◽  
Half Life ◽  
Cost Effective ◽  
Binding Domain ◽  
Humanized Mice ◽  
Receptor Binding Domain ◽  
Neutralizing Activity ◽  
High Affinity

ABSTRACT The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics. IMPORTANCE Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.

scholarly journals Plant-Produced Glycosylated and In Vivo Deglycosylated Receptor Binding Domain Proteins of SARS-CoV-2 Induce Potent Neutralizing Responses in Mice

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1595
Author(s):  
Tarlan Mamedov ◽  
Damla Yuksel ◽  
Merve Ilgın ◽  
Irem Gurbuzaslan ◽  
Burcu Gulec ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Plant Biomass ◽  
Specific Binding ◽  
High Titer ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
S Protein ◽  
Vaccine Candidates ◽  
Neutralizing Activity

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.

scholarly journals High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Thomas J. Esparza ◽  
Negin P. Martin ◽  
George P. Anderson ◽  
Ellen R. Goldman ◽  
David L. Brody
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Hek293 Cells ◽  
Scale Production ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
High Affinity ◽  
Diagnostic Potential

AbstractThere are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12–15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1–5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

Receptor-binding domain of human α2-macroglobulin Expression, folding and biochemical characterization of a high-affinity recombinant derivative

FEBS Letters ◽  
1994 ◽  
Vol 344 (2-3) ◽  
pp. 242-246 ◽  
Author(s):  
Thor Las Holtet ◽  
Kåre Lehmann Nielsen ◽  
Michael Etzerodt ◽  
Søren Kragh Moestrup ◽  
Jørgen Gliemann ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Biochemical Characterization ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
High Affinity ◽  
Α2 Macroglobulin

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.

scholarly journals Expression and refolding of a high-affinity receptor binding domain from rat α 1 -macroglobulin

FEBS Letters ◽  
1995 ◽  
Vol 373 (3) ◽  
pp. 296-298 ◽  
Author(s):  
Kåre L. Nielsen ◽  
Lars Sottrup-Jensen ◽  
Georg H. Fey ◽  
Hans C. Thøgersen
Keyword(s):  
Receptor Binding ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Affinity Receptor

scholarly journals Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

2021 ◽  
Author(s):  
Alexandra C Walls ◽  
Marcos C Miranda ◽  
Minh N Pham ◽  
Alexandra Schaefer ◽  
Allison Greaney ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Polyclonal Antibodies ◽  
Clinical Stage ◽  
Binding Domain ◽  
Single Shot ◽  
Receptor Binding Domain ◽  
Negative Consequences ◽  
Neutralizing Activity

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.

scholarly journals A Recombinant Protein SARS-CoV-2 Candidate Vaccine Elicits High-titer Neutralizing Antibodies in Macaques.

2021 ◽  
Author(s):  
Gary Baisa ◽  
David Rancour ◽  
Keith Mansfield ◽  
Monika Burns ◽  
Lori Martin ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
High Titer ◽  
Binding Domain ◽  
Pseudotyped Virus ◽  
Receptor Binding Domain ◽  
Binding Assays ◽  
Neutralizing Activity ◽  
Terminal Domain

Abstract BackgroundVaccines that generate robust and long-lived protective immunity against SARS-CoV-2 infection are urgently required. MethodsWe assessed the potential of vaccine candidates based on the SARS-CoV-2 spike in cynomolgus macaques (M. fascicularis) by examining their ability to generate spike binding antibodies with neutralizing activity. Antigens were derived from two distinct regions of the spike S1 subunit, either the N-terminal domain or an extended C-terminal domain containing the receptor-binding domain and were fused to the human IgG1 Fc domain. Three groups of 2 animals each were immunized with either antigen, alone or in combination. The development of antibody responses was evaluated through 20 weeks post-immunization. ResultsA robust IgG response to the spike protein was detected as early as 2 weeks after immunization with either protein and maintained for over 20 weeks. Sera from animals immunized with antigens derived from the RBD were able to prevent binding of soluble spike proteins to the ACE2 receptor, shown by in vitro binding assays, while sera from animals immunized with the N-terminal domain alone lacked this activity. Crucially, sera from animals immunized with the extended receptor binding domain but not the N-terminal domain had potent neutralizing activity against SARS-CoV-2 pseudotyped virus, with titers in excess of 10,000, greatly exceeding that typically found in convalescent humans. Neutralizing activity persisted for more than 20 weeks. ConclusionsThese data support the utility of spike subunit-based antigens as a vaccine for use in humans.

scholarly journals Antibody responses to BNT162b2 vaccination in Japan: Monitoring vaccine efficacy by measuring IgG antibodies against the receptor binding domain of SARS-CoV-2

2021 ◽  
Author(s):  
Hidetsugu Fujigaki ◽  
Yasuko Yamamoto ◽  
Takenao Koseki ◽  
Sumi Banno ◽  
Tatsuya Ando ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Vaccine Efficacy ◽  
Vaccination Strategy ◽  
Binding Domain ◽  
Enzyme Linked Immunosorbent Assay ◽  
Receptor Binding Domain ◽  
Neutralizing Activity ◽  
Before And After ◽  
Reliable Performance ◽  
Antibody Levels

BACKGROUND: To fight severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), mass vaccination has begun in many countries. To investigate the usefulness of a serological assay to predict vaccine efficacy, we analyzed the levels of IgG, IgM, and IgA against the receptor binding domain (RBD) of SARS-CoV-2 in the sera from BNT162b2 vaccinated individuals in Japan. METHODS: This study included 219 individuals who received two doses of BNT162b2. The levels of IgG, IgM, and IgA against RBD were measured by enzyme-linked immunosorbent assay before and after the first and second vaccination, respectively. The relationship between antibody levels and several factors including age, gender, and hypertension were analyzed. Virus-neutralizing activity in sera was measured to determine the correlation with the levels of antibodies. A chemiluminescent enzyme immunoassay (CLEIA) method to measure IgG against RBD was developed and validated for the clinical setting. RESULTS: The levels of all antibody isotypes were increased after vaccination. Among them, RBD-IgG was dramatically increased after the second vaccination. The IgG levels in females were significantly higher than in males. There was a negative correlation between age and IgG levels in males. The IgG levels significantly correlated with the neutralizing activity. The CLEIA assay measuring IgG against RBD showed a reliable performance and a high correlation with neutralizing activity. CONCLUSIONS: Monitoring of IgG against RBD is a powerful tool to predict the efficacy of SARS-CoV-2 vaccination and provides useful information in considering a personalized vaccination strategy for COVID-19.

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