In vivo evaluation of pathogenicity and transmissibility of influenza A(H1N1)pdm09 hemagglutinin receptor binding domain 222 intrahost variants isolated from a single immunocompromised patient

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.

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SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

PLoS Pathogens ◽

10.1371/journal.ppat.1010175 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1010175

Author(s):

Abigael Eva Chaouat ◽

Hagit Achdout ◽

Inbal Kol ◽

Orit Berhani ◽

Gil Roi ◽

...

Keyword(s):

Virus Infection ◽

Enzymatic Activity ◽

Receptor Binding ◽

Viral Entry ◽

Surface Expression ◽

Binding Domain ◽

Host Cells ◽

Receptor Binding Domain ◽

High Virus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus membrane binds to the angiotensin converting enzyme 2) ACE2 (receptor on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.

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Cysteine residues in a synthetic peptide corresponding to human follicle-stimulating hormone β-subunit receptor-binding domain 81–95 [hFSH-β-(81–95)] modulate the in vivo effects of hFSH-β-(81–95) on the mouse estrous cycle

Regulatory Peptides ◽

10.1016/s0167-0115(99)00022-1 ◽

1999 ◽

Vol 81 (1-3) ◽

pp. 67-71 ◽

Cited By ~ 2

Author(s):

Patricia Grasso ◽

Marina Rozhavskaya-Arena ◽

Leo E Reichert

Keyword(s):

Estrous Cycle ◽

Receptor Binding ◽

Synthetic Peptide ◽

Follicle Stimulating Hormone ◽

Binding Domain ◽

Cysteine Residues ◽

Receptor Binding Domain ◽

Β Subunit ◽

In Vivo Effects

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Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli

Protein Expression and Purification ◽

10.1016/j.pep.2017.11.001 ◽

2018 ◽

Vol 143 ◽

pp. 77-82 ◽

Cited By ~ 1

Author(s):

Murad Mollaev ◽

Neonila Gorokhovets ◽

Elena Nikolskaya ◽

Maria Faustova ◽

Arthur Zabolotsky ◽

...

Keyword(s):

Receptor Binding ◽

Binding Domain ◽

Alpha Fetoprotein ◽

Receptor Binding Domain ◽

E Coli

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Murine Coronavirus Evolution In Vivo: Functional Compensation of a Detrimental Amino Acid Substitution in the Receptor Binding Domain of the Spike Glycoprotein

Journal of Virology ◽

10.1128/jvi.79.12.7629-7640.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7629-7640 ◽

Cited By ~ 18

Author(s):

Sonia Navas-Martin ◽

Susan T. Hingley ◽

Susan R. Weiss

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Receptor Binding ◽

Amino Acid Substitution ◽

Binding Domain ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Functional Compensation

ABSTRACT Murine coronavirus A59 strain causes mild to moderate hepatitis in mice. We have previously shown that mutants of A59, unable to induce hepatitis, may be selected by persistent infection of primary glial cells in vitro. These in vitro isolated mutants encoded two amino acids substitutions in the spike (S) gene: Q159L lies in the putative receptor binding domain of S, and H716D, within the cleavage signal of S. Here, we show that hepatotropic revertant variants may be selected from these in vitro isolated mutants (Q159L-H716D) by multiple passages in the mouse liver. One of these mutants, hr2, was chosen for more in-depth study based on a more hepatovirulent phenotype. The S gene of hr2 (Q159L- R654H -H716D- E1035D ) differed from the in vitro isolates (Q159L-H716D) in only 2 amino acids (R654H and E1035D). Using targeted RNA recombination, we have constructed isogenic recombinant MHV-A59 viruses differing only in these specific amino acids in S (Q159L-R654H-H716D-E1035D). We demonstrate that specific amino acid substitutions within the spike gene of the hr2 isolate determine the ability of the virus to cause lethal hepatitis and replicate to significantly higher titers in the liver compared to wild-type A59. Our results provide compelling evidence of the ability of coronaviruses to rapidly evolve in vivo to highly virulent phenotypes by functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.

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The Receptor Binding Domain of Botulinum Neurotoxin Serotype A (BoNT/A) Inhibits BoNT/A and BoNT/E IntoxicationsIn Vivo

Clinical and Vaccine Immunology ◽

10.1128/cvi.00268-13 ◽

2013 ◽

Vol 20 (8) ◽

pp. 1266-1273 ◽

Cited By ~ 7

Author(s):

Alon Ben David ◽

Eran Diamant ◽

Ada Barnea ◽

Osnat Rosen ◽

Amram Torgeman ◽

...

Keyword(s):

Receptor Binding ◽

Botulinum Neurotoxin ◽

Expression System ◽

Lethal Dose ◽

Binding Domain ◽

Receptor Binding Domain ◽

Time To Death ◽

Order Of Magnitude ◽

Botulinum Neurotoxin Serotype A

ABSTRACTThe receptor binding domain of botulinum neurotoxin (BoNT), also designated the C terminus of the heavy chain (HC), is a promising vaccine candidate against botulism. In this study, a highly efficient expression system for the protein was developed inEscherichia coli, which provided yields that were 1 order of magnitude higher than those reported to date (350 mg HCper liter). The product was highly immunogenic, protecting mice from a challenge with 10550% lethal dose (LD50) after a single vaccination and generating a neutralizing titer of 49.98 IU/ml after three immunizations. In addition, a single boost with HCincreased neutralizing titers by up to 1 order of magnitude in rabbits hyperimmunized against toxoid. Moreover, we demonstrate here for the first timein vivoinhibition of BoNT/A intoxication by HC/A, presumably due to a blockade of the neurotoxin protein receptor SV2. Administration of HC/A delayed the time to death from 10.4 to 27.3 h in mice exposed to a lethal dose of BoNT/A (P= 0.0005). Since BoNT/A and BoNT/E partially share SV2 isoforms as their protein receptors, the ability of HC/A to cross-inhibit BoNT/E intoxication was evaluated. The administration of HC/A together with BoNT/E led to 50% survival and significantly delayed the time to death for the nonsurviving mice (P= 0.003). Furthermore, a combination of HC/A and a subprotective dose of antitoxin E fully protected mice against 850 mouse LD50of BoNT/E, suggesting complementary mechanisms of protection consisting of toxin neutralization by antibodies and receptor blocking by HC/A.

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An ultrapotent pan-β-coronavirus lineage B (β-CoV-B) neutralizing antibody locks the receptor-binding domain in closed conformation by targeting its conserved epitope

Protein & Cell ◽

10.1007/s13238-021-00871-6 ◽

2021 ◽

Author(s):

Zezhong Liu ◽

Wei Xu ◽

Zhenguo Chen ◽

Wangjun Fu ◽

Wuqiang Zhan ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibody ◽

Cross Reactivity ◽

Binding Domain ◽

Receptor Binding Domain ◽

Binding Antibody ◽

Neutralizing Epitopes ◽

Conserved Epitope ◽

Induce Antibody

AbstractNew threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes β-coronavirus lineage B (β-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional “down” conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD “up”. Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-β-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against β-CoV-B and newly emerging SARS-CoV-2 variants of concern.

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

Viruses ◽

10.3390/v13081595 ◽

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.

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