scholarly journals Development of Spike Receptor-Binding Domain Nanoparticles as a Vaccine Candidate against SARS-CoV-2 Infection in Ferrets

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Young-Il Kim ◽  
Dokyun Kim ◽  
Kwang-Min Yu ◽  
Hogyu David Seo ◽  
Shin-Ae Lee ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Clinical Symptoms ◽  
Binding Domain ◽  
Host Cells ◽  
Antigen Delivery ◽  
Receptor Binding Domain ◽  
Protein Vaccine

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of the CoV disease 2019 (COVID-19) pandemic, enters host cells via the interaction of its receptor-binding domain (RBD) of the spike protein with host angiotensin-converting enzyme 2 (ACE2). Therefore, the RBD is a promising vaccine target to induce protective immunity against SARS-CoV-2 infection. In this study, we report the development of an RBD protein-based vaccine candidate against SARS-CoV-2 using self-assembling Helicobacter pylori-bullfrog ferritin nanoparticles as an antigen delivery system. RBD-ferritin protein purified from mammalian cells efficiently assembled into 24-mer nanoparticles. Sixteen- to 20-month-old ferrets were vaccinated with RBD-ferritin nanoparticles (RBD nanoparticles) by intramuscular or intranasal inoculation. All vaccinated ferrets with RBD nanoparticles produced potent neutralizing antibodies against SARS-CoV-2. Strikingly, vaccinated ferrets demonstrated efficient protection from SARS-CoV-2 challenge, showing no fever, body weight loss, or clinical symptoms. Furthermore, vaccinated ferrets showed rapid clearance of infectious virus in nasal washes and lungs as well as of viral RNA in respiratory organs. This study demonstrates that spike RBD-nanoparticles are an effective protein vaccine candidate against SARS-CoV-2.

scholarly journals Development of spike receptor-binding domain nanoparticle as a vaccine candidate against SARS-CoV-2 infection in ferrets

2021 ◽  
Author(s):  
Young-Il Kim ◽  
Dokyun Kim ◽  
Kwang-Min Yu ◽  
Hogyu David Seo ◽  
Shin-Ae Lee ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Clinical Symptoms ◽  
Binding Domain ◽  
Host Cells ◽  
Antigen Delivery ◽  
Receptor Binding Domain ◽  
Protein Vaccine

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of COVID-19 pandemic, enters host cells via the interaction of its Receptor-Binding Domain (RBD) of Spike protein with host Angiotensin-Converting Enzyme 2 (ACE2). Therefore, RBD is a promising vaccine target to induce protective immunity against SARS-CoV-2 infection. In this study, we report the development of RBD protein-based vaccine candidate against SARS-CoV-2 using self-assembling H. pylori-bullfrog ferritin nanoparticles as an antigen delivery. RBD-ferritin protein purified from mammalian cells efficiently assembled into 24-mer nanoparticles. 16-20 months-old ferrets were vaccinated with RBD-ferritin nanoparticles (RBD-nanoparticles) by intramuscular or intranasal inoculation. All vaccinated ferrets with RBD-nanoparticles produced potent neutralizing antibodies against SARS-CoV-2. Strikingly, vaccinated ferrets demonstrated efficient protection from SARS-CoV-2 challenge, showing no fever, body weight loss and clinical symptoms. Furthermore, vaccinated ferrets showed rapid clearance of infectious viruses in nasal washes and lungs as well as viral RNA in respiratory organs. This study demonstrates the Spike RBD-nanoparticle as an effective protein vaccine candidate against SARS-CoV-2.

scholarly journals Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate

2021 ◽  
Author(s):  
Wen-Hsiang Chen ◽  
Jeroen Pollet ◽  
Ulrich Strych ◽  
Jungsoon Lee ◽  
Zhuyun Liu ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Total Yield ◽  
Development Program ◽  
Exchange Chromatography ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Protein Vaccine ◽  
Protein Subunit

Background: SARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice. Method: The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice. Results, Conclusions, and Significance: The RBD203-N1 production process yielded 492.9 ± 3.0 mg/L of protein in the fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.

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 Xeno-Nucleic Acid (XNA) 2’-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1983
Author(s):  
Irani Alves Ferreira-Bravo ◽  
Jeffrey J. DeStefano
Keyword(s):  
Nucleic Acid ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Dissociation Constants ◽  
High Specificity ◽  
Binding Domain ◽  
Host Cells ◽  
Receptor Binding Domain ◽  
S Protein ◽  
Equilibrium Dissociation Constants

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2ʹ-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10–20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

scholarly journals Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Keyword(s):  
Pichia Pastoris ◽  
Receptor Binding ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Binding Domain ◽  
Size Exclusion ◽  
Scale Production ◽  
Receptor Binding Domain ◽  
Serological Tests

AbstractThe yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

scholarly journals Comparative Immunomodulatory Evaluation of the Receptor Binding Domain of the SARS-CoV-2 Spike Protein; a Potential Vaccine Candidate Which Imparts Potent Humoral and Th1 Type Immune Response in a Mouse Model

2021 ◽  
Vol 12 ◽  
Author(s):  
Tripti Shrivastava ◽  
Balwant Singh ◽  
Zaigham Abbas Rizvi ◽  
Rohit Verma ◽  
Sandeep Goswami ◽  
...  
Keyword(s):  
Immune Response ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Unmet Need ◽  
Binding Domain ◽  
Spike Protein ◽  
Cell Immune Response ◽  
Receptor Binding Domain

The newly emerged novel coronavirus, SARS-CoV-2, the causative agent of COVID-19 has proven to be a threat to the human race globally, thus, vaccine development against SARS-CoV-2 is an unmet need driving mass vaccination efforts. The receptor binding domain of the spike protein of this coronavirus has multiple neutralizing epitopes and is associated with viral entry. Here we have designed and characterized the SARS-CoV-2 spike protein fragment 330-526 as receptor binding domain 330-526 (RBD330-526) with two native glycosylation sites (N331 and N343); as a potential subunit vaccine candidate. We initially characterized RBD330-526 biochemically and investigated its thermal stability, humoral and T cell immune response of various RBD protein formulations (with or without adjuvant) to evaluate the inherent immunogenicity and immunomodulatory effect. Our result showed that the purified RBD immunogen is stable up to 72 h, without any apparent loss in affinity or specificity of interaction with the ACE2 receptor. Upon immunization in mice, RBD generates a high titer humoral response, elevated IFN-γ producing CD4+ cells, cytotoxic T cells, and robust neutralizing antibodies against live SARS-CoV-2 virus. Our results collectively support the potential of RBD330-526 as a promising vaccine candidate against SARS-CoV-2.

scholarly journals The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients

2020 ◽  
Vol 5 (48) ◽  
pp. eabc8413 ◽  
Author(s):  
Lakshmanane Premkumar ◽  
Bruno Segovia-Chumbez ◽  
Ramesh Jadi ◽  
David R. Martinez ◽  
Rajendra Raut ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Clinical Symptoms ◽  
Severe Disease ◽  
Population Level ◽  
Respiratory Illness ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Specific Antibodies

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.

scholarly journals A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern

2021 ◽  
Vol 12 ◽  
Author(s):  
Shahbaz Ahmed ◽  
Mohammad Suhail Khan ◽  
Savitha Gayathri ◽  
Randhir Singh ◽  
Sahil Kumar ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
High Titer ◽  
Binding Domain ◽  
High Yield ◽  
High Dose ◽  
Receptor Binding Domain ◽  
Triple Mutant ◽  
Water Emulsion

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

scholarly journals Human basigin (CD147) does not directly interact with SARS-CoV-2 spike glycoprotein

2021 ◽  
Author(s):  
Robert J. Ragotte ◽  
David Pulido ◽  
Francesca R. Donnellan ◽  
Giacomo Gorini ◽  
Hannah Davies ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Therapeutic Target ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Binding Domain ◽  
Host Cells ◽  
Size Exclusion ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
Mortality And Morbidity

AbstractBasigin, or CD147, has been reported as a co-receptor used by SARS-CoV-2 to invade host cells. Basigin also has a well-established role in Plasmodium falciparum malaria infection of human erythrocytes where it is bound by one of the parasite’s invasion ligands, reticulocyte binding protein homolog 5 (RH5). Here, we sought to validate the claim that the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein can form a complex with basigin, using RH5-basigin as a positive control. Using recombinantly expressed proteins, size exclusion chromatography and surface plasmon resonance, we show that neither RBD nor full-length spike glycoprotein bind to recombinant human basigin (either expressed in E. coli or mammalian cells). Given the immense interest in SARS-CoV-2 therapeutic targets, we would caution the inclusion of basigin in this list on the basis of its reported direct interaction with SARS-CoV-2 spike glycoprotein.ImportanceReducing the mortality and morbidity associated with COVID-19 remains a global health priority. Critical to these efforts is the identification of host factors that are essential to viral entry and replication. Basigin, or CD147, was previously identified as a possible therapeutic target based on the observation that it may act as a co-receptor for SARS-COV-2, binding to the receptor binding domain of the spike protein. Here, we show that there is no direct interaction between the RBD and basigin, casting doubt on its role as a co-receptor and plausibility as a therapeutic target.

scholarly journals Xeno-nucleic Acid (XNA) 2'-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding

2021 ◽  
Author(s):  
Irani Alves Ferreira-Bravo ◽  
Jeffrey J. DeStefano
Keyword(s):  
Nucleic Acid ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Dissociation Constants ◽  
High Specificity ◽  
Binding Domain ◽  
Host Cells ◽  
Receptor Binding Domain ◽  
S Protein ◽  
Equilibrium Dissociation Constants

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor binding domain (RBD) on the viral S protein with angiotensin converting enzyme 2 (ACE2) on the surface of human host cells. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2′-fluoroarabinonucleic acid (FANA). The best selected ~ 79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~ 10-20 nM and a binding half-life for the RBD of 53 ± 18 minutes. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

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