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 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 Rapid High-Yield Production of Functional SARS-CoV-2 Receptor Binding Domain by Viral and Non-Viral Transient Expression for Pre-Clinical Evaluation

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 654
Author(s):  
Omar Farnós ◽  
Alina Venereo-Sánchez ◽  
Xingge Xu ◽  
Cindy Chan ◽  
Shantoshini Dash ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Binding Domain ◽  
Added Value ◽  
High Yield ◽  
Conformational Structure ◽  
Receptor Binding Domain ◽  
Sialic Acid Content ◽  
Production Methods ◽  
Adenoviral Infection

Vaccine design strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the Spike protein or its subunits as the main antigen target of neutralizing antibodies. In this work, we propose rapid production methods of an extended segment of the Spike Receptor Binding Domain (RBD) in HEK293SF cells cultured in suspension, in serum-free media, as a major component of a COVID-19 subunit vaccine under development. The expression of RBD, engineered with a sortase-recognition motif for protein-based carrier coupling, was achieved at high yields by plasmid transient transfection or human type-5-adenoviral infection of the cells, in a period of only two and three weeks, respectively. Both production methods were evaluated in 3L-controlled bioreactors with upstream and downstream bioprocess improvements, resulting in a product recovery with over 95% purity. Adenoviral infection led to over 100 µg/mL of RBD in culture supernatants, which was around 7-fold higher than levels obtained in transfected cultures. The monosaccharide and sialic acid content was similar in the RBD protein from the two production approaches. It also exhibited a proper conformational structure as recognized by monoclonal antibodies directed against key native Spike epitopes. Efficient direct binding to ACE2 was also demonstrated at similar levels in RBD obtained from both methods and from different production lots. Overall, we provide bioprocess-related data for the rapid, scalable manufacturing of low cost RBD based vaccines against SARS-CoV-2, with the added value of making a functional antigen available to support further research on uncovering mechanisms of virus binding and entry as well as screening for potential COVID-19 therapeutics.

scholarly journals Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Romain Marlin ◽  
Veronique Godot ◽  
Sylvain Cardinaud ◽  
Mathilde Galhaut ◽  
Severin Coleon ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Viral Vector ◽  
Binding Domain ◽  
Long Term Memory ◽  
High Dose ◽  
Receptor Binding Domain ◽  
Humanized Mouse Model ◽  
Subunit Vaccines ◽  
Antigen Presenting

AbstractAchieving sufficient worldwide vaccination coverage against SARS-CoV-2 will require additional approaches to currently approved viral vector and mRNA vaccines. Subunit vaccines may have distinct advantages when immunizing vulnerable individuals, children and pregnant women. Here, we present a new generation of subunit vaccines targeting viral antigens to CD40-expressing antigen-presenting cells. We demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with long-term memory phenotypes, in a humanized mouse model. Additionally, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Vaccine-elicited antibodies cross-neutralize different SARS-CoV-2 variants, including D614G, B1.1.7 and to a lesser extent B1.351. Such vaccination significantly improves protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals.

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 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 Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques

2021 ◽  
Author(s):  
Romain Marlin ◽  
Véronique Godot ◽  
Sylvain Cardinaud ◽  
Mathilde Galhaut ◽  
Severin Coleon ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
High Efficiency ◽  
Viral Vector ◽  
Binding Domain ◽  
Long Term Memory ◽  
High Dose ◽  
Human Populations ◽  
Receptor Binding Domain ◽  
Subunit Vaccines

Abstract Controlling the circulation of the recently emerged SARS-CoV-2 in the human populations requires massive vaccination campaigns. Achieving sufficient worldwide vaccination coverage will require additional approaches to first generation of approved viral vector and mRNA vaccines. Subunit vaccines have excellent safety and efficacy records and may have distinct advantages, in particular when immunizing individuals with vulnerabilities or when considering the vaccination of children and pregnant women.. We have developed a new generation of subunit vaccines with enhanced immunogenicity by the targeting of viral antigens to CD40-expressing antigen-presenting cells, thus harnessing their intrinsic immune-stimulant properties. Here, we demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with a long-term memory phenotype, in a humanized mouse model. In addition, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Such vaccination thus significantly improved protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals. Viral dynamics modelling showed the high efficiency of the vaccine at controlling the viral dissemination.

scholarly journals A highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative elicits high titers of neutralizing antibodies

2021 ◽  
Author(s):  
Sameer Kumar Malladi ◽  
Unnatiben Rajeshbhai Patel ◽  
Randhir Singh ◽  
Suman Pandey ◽  
Sahil Kumar ◽  
...  
Keyword(s):  
Mammalian Cell ◽  
Receptor Binding ◽  
Guinea Pigs ◽  
Neutralizing Antibodies ◽  
Matrix Protein ◽  
Geometric Mean ◽  
Binding Domain ◽  
Cold Chain ◽  
High Yield ◽  
Receptor Binding Domain

AbstractThe Receptor Binding Domain of SARS-CoV-2 is the primary target of neutralizing antibodies. We fused our previously described, highly thermotolerant glycan engineered monomeric RBD to a heterologous non-immunogenic trimerization domain derived from cartilage matrix protein. The protein was expressed at a good yield of ∼80-100 mg/liter in Expi293 cells, as well as in both CHO and HEK293 stable cell lines. The designed trimeric RBD was observed to form homogeneous disulfide-linked trimers. When lyophilized, the trimer possessed remarkable functional stability to transient thermal stress of upto 100 °C and was stable to long term storage of over 4 weeks at 37 °C. Two immunizations with an AddaVax adjuvanted formulation elicited antibodies with high endpoint neutralizing titers against replicative virus with geometric mean titers of ∼1114 and 1940 in guinea pigs and mice respectively. In pseudoviral assays, corresponding titers were ∼3600 and ∼16050, while the corresponding value for human convalescent sera was 137. Similar results were obtained with an Alhydrogel, CpG combination adjuvant. The same immunogen was expressed in Pichia pastoris, but this formed high molecular weight aggregates and elicited much lower ACE2 competing antibodies than mammalian cell expressed protein. The excellent thermotolerance, high yield, and robust immunogenicity of such trimeric RBD immunogens suggest that they are a promising modality to combat COVID-19.ImportanceSARS-CoV-2 is the causative agent of the ongoing COVID-19 pandemic. The viral surface exposed Spike glycoprotein is the target of neutralizing antibodies of which a major fraction targets the receptor binding domain (RBD). Thus RBD derived immunogens are attractive vaccine candidates. Monomeric, mammalian cell expressed RBD protein elicits low to moderate titers of neutralizing antibodies. We designed a highly expressed, trimeric RBD derivative with a non-immunogenic trimerization domain. In guinea pigs and mice respectively, this derivative induces 20-300 fold higher neutralizing antibody titers relative to convalescent human sera, while remaining conformationally intact after incubation for over four weeks at 37 °C and for ninety minutes at 100 °C when lyophilized. Such trimeric RBD formulations should not require a cold chain. Additionally, the high titers of neutralizing antibodies should buffer against viral sequence variation. These are both highly desirable attributes for a COVID-19 vaccine, especially in resource limited settings.

scholarly journals Covalent coupling of Spike’s receptor binding domain to a multimeric carrier produces a high immune response against SARS-CoV-2

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
◽  
Paula M. Berguer ◽  
Matías Blaustein ◽  
Luis M. Bredeston ◽  
Patricio O. Craig ◽  
...  
Keyword(s):  
Immune Response ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
High Titer ◽  
Binding Domain ◽  
Efficient Production ◽  
Receptor Binding Domain ◽  
Promising Candidate ◽  
Humoral Immune ◽  
Lumazine Synthase

AbstractThe receptor binding domain (RBD) of the Spike protein from SARS-CoV-2 is a promising candidate to develop effective COVID-19 vaccines since it can induce potent neutralizing antibodies. We have previously reported the highly efficient production of RBD in Pichia pastoris, which is structurally similar to the same protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer with the purpose of increasing its immunogenicity. We produced multimeric particles by a transpeptidation reaction between RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric 170 kDa protein. Such particles were used to vaccinate mice with two doses 30 days apart. When the particles ratio of RBD to BLS units was high (6–7 RBD molecules per BLS decamer in average), the humoral immune response was significantly higher than that elicited by RBD alone or by RBD-BLS particles with a lower RBD to BLS ratio (1–2 RBD molecules per BLS decamer). Remarkably, multimeric particles with a high number of RBD copies elicited a high titer of neutralizing IgGs. These results indicate that multimeric particles composed of RBD covalent coupled to BLS possess an advantageous architecture for antigen presentation to the immune system, and therefore enhancing RBD immunogenicity. Thus, multimeric RBD-BLS particles are promising candidates for a protein-based vaccine.

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 Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells

2020 ◽  
Author(s):  
◽  
Claudia R. Arbeitman ◽  
Gabriela Auge ◽  
Matías Blaustein ◽  
Luis Bredeston ◽  
...  
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 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 HPLC, 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.

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