scholarly journals Engineering, production and characterization of Spike and Nucleocapsid structural proteins of SARS–CoV-2 in Nicotiana benthamiana as vaccine candidates against COVID-19

2020 ◽  
Author(s):  
Tarlan Mamedov ◽  
Damla Yuksel ◽  
Merve Ilgın ◽  
İrem Gürbüzaslan ◽  
Burcu Gulec ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Plant Biomass ◽  
Specific Binding ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Protein S ◽  
Global Public Health ◽  
S Protein ◽  
Vaccine Candidates ◽  
Protein Variants

AbstractThe COVID-19 pandemic, which is caused by SARS-CoV-2 has rapidly spread to more than 216 countries and has put global public health at high risk. The world urgently needs a cost-effective and safe SARS-CoV-2 coronavirus vaccine, antiviral and therapeutic drugs to control the COVID-19 pandemic. In this study, we engineered the Nucleocapsid (N) and Spike protein (S) variants (Receptor binding domain, RBD and S1 domain) of SARS-CoV-2 genes and produced in Nicotiana benthamiana plant. The purification yields were at least 20 mg of pure protein/kg of plant biomass for each target protein. The S protein variants of SARS-CoV-2 showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. The purified plant produced N and S variants were recognized by N and S protein specific monoclonal and polyclonal antibodies demonstrating specific reactivity of mAb to plant produced N and S protein variants. In addition, IgG responses of plant produced N and S antigens elicited significantly high titers of antibody in mice. This is the first report demonstrating production of functional active S1 domain and Nucleocapsid protein of SARC-CoV-2 in plants. In addition, in this study, for the first time, we report the co-expression of RBD with N protein to produce a cocktail antigen of SARS-CoV-2, which elicited high-titer antibodies compared to RBD or N proteins. Thus, obtained data support that plant produced N and S antigens, developed in this study, are promising vaccine candidates against COVID-19.

scholarly journals Production and Characterization of Nucleocapsid and RBD Cocktail Antigens of SARS-CoV-2 in Nicotiana benthamiana Plant as a Vaccine Candidate against COVID-19

Vaccines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1337
Author(s):  
Tarlan Mamedov ◽  
Damla Yuksel ◽  
Merve Ilgın ◽  
Irem Gürbüzaslan ◽  
Burcu Gulec ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Vaccine Candidate ◽  
Plant Biomass ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Benthamiana Plant ◽  
Global Public Health ◽  
Neutralizing Activity ◽  
N Protein ◽  
The World

The COVID-19 pandemic has put global public health at high risk, rapidly spreading around the world. Although several COVID-19 vaccines are available for mass immunization, the world still urgently needs highly effective, reliable, cost-effective, and safe SARS-CoV-2 coronavirus vaccines, as well as antiviral and therapeutic drugs, to control the COVID-19 pandemic given the emerging variant strains of the virus. Recently, we successfully produced receptor-binding domain (RBD) variants in the Nicotiana benthamiana plant as promising vaccine candidates against COVID-19 and demonstrated that mice immunized with these antigens elicited a high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2. In this study, we engineered the nucleocapsid (N) protein and co-expressed it with RBD of SARS-CoV-2 in Nicotiana benthamiana plant to produce an antigen cocktail. The purification yields were about 22 or 24 mg of pure protein/kg of plant biomass for N or N+RBD antigens, respectively. The purified plant produced N protein was recognized by N protein-specific monoclonal and polyclonal antibodies demonstrating specific reactivity of mAb to plant-produced N protein. In this study, for the first time, we report the co-expression of RBD with N protein to produce a cocktail antigen of SARS-CoV-2, which elicited high-titer antibodies with potent neutralizing activity against SARS-CoV-2. Thus, obtained data support that a plant-produced antigen cocktail, developed in this study, is a promising vaccine candidate against COVID-19.

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 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 Immunotherapeutic Efficacy of IgY Antibodies Targeting the Full-Length Spike Protein in an Animal Model of Middle East Respiratory Syndrome Coronavirus Infection

2021 ◽  
Vol 14 (6) ◽  
pp. 511
Author(s):  
Sherif A. El-Kafrawy ◽  
Aymn T. Abbas ◽  
Sayed S. Sohrab ◽  
Ashraf A. Tabll ◽  
Ahmed M. Hassan ◽  
...  
Keyword(s):  
Middle East ◽  
Specific Binding ◽  
High Titer ◽  
Egg Yolk ◽  
Middle East Respiratory Syndrome ◽  
Therapeutic Interventions ◽  
Dot Blot ◽  
S Protein

Identified in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and often fatal acute respiratory illness in humans. No approved prophylactic or therapeutic interventions are currently available. In this study, we developed chicken egg yolk antibodies (IgY Abs) specific to the MERS-CoV spike (S) protein and evaluated their neutralizing efficiency against MERS-CoV infection. S-specific IgY Abs were produced by injecting chickens with the purified recombinant S protein of MERS-CoV at a high titer (4.4 mg/mL per egg yolk) at week 7 post immunization. Western blotting and immune-dot blot assays demonstrated specific binding to the MERS-CoV S protein. In vitro neutralization of the generated IgY Abs against MERS-CoV was evaluated and showed a 50% neutralizing concentration of 51.42 μg/mL. In vivo testing using a human-transgenic mouse model showed a reduction of viral antigen positive cells in treated mice, compared to the adjuvant-only controls. Moreover, the lung cells of the treated mice showed significantly reduced inflammation, compared to the controls. Our results show efficient neutralization of MERS-CoV infection both in vitro and in vivo using S-specific IgY Abs. Clinical trials are needed to evaluate the efficiency of the IgY Abs in camels and humans.

scholarly journals Antibodies and Vaccines Target RBD of SARS-CoV-2

2021 ◽  
Vol 8 ◽  
Author(s):  
Long Min ◽  
Qiu Sun
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Protein S ◽  
The Novel ◽  
Global Public Health ◽  
Human Coronavirus ◽  
S Protein ◽  
Important Target ◽  
A Subunit

The novel human coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which gives rise to the coronavirus disease 2019 (COVID-19), has caused a serious threat to global public health. On March 11, 2020, the WHO had officially announced COVID-19 as a pandemic. Therefore, it is vital to find effective and safe neutralizing antibodies and vaccines for COVID-19. The critical neutralizing domain (CND) that is contained in the receptor-binding domain (RBD) of the spike protein (S protein) could lead to a highly potent neutralizing antibody response as well as the cross-protection of other strains of SARS. By using RBD as an antigen, many neutralizing antibodies are isolated that are essential to the therapeutics of COVID-19. Furthermore, a subunit vaccine, which is based on the RBD, is expected to be safer than others, thus the RBD in the S protein is a more important target for vaccine development. In this review, we focus on neutralizing antibodies that are targeting RBD as well as the vaccine based on RBD under current development.

scholarly journals SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Nikolaos C. Kyriakidis ◽  
Andrés López-Cortés ◽  
Eduardo Vásconez González ◽  
Alejandra Barreto Grimaldos ◽  
Esteban Ortiz Prado
Keyword(s):  
Neutralizing Antibodies ◽  
Large Scale ◽  
Vaccine Development ◽  
Rna Virus ◽  
Protein S ◽  
Effective Vaccine ◽  
Phase 3 ◽  
Vaccine Candidates ◽  
Advantages And Disadvantages ◽  
The World

AbstractThe new SARS-CoV-2 virus is an RNA virus that belongs to the Coronaviridae family and causes COVID-19 disease. The newly sequenced virus appears to originate in China and rapidly spread throughout the world, becoming a pandemic that, until January 5th, 2021, has caused more than 1,866,000 deaths. Hence, laboratories worldwide are developing an effective vaccine against this disease, which will be essential to reduce morbidity and mortality. Currently, there more than 64 vaccine candidates, most of them aiming to induce neutralizing antibodies against the spike protein (S). These antibodies will prevent uptake through the human ACE-2 receptor, thereby limiting viral entrance. Different vaccine platforms are being used for vaccine development, each one presenting several advantages and disadvantages. Thus far, thirteen vaccine candidates are being tested in Phase 3 clinical trials; therefore, it is closer to receiving approval or authorization for large-scale immunizations.

INTERACTION OF S PROTEIN/VITRONECTIN WITH CULTURED ENDOTHELIAL CELLS: PROMOTION OF ATTACHMENT AND SPECIFIC BINDING

1987 ◽  
Author(s):  
K T Preissner ◽  
E Anders ◽  
G Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Cell Attachment ◽  
Specific Binding ◽  
Umbilical Vein ◽  
Attachment Site ◽  
Cell Types ◽  
S Protein ◽  
Specific Association ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Proteins

The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.

scholarly journals Plant-Produced Antigen Displaying Virus-Like Particles Evokes Potent Antibody Responses against West Nile Virus in Mice

Vaccines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 60
Author(s):  
Junyun He ◽  
Huafang Lai ◽  
Adrian Esqueda ◽  
Qiang Chen
Keyword(s):  
West Nile Virus ◽  
Fusion Protein ◽  
Nicotiana Benthamiana ◽  
Specific Binding ◽  
Low Cost ◽  
Gradient Centrifugation ◽  
Conformational Epitope ◽  
Protein Domain ◽  
Core Antigen ◽  
West Nile

In this study, we developed a hepatitis B core antigen (HBcAg)-based virus-like particle (VLP) that displays the West Nile virus (WNV) Envelope protein domain III (wDIII) as a vaccine candidate for WNV. The HBcAg-wDIII fusion protein was quickly produced in Nicotiana benthamiana plants and reached a high expression level of approximately 1.2 mg of fusion protein per gram of leaf fresh weight within six days post gene infiltration. Electron microscopy and gradient centrifugation analysis indicated that the introduction of wDIII did not interfere with VLP formation and HBcAg-wDIII successfully assembled into VLPs. HBcAg-wDIII VLPs can be easily purified in large quantities from Nicotiana benthamiana leaves to >95% homogeneity. Further analysis revealed that the wDIII was displayed properly and demonstrated specific binding to an anti-wDIII monoclonal antibody that recognizes a conformational epitope of wDIII. Notably, HBcAg-wDIII VLPs were shown to be highly immunogenic and elicited potent humoral responses in mice with antigen-specific IgG titers equivalent to that of protective wDIII antigens in previous studies. Thus, our wDIII-based VLP vaccine offers an attractive option for developing effective, safe, and low-cost vaccines against WNV.

LOW TOTAL PROTEIN S ANTIGEN BUT HIGH PROTEIN S ACTIVITY DUE TO DECREASED C4b-BINDING PROTEIN (C4b-BP) LEVELS IN NEWBORNS

1987 ◽  
Author(s):  
H P Schwarz ◽  
W Muntean
Keyword(s):  
Vitamin K ◽  
Limit Of Detection ◽  
Polyclonal Antibodies ◽  
Regulatory Protein ◽  
Activated Protein C ◽  
Protein S ◽  
Bleeding Risk ◽  
Free Form ◽  
Classical Complement Pathway ◽  
Coagulation Proteins

Vitamin K-dependent coagulation proteins are known to be decreased in the neonatal period. So far no data have been published on protein S (PS), the vitamin K-dependent cofactor for the antithrombotic enzyme, activated protein C (APC) in this period. We determined, therefore, PS antigen, PS activity and C4b-BP,a regulatory protein of the classical complement pathway to which PS is complexed, in 36 neonates. Total PS antigen in newborns was below the range associated with thromboembolism in patients congenitally deficient in this protein (22±9.6%, mean±SD). None of these infants had clinical or laboratory evidence of thromboembolism or DIC. In contrast to the PS antigen level PS activity measured by the ability of APC to prolong the clotting time of a modified APTT assay using PS-immunodep1eted plasma was significantly higher (77.6±14%, mean±SD, p< 0,001), suggesting a shift in PS to the free form. In fact two dimensional immunoe1ectrophoresis studies revealed the absence of protein S-C4b-BP complexes and only one precipitation indicating free PS was seen in 15 out of the 36 infants. In these 15 neonates C4b-BP was below the limit of detection by sensitive quantitative immunob1otting techniques using monoclonal or polyclonal antibodies. In the remaining 21 infants PS-C4b-BP complexes were detected, but in contrast to adult normal plasma approximately 80% of PS was found in the free form. Mixing experiments with normal human plasma and newborn’s plasma indicate that PS in neonate deficient of C4b-BP can bind normally to C4bp. Absence of C4b-BP did not correlate to gestational age. If an equilibrium distribution of PS between bound and free form regulates the cofactor activity of PS for the anticoagulant and profibrino 1ytic properties of APC in normal adults, our study demonstrates that the absence of PS-C4b-BP complexes in newborns and the presence of free PS only may contribute to the increased bleeding risk of premature infants.

scholarly journals A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies

2005 ◽  
Vol 86 (5) ◽  
pp. 1435-1440 ◽  
Author(s):  
Milosz Faber ◽  
Elaine W. Lamirande ◽  
Anjeanette Roberts ◽  
Amy B. Rice ◽  
Hilary Koprowski ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Protein S ◽  
S Protein ◽  
Cellular Immune Responses ◽  
Glycoprotein G ◽  
Cellular Immune ◽  
Animal Reservoirs ◽  
S Genes

Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.

Sign in / Sign up

Export Citation Format

Share Document