Engineering, production and characterization of Spike and Nucleocapsid structural proteins of SARS–CoV-2 in Nicotiana benthamiana as vaccine candidates against COVID-19
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
The 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.
Article activity feed
-
SciScore for 10.1101/2020.12.29.424779: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Mice studies were carried out at Akdeniz University Experimental Animal CareUnit under permission of the Local Ethics Committee for Animal Experiments at Akdeniz University with thesupervision of a veterinarian. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources After transfer, Western blot membranes were blocked with I-Block (Applied Biosystems, Carlsbad, CA) and recombinant proteins N, RBD, S1 and N+RBD were detected with anti-DYKDDDDK antibody (cat. no. 651503, BioLegend, for N, RBD or N+RBD), anti-DYKDDDDKsuggested: Noneanti-His antibody (for … SciScore for 10.1101/2020.12.29.424779: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Mice studies were carried out at Akdeniz University Experimental Animal CareUnit under permission of the Local Ethics Committee for Animal Experiments at Akdeniz University with thesupervision of a veterinarian. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources After transfer, Western blot membranes were blocked with I-Block (Applied Biosystems, Carlsbad, CA) and recombinant proteins N, RBD, S1 and N+RBD were detected with anti-DYKDDDDK antibody (cat. no. 651503, BioLegend, for N, RBD or N+RBD), anti-DYKDDDDKsuggested: Noneanti-His antibody (for S1) and anti-RBD of S protein of SARC-CoV-2 monoclonal antibody (cat. no. MBS7135930, for RBD) or Human Novel Coronavirus Nucleoprotein (N) (1-419aa) monoclonal Antibody (MyBioSource, cat. no. MBS7135930). 1-419aasuggested: NoneAfter washing, wells were incubated with anti-rabbit IgG antibody (Cat. no. MBS440123, MyBioSource, USA). anti-rabbit IgGsuggested: (MyBioSource Cat# MBS440123, RRID:AB_10571491)Serum samples were collected on days −1 (pre-bleed) and 42 (post 2nd vaccination) and assessed for anti-N, anti-RBD, anti-N+RBD and anti-S1 domain antibody responses by an IgG ELISA. anti-Nsuggested: Noneanti-RBDsuggested: Noneanti-N+RBDsuggested: Noneanti-S1 domainsuggested: NoneAfter transfer, Western blot membranes were blocked with I-Block (Applied Biosystems, Carlsbad, CA) and recombinant proteins detected with an anti-FLAG (N, S1 and N+S1) or anti-His Antibody (S2), or anti-SARS-COV2 COVID 19 Spike Protein Coronavirus Monoclonal Antibody (MyBioSource, cat. no. MBS2563837). anti-FLAGsuggested: Noneanti-Hissuggested: Noneanti-SARS-COV2 COVID 19 Spike Protein Coronavirussuggested: NoneSoftware and Algorithms Sentences Resources Total protein content was estimated using the BioDrop and then analyzed by SDS-PAGE and western blot. BioDropsuggested: NoneThe degradation of gRBD, dRBD or gS1 protein bands were calculated and quantified based on SDS-PAGE and WB analysis using highly sensitive Gene Tools software (Syngene Bioimaging, UK) and ImageJ software as described previously21. ImageJsuggested: (ImageJ, RRID:SCR_003070)GraphPad Prism software was used for all statistical analyses. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-