Genetic Modification to Design a Stable Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain as a COVID-19 Vaccine Candidate
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Abstract
Background
Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed.
Method
We constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a “wild type” RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs.
Results and conclusions
These three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation.
General significance
By genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.
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SciScore for 10.1101/2020.11.09.373449: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources The final buffer for these three proteins was TBS (20 mM Tris, 150 mM NaCl, pH 7.5). 2.3. SDS-PAGE and Western Blot: RBD219-WT, RBD219-N1+His, and RBD219-N1C1 were loaded on 4-20% Tris-glycine gels, and stained with Coomassie Blue or transferred to a polyvinylidene difluoride membrane and probed with a monoclonal anti-SARS-CoV-2 Spike rabbit antibody recognizing the RBD region (Sino Biological, Beijing, China; Cat # 40150-R007) to evaluate the size and confirm the identity. RBD219-N1+Hissuggested: Noneanti-SARS-CoV-2suggested: NoneAfter this binding step, the plates were washed with PBST four times … SciScore for 10.1101/2020.11.09.373449: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources The final buffer for these three proteins was TBS (20 mM Tris, 150 mM NaCl, pH 7.5). 2.3. SDS-PAGE and Western Blot: RBD219-WT, RBD219-N1+His, and RBD219-N1C1 were loaded on 4-20% Tris-glycine gels, and stained with Coomassie Blue or transferred to a polyvinylidene difluoride membrane and probed with a monoclonal anti-SARS-CoV-2 Spike rabbit antibody recognizing the RBD region (Sino Biological, Beijing, China; Cat # 40150-R007) to evaluate the size and confirm the identity. RBD219-N1+Hissuggested: Noneanti-SARS-CoV-2suggested: NoneAfter this binding step, the plates were washed with PBST four times followed by adding 100 μL 1:10,000 diluted HRP conjugated anti-human IgG antibodies (GenScript, Piscataway, NJ, USA; Cat # A00166) and incubating for 1 h at room temperature. anti-human IgGsuggested: NoneResults 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 did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 23. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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.
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SciScore for 10.1101/2020.11.09.373449: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources The final buffer for these three proteins was TBS (20 mM Tris, 150 mM NaCl, pH 7.5). 2.3. SDS-PAGE and Western Blot RBD219-WT, RBD219-N1+His, and RBD219-N1C1 were loaded on 4-20% Tris-glycine gels, and stained with Coomassie Blue or transferred to a polyvinylidene difluoride membrane and probed with a monoclonal anti-SARS-CoV-2 Spike rabbit antibody recognizing the RBD region (Sino Biological, Beijing, China; Cat # 40150-R007) to evaluate the size and confirm the identity. …RBD219-N1+Hissuggested: NoneSciScore for 10.1101/2020.11.09.373449: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources The final buffer for these three proteins was TBS (20 mM Tris, 150 mM NaCl, pH 7.5). 2.3. SDS-PAGE and Western Blot RBD219-WT, RBD219-N1+His, and RBD219-N1C1 were loaded on 4-20% Tris-glycine gels, and stained with Coomassie Blue or transferred to a polyvinylidene difluoride membrane and probed with a monoclonal anti-SARS-CoV-2 Spike rabbit antibody recognizing the RBD region (Sino Biological, Beijing, China; Cat # 40150-R007) to evaluate the size and confirm the identity. RBD219-N1+Hissuggested: Noneanti-SARS-CoV-2suggested: (Abcam Cat# ab272854, RRID:AB_2847844)After this binding step, the plates were washed with PBST four times followed by adding 100 µL 1:10,000 diluted HRP conjugated anti-human IgG antibodies (GenScript, Piscataway, NJ, USA; Cat # A00166) and incubating for 1 h at room temperature. anti-human IgGsuggested: NoneResults 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: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04466085 Recruiting Clinical Study of Recombinant Novel Coronavirus Vaccine NCT04522089 Recruiting A Study to Evaluate the Safety and Immunogenicity of COVID-1... Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 23, 23 and 23. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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