BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Effective vaccine against SARS-CoV-2 is the utmost importance in the current world. More than 1 million deaths are accounted for relevant pandemic disease COVID-19. Recent data showed that D614G genotype of the virus is highly infectious and responsible for almost all infection for 2 nd wave. Despite of multiple vaccine development initiatives, there are currently no report that has addressed this critical variant D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine considering the D614G variant and characterization of the vaccine in preclinical trial. The surface plasmon resonance (SPR) data with spike protein as probe and competitive neutralization with RBD and S2 domain revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice on day 7 and 14 neutralized SARS-CoV-2 pseudovirus in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, immunization protected mice lungs from pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4 + cells with a Th1 bias. The IgG2a to IgG1 and (IgG2a+IgG2b) to (IgG1+IgG3) ratios were found 1±0.2 and 1.24±0.1, respectively. These values are comparatively higher than relevant values for other published SARS-CoV-2 vaccine in development, 1, 2 and suggesting higher viral clearance capacity for our vaccine. The data suggested great promise for immediate translation of the technology to the clinic.
Article activity feed
-
SciScore for 10.1101/2020.09.29.319061: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The study plan and procedures were approved by the internal ethical review board, which is complied with local ethical regulation. Randomization Target gene and vector cloning: mRNA production: Formulation of mRNA: Safety and efficacy in mice: A total number of 50 BALB/c swiss albino mice (male and female) of 6-8 weeks old, were selected randomly and isolated 5 days before immunization. Blinding No treatment randomization and blinding methods were used in the study and sample sizes were determined by the resource equation method. Power Analysis not detected. Sex as a biological variable Target gene and vector cloning: mRNA production: Formulation of mRNA: Safety … SciScore for 10.1101/2020.09.29.319061: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The study plan and procedures were approved by the internal ethical review board, which is complied with local ethical regulation. Randomization Target gene and vector cloning: mRNA production: Formulation of mRNA: Safety and efficacy in mice: A total number of 50 BALB/c swiss albino mice (male and female) of 6-8 weeks old, were selected randomly and isolated 5 days before immunization. Blinding No treatment randomization and blinding methods were used in the study and sample sizes were determined by the resource equation method. Power Analysis not detected. Sex as a biological variable Target gene and vector cloning: mRNA production: Formulation of mRNA: Safety and efficacy in mice: A total number of 50 BALB/c swiss albino mice (male and female) of 6-8 weeks old, were selected randomly and isolated 5 days before immunization. Table 2: Resources
Experimental Models: Organisms/Strains Sentences Resources Target gene and vector cloning: mRNA production: Formulation of mRNA: Safety and efficacy in mice: A total number of 50 BALB/c swiss albino mice (male and female) of 6-8 weeks old, were selected randomly and isolated 5 days before immunization. BALB/c swiss albinosuggested: NoneSoftware and Algorithms Sentences Resources Repeat the step. Repeatsuggested: (ProRepeat, RRID:SCR_006113)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 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 40. 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.
-
SciScore for 10.1101/2020.09.29.319061: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement The study plan and procedures were approved by the internal ethical review board, which is complied with local ethical regulation. Randomization Safety and efficacy in mice A total number of 50 BALB/c swiss albino mice (male and female) of 6-8 weeks old, were selected randomly and isolated 5 days before immunization. Blinding No treatment randomization and blinding methods were used in the study and sample sizes were determined by the resource equation method. Power Analysis not detected. Sex as a biological variable Target amplification Nasopharyngeal and oropharyngeal swab sample were collected from a COVID-19 positive male patient. Cell Line Authentication not detected. T…
SciScore for 10.1101/2020.09.29.319061: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement The study plan and procedures were approved by the internal ethical review board, which is complied with local ethical regulation. Randomization Safety and efficacy in mice A total number of 50 BALB/c swiss albino mice (male and female) of 6-8 weeks old, were selected randomly and isolated 5 days before immunization. Blinding No treatment randomization and blinding methods were used in the study and sample sizes were determined by the resource equation method. Power Analysis not detected. Sex as a biological variable Target amplification Nasopharyngeal and oropharyngeal swab sample were collected from a COVID-19 positive male patient. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The immobilization procedure was performed by using running buffer HBS-EP, pH 7.4 (GE Healthcare, USA). samples containing 1 µL mouse serum each were analyzed using surface plasmon resonance (SPR) technology to analyze the binding affinity of the antibody pool. SPRsuggested: NoneIntracellular cytokine staining of cells were stained with following antibodies with maintaining supplier’s instructions: V500 anti-mouse CD45 (BD Bioscience, USA) anti-mouse CD45suggested: NoneAlexa Fluor® 594 conjugate secondary antibody (ThermoFisher, USA), in-house developed TNF alpha fusion protein, anti Fc primary antibody (ThermoFisher, USA) anti Fcsuggested: NoneIL-2 and Il-6 titer ELISA plate (Corning) was coated with 1µg/mL IL-2 polyclonal antibody (ThermoFisher, USA) in Dulbecco’s phosphate-buffered saline (DPBS) (ThermoFisher, USA) for 2 hours at room temperature. IL-2suggested: NoneAfter washing for 3 times, the plate was again incubated with Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (ThermoFisher, USA) for 50 min at room temperature. anti-Mouse IgGsuggested: NoneTo check whether the immunization have generated antibody pool spanning for the whole antigen or for any specific domain (S1 or S2), we have chosen surface plasmon resonance (SPR) experiment. S1suggested: NoneS2suggested: NoneExperimental Models: Cell Lines Sentences Resources In-vitro neutralization ACE2 overexpressing HEK293 cell (Innoprot, Spain) were seeded in a two 96 well TC treated plate at a concentration of 2.2 × 104 cells per well and overnight incubation was performed. HEK293suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Safety and efficacy in mice A total number of 50 BALB/c swiss albino mice (male and female) of 6-8 weeks old, were selected randomly and isolated 5 days before immunization. BALB/c swiss albinosuggested: NoneImmunogenicity The immunogenicity of BANCOVID was evaluated in BALB/c mice, post administration to the quadriceps muscle. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)Software and Algorithms Sentences Resources Amplified S-gene and polymerase chain reaction (PCR) engineered pET31b(+) (Novagen, Germany) bacterial expression vector were amplified using 0570F and 0571R primers, excised and extracted from agarose gel using GeneJET Gel Extraction and DNA Cleanup Micro Kit (ThermoFisher, USA), using Clustal alignment was Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) identified (data not shown) using EMBOSS showed Cons and assembled together using NEBuilder® HiFi DNA Assembly Master Mix (NEB, USA). https://www.ebi.ac.uk/Tools/msa/clustalo/suggested: (Clustal Omega, RRID:SCR_001591)EMBOSSsuggested: (EMBOSS, RRID:SCR_008493)mRNA production mRNA synthesis The in-vitro (IVT) mRNA synthesis reaction was performed using MEGAscript™ T7 Transcription Kit (ThermoFisher, USA), and Ribonucleotide Solution Set (NEB, USA). MEGAscript™suggested: NoneCellular immunogenicity SARS-CoV-2 surface glycoprotein peptide mapping SARS-CoV-2 Spike S1+S2 ECD His recombinant protein (Sino Biological, China), S2 ECD- His Recombinant Protein (Sino Biological, China), and RBD-His Recombinant Protein (Sino Biological, China) were digested and purified according to ThermoFisher Pierce Trypsin Protease, MS grade instructions (supplementary method 2). ThermoFisher Pierce Trypsin Proteasesuggested: NoneAfter getting raw data from mass spectrometry system, data analysis was performed in BioPharma Finder (ThermoFisher, USA) using variable parameters to get confident data, and then data were combined in one map to visualize complete fragmentation (supplementary figure 6). BioPharma Findersuggested: NoneOn the other hand, full length SARS-CoV-2 surface glycoprotein was digested computationally (ExPASy PeptideMass: https://web.expasy.org/peptide_mass/) via trypsin (supplementary figure 7) ExPASysuggested: NoneAfter hours, cell events were acquired using an FACS Lyric (BD Biosciences), followed by FlowJo software (FlowJo LLC, Ashland, OR) analysis (supplementary figure 8, 9, 10). FlowJosuggested: (FlowJo, RRID:SCR_008520)Results from OddPub: Thank you for sharing your data.
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: Please consider improving the rainbow (“jet”) colormap used on page 40. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
-
