Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine
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Abstract
Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We developed a novel vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we used this novel vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. Mice immunized with these sMVA vectors developed robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a novel vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.
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SciScore for 10.1101/2020.07.01.183236: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Mouse immunization: The Institutional Animal Care and Use Committee (IACUC) of the Beckman Research Institute of City of Hope (COH) approved protocol 20013 assigned for this study. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological) anti-SARS-CoV-1suggested: Noneanti-SARS-CoV1 NPsuggested: NoneAnti-rabbi… SciScore for 10.1101/2020.07.01.183236: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Mouse immunization: The Institutional Animal Care and Use Committee (IACUC) of the Beckman Research Institute of City of Hope (COH) approved protocol 20013 assigned for this study. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological) anti-SARS-CoV-1suggested: Noneanti-SARS-CoV1 NPsuggested: NoneAnti-rabbit polyclonal antibody conjugated with horseradish peroxidase (Sigma-Aldrich) was used as a secondary antibody and protein bands were visualized with chemiluminescent substrate (ThermoFisher). Anti-rabbitsuggested: NoneAnti-SARS-CoV-1 S1 mouse (40150-R007, Sino Biological) and S2 rabbit (GTX632604, GeneTex) monoclonal antibodies, anti-SARS-CoV-1 N rabbit monoclonal antibody (40143-R001, Sino Biological), and anti-vaccinia rabbit polyclonal antibody (9503-2057, Bio Rad) were used in dilution 1:2,000. GTX632604suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)anti-SARS-CoV-1 Nsuggested: Noneanti-vacciniasuggested: NoneOne hour later anti-mouse or anti-rabbit Alexa Fluor 488-conjugated secondary antibodies (A11001, A21206; Invitrogen) were added to the cells at a dilution of 1:4,000. anti-mousesuggested: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)After washing the plate, 1:3,000 dilution of HRP-conjugated anti-mouse IgG secondary antibody (W402B, Promega) was added and incubated for one additional hour. anti-mouse IgGsuggested: NoneThe assay was performed as described above except for the secondary antibodies (1:2,000. goat Anti-Mouse IgG2a cross absorbed HRP antibody, Southern biotech, 1083-05; Goat anti-Mouse IgG1 cross absorbed HRP antibody, Thermo Fisher, A10551). Anti-Mouse IgG2asuggested: (Thermo Fisher Scientific Cat# A10551, RRID:AB_2534048)anti-Mouse IgG1suggested: (Thermo Fisher Scientific Cat# A10551, RRID:AB_2534048)A10551suggested: NoneThe IgG2a/IgG1 ratio was calculated by dividing the absorbance read in the well incubated with the IgG2a secondary antibody divided the absorbance for the same sample incubated with the IgG1 antibody. IgG2asuggested: NoneIgG1suggested: NoneCross-adsorbed goat anti-human IgG (H+L) secondary antibody (A18811, Invitrogen) was used at a dilution of 1:3,000. anti-human IgGsuggested: (Thermo Fisher Scientific Cat# A18811, RRID:AB_2535588)A18811suggested: NoneAnti-mouse CD28 and CD49d antibodies (1μg/ml; BioLegend) were added as co-stimulation. Anti-mouse CD28suggested: NoneCD49dsuggested: NoneIn experiments testing double recombinants SARS-CoV2 vectors IL-2 antibody was not included and PE-CF594 anti-mouse IL-4 (clone 11B11, 562450, BD) and BV421 rat anti mouse IL-10 (clone JES5-16E3, 563276, BD) were added. IL-2suggested: Noneanti-mouse IL-4suggested: (Thermo Fisher Scientific Cat# 88-7771-88, RRID:AB_476398)BV421 rat anti mouse IL-10suggested: (BioLegend Cat# 505022, RRID:AB_2563240)antisuggested: NoneExperimental Models: Cell Lines Sentences Resources Cells and Viruses: BHK-21 (CCL-10), A549 (CCL-185), HeLa (CCL-2), 293T ( BHK-21suggested: NoneHeLasuggested: NoneCRL-1573), 143B (CRL-8303), MRC-5 (CCL-171), HEK293/17 (CRL11268), THP-1 (TIB-202), ARPE-19 (CRL-2302) were purchased from the American Type Culture Collection (ATCC) and grown according to ATCC recommendations. THP-1suggested: NoneHEK293T/ACE2 were a kind gift of Pamela J. HEK293T/ACE2suggested: NoneFully infected BHK cell monolayers were usually visible at 8-12 days post transfection/infection. sMVA virus from infected BHK cell monolayers was prepared by conventional freeze/thaw method and passaged once on BHK cells before producing ultra-purified virus stocks on CEF. sMVA or recombinant sMVA-CoV-2 vectors were reconstituted either with FPV HP1.441 (sMVA hp, sMVA-N/S, BHKsuggested: NoneHost cell range: sMVA and wtMVA host cell range using various human cell lines (HeLa, 293T, MRC-5, A549, and 143B) BHK cells, and CEF was determined as follows. 293Tsuggested: NoneMRC-5suggested: NoneA549suggested: NoneMVA neutralization assay: ARPE-19 cells were seeded in 96 well plates (1.5×104 cells/well). ARPE-19suggested: NoneHEK293T cells overexpressing ACE-2 receptor were seeded the day before transduction at a density of 2×105 cells per well in a 96-well plate in complete DMEM (47). HEK293Tsuggested: NoneFor the ADE assay, THP1 cells were seeded at a confluency of 2×106 cells/mL in a 96 well plate and co-incubated for 48 hours with serum samples diluted at 1:5,000 or 1:50,000 in the presence of SARS-CoV-2-Spike pseudotyped or VSV-G luciferase lentiviral vector, normalized to 100 ng/mL of p24 antigen. THP1suggested: NoneBriefly, HeLa-ACE2 cells were seeded in 12 μL complete DMEM at a density of 2×103 cells per well. HeLa-ACE2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources 6 weeks old C57BL/6 (C57BL/6J, 000664) or Balb/c (BALB/cJ, 000651) were purchased from the Jackson laboratories. C57BL/6suggested: NoneC57BL/6Jsuggested: RRID:IMSR_JAX:000664)Balb/csuggested: RRID:IMSR_JAX:000651)BALB/cJsuggested: RRID:IMSR_JAX:000651)Software and Algorithms Sentences Resources De novo assembly was done using either canu v1.7.1 or wtdbg2 v2.5. canusuggested: (Canu, RRID:SCR_015880)Images were acquired and processed using Zen software (Zeiss) Zensuggested: NonePictures from each well were processed using Image-Pro Premier (Media Cybernetics) and the fluorescent area corresponding to the area covered by MVA-Venus infected cells was calculated. Image-Pro Premiersuggested: (Image-Pro Premier , RRID:SCR_016497)Analysis was performed using FlowJo. FlowJosuggested: (FlowJo, RRID:SCR_008520)Statistics: Statistical evaluation was pursued using GraphPad Prism (v8.3.0). 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 did not find any issues relating to the usage of bar graphs.
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.
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