Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response
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Abstract
There is a great need for the development of vaccines for preventing SARS-CoV-2 infection and mitigating the COVID-19 pandemic. Here, we developed two modified vaccinia Ankara (MVA) based vaccines which express either a membrane anchored full-length spike protein (MVA/S) stabilized in a prefusion state or the S1 region of the spike (MVA/S1) which forms trimers and is secreted. Both immunogens contained the receptor-binding domain (RBD) which is a known target of antibody-mediated neutralization. Following immunizations with MVA/S or MVA/S1, both spike protein recombinants induced strong IgG antibodies to purified full-length SARS-CoV-2 spike protein. The MVA/S induced a robust antibody response to purified RBD, S1 and S2 whereas MVA/S1 induced an antibody response to the S1 region outside of the RBD region. Both vaccines induced an antibody response in the lung and that was associated with induction of bronchus-associated lymphoid tissue. MVA/S but not MVA/S1 vaccinated mice generated robust neutralizing antibody responses against SARS-CoV-2 that strongly correlated with RBD antibody binding titers. Mechanistically, S1 binding to ACE-2 was strong but reduced following prolonged pre-incubation at room temperature suggesting confirmation changes in RBD with time. These results demonstrate MVA/S is a potential vaccine candidate against SARS-CoV-2 infection.
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SciScore for 10.1101/2020.06.27.175166: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Emory University. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Briefly, 6–8-week-old female BALB/c mice (n=5) were immunized with 10^7 plaque-forming-units (pfu) of rMVA/S or rMVA/S1 by intramuscular (i.m.) route on weeks 0 and 3. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The expression of spike on the MVA-infected cells from MVA/S construct was determined using anti-SARS-CoV-2 spike antibody (Cat #135356, GeneTex). anti-SARS-CoV-2suggested: NoneFor MVA/S1, anti … SciScore for 10.1101/2020.06.27.175166: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Emory University. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Briefly, 6–8-week-old female BALB/c mice (n=5) were immunized with 10^7 plaque-forming-units (pfu) of rMVA/S or rMVA/S1 by intramuscular (i.m.) route on weeks 0 and 3. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The expression of spike on the MVA-infected cells from MVA/S construct was determined using anti-SARS-CoV-2 spike antibody (Cat #135356, GeneTex). anti-SARS-CoV-2suggested: NoneFor MVA/S1, anti SARS-CoV-2 RBD antibody (Cat# 40592-T62, Sino Biological) was used to stain intracellularly. anti SARS-CoV-2 RBDsuggested: NoneMVA/S infected cells were harvested, stained with live dead marker and anti SARS-CoV-2 spike antibody (#GTX135356, GeneTex) followed by donkey anti-rabbit IgG coupled to PE (# 406421, BioLegend) on the surface. anti SARS-CoV-2suggested: Noneanti-rabbit IgGsuggested: (BioLegend Cat# 406421, RRID:AB_2563484)The membrane was washed in PBS containing Tween-20 (0.05%) and was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Southern Biotech) diluted 1:20,000 accordingly. anti-mousesuggested: Noneanti-rabbitsuggested: NonePlates were then incubated with serial dilutions of mouse sera for 2h at RT, washed 6 times and then incubated with 1: 6000 dilution of horseradish peroxidase (HRP) conjugated anti-mouse IgG secondary antibody (Southern Biotech; Birmingham, AL, USA) for 1 h at RT. anti-mouse IgGsuggested: NoneFor investigating the formation of iBALT B cell and T cells were stained in lungs using primary antibody cocktail containing rat anti-mouse CD45R/B220 (BioLegend, Cat#103202) and Hamster anti mouse CD3 (BD, Cat#550277) antibodies. anti-mouse CD45R/B220suggested: (Fluidigm Cat# 3144011B, RRID:AB_2811239)anti mouse CD3suggested: NoneSecondary antibodies cocktail contained goat antirat IgG-Alexa488 (abcam, Cat#ab150157) and goat anti-hamster IgG-Alexa546 (Thermofisher, Cat#A21111). antirat IgG-Alexa488suggested: Noneanti-hamster IgG-Alexa546suggested: NoneSystems serology by Luminex analysis: For relative quantification of antigen-specific antibody titers, a customized multiplexed approach was applied, as previously described (30). antigen-specificsuggested: NoneAnti-mouse IgG-, IgG2a-, IgG3-, IgA- and IgM-PE coupled (Southern Biotech) detection antibodies were diluted in Luminex assay buffer to 0.65ug/ml. Anti-mouse IgG-suggested: NoneFollowing washing of stained immune complexes, a tertiary goat anti-mouse IgG-PE antibody (Southern Biotech) was added and incubated for 1h at RT on a shaker. anti-mouse IgG-PEsuggested: NoneExperimental Models: Cell Lines Sentences Resources Similarly human ACE2 binding to surface expressed spike on MVA/S infected DF-1 cells was done using biotinylated human ACE2 protein (#10108-H08H-B, Sino Biological) followed by streptavidin-PE (BD Pharmingen) and intracellularly for MVA as described earlier. DF-1suggested: NoneProtein expression and purification: RBD-His and S1 proteins were produced in Amara laboratory by transfecting HEK293 cells using plasmids pCAGGS-RBD-His and pGA8-S1, respectively. HEK293suggested: NoneBriefly, HEK 293F cells were seeded at a density of 2×106 cells/ml in Expi293 expression medium and incubated in an orbital shaking incubator at 37°C and 127 rpm with 8% CO2 overnight. HEK 293Fsuggested: RRID:CVCL_6642)The stock virus was passaged twice (P2) and viral titers were determined by plaque assay on Vero E6 cells (ATCC). Vero E6suggested: RRID:CVCL_XD71)Vero cells were cultured in complete DMEM medium consisting of 1x DMEM (Corning Cellgro), 10% FBS, 25mM HEPES Buffer (Corning Cellgro), 2mM L-glutamine, 1mM sodium pyruvate, 1x Non-essential Amino Acids, and 1x antibiotics. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Experimental Models: Organisms/Strains Sentences Resources Briefly, 6–8-week-old female BALB/c mice (n=5) were immunized with 10^7 plaque-forming-units (pfu) of rMVA/S or rMVA/S1 by intramuscular (i.m.) route on weeks 0 and 3. BALB/csuggested: NoneSoftware and Algorithms Sentences Resources Briefly, Nunc high-binding ELISA plates (Thermo Fisher Scientific; Waltham, MA, USA) were coated with 2 μg/ml of recombinant SARS-CoV-2 proteins (S1 and RBD-His proteins were produced in the lab and S1 +S2 ECD spike protein was purchased from Sino Biologicals) in Dulbecco’s phosphate-buffered saline (DPBS) and incubated overnight at 4 °C. Thermo Fisher Scientificsuggested: (Thermo Fisher Scientific, RRID:SCR_008452)FRNT50 curves were generated by non-linear regression analysis using the 4PL sigmoidal dose curve equation on Prism 8 (Graphpad Software). Graphpadsuggested: (GraphPad, RRID:SCR_000306)The slides were imaged by Olympus FV1000 confocal imaging using 20X objective and images were analyzed using ImageJ. ImageJsuggested: (ImageJ, RRID:SCR_003070)Data analysis and statistical procedures: GraphPad Prism v7.0 was used to perform data analysis and statistics. 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 17. 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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- No protocol registration statement was detected.
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