Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19
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Abstract
The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 is a worldwide health emergency. The immense damage done to public health and economies has prompted a global race for cures and vaccines. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein by adding two proline substitutions at the top of the central helix (S-2P). To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P was combined with various adjuvants, including CpG 1018, and administered to mice to test its effectiveness in eliciting anti-SARS-CoV-2 neutralizing antibodies. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of spike-specific antibodies in sera of immunized mice. The neutralizing abilities in pseudotyped lentivirus reporter or live wild-type SARS-CoV-2 were measured with reciprocal inhibiting dilution (ID 50 ) titers of 5120 and 2560, respectively. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th-1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens with up to 50 μg of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018/alum as a candidate vaccine to prevent COVID-19 disease.
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SciScore for 10.1101/2020.08.11.245704: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal work was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Female BALB/cJ mice aged 6–9 weeks were immunized twice at 3 weeks apart as previously described [8]. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Pseudovirus production and titration: To produce SARS-CoV-2 pseudoviruses, a plasmid expressing full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein was cotransfected into HEK293T cells with packaging and reporter plasmids pCMVΔ8.91 and … SciScore for 10.1101/2020.08.11.245704: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal work was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Female BALB/cJ mice aged 6–9 weeks were immunized twice at 3 weeks apart as previously described [8]. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Pseudovirus production and titration: To produce SARS-CoV-2 pseudoviruses, a plasmid expressing full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein was cotransfected into HEK293T cells with packaging and reporter plasmids pCMVΔ8.91 and pLAS2w.FLuc. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)In brief, HEK-293T cells stably expressing human ACE2 gene were plated on 96-well plate one day before lentivirus transduction. HEK-293Tsuggested: NonePseudovirus-based neutralization assay: HEK293-hAce2 cells (2×104 cells/well) were seeded in 96-well white isoplates and incubated for overnight. HEK293-hAce2suggested: NoneVero E6 cells (2.5×104 cells/well) were seeded in 96-well plates and incubated overnight. Vero E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Pseudovirus production and titration: To produce SARS-CoV-2 pseudoviruses, a plasmid expressing full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein was cotransfected into HEK293T cells with packaging and reporter plasmids pCMVΔ8.91 and pLAS2w.FLuc. Wuhan-Hu-1suggested: NoneFemale BALB/cJ mice aged 6–9 weeks were immunized twice at 3 weeks apart as previously described [8]. BALB/cJsuggested: RRID:IMSR_JAX:000651)Dose range finding study for single and repeat-dose intramuscular injection (IM) in Sprague Dawley (SD) Rats: To investigate the safety of SARS-CoV-2 S-2P protein adjuvanted with CpG 1018 alone or combined with aluminum hydroxide, pilot toxicity studies were conducted for dose range finding. Sprague Dawleysuggested: NoneSoftware and Algorithms Sentences Resources The purified S-2P proteins produced from Expi293 and ExpiCHO-S cells were quantified by BCA assay (ThermoFisher), flash frozen in liquid nitrogen and then stored at −80 °C. ThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)Animals: BALB/cJ mice were obtained from the National Laboratory Animal Center, Academia Sinica, Taiwan and BioLASCO Taiwan Co. Ltd. Crl:CD® Sprague Dawley (SD) rats were obtained from BioLASCO Taiwan Co. Ltd. BioLASCOsuggested: NoneMann-Whitney U-test included in the analysis package in Prism 6.01 (GraphPad) was used to compare between two experimental groups. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 GraphPadsuggested: (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.
- No funding statement was detected.
- No protocol registration statement was detected.
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