Broad neutralization of SARS-CoV-2 variants by circular mRNA producing VFLIP-X spike in mice
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X elicited neutralizing antibodies for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in T H 1 and T H 2 responses was achieved by immunization with VFLIP-X. Our results indicate that the VFLIP-X delivered by circRNA confers humoral and cellular immune responses, as well as neutralizing activity against broad SARS-CoV-2 variants.
Article activity feed
-
-
SciScore for 10.1101/2022.03.17.484759: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Mouse immunization: Mouse experiments were performed under the Animal Ethics approved by Faculty of Science, Mahidol University (MUSC63-016-524). Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Blotted proteins were detected with a rabbit polyclonal antibody that recognizes SARS-CoV-2 spike RBD (40592-T62, SinoBiological) and a donkey anti-rabbit IgG-HRP antibody (sc-2077, Santa Cruz Biotechnology). anti-rabbit IgG-HRPsuggested: (Santa Cruz Biotechnology Cat# sc-2077, RRID:AB_631745)sc-2077suggested: NoneFlow cytometry: To detect surface … SciScore for 10.1101/2022.03.17.484759: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Mouse immunization: Mouse experiments were performed under the Animal Ethics approved by Faculty of Science, Mahidol University (MUSC63-016-524). Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Blotted proteins were detected with a rabbit polyclonal antibody that recognizes SARS-CoV-2 spike RBD (40592-T62, SinoBiological) and a donkey anti-rabbit IgG-HRP antibody (sc-2077, Santa Cruz Biotechnology). anti-rabbit IgG-HRPsuggested: (Santa Cruz Biotechnology Cat# sc-2077, RRID:AB_631745)sc-2077suggested: NoneFlow cytometry: To detect surface protein expression, transfected HEK293T cells were stained with a rabbit polyclonal antibody that recognizes SARS-CoV-2 spike RBD (40592-T62, SinoBiological) and an Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11034, Invitrogen). A11034suggested: NoneCells were then blocked with 20% FBS in PBS and incubated with a rabbit polyclonal antibody that recognizes SARS-CoV-2 spike RBD (40592-T62, SinoBiological) followed by an Alexa Fluor 594-conjugated goat anti-rabbit IgG (A11037, Invitrogen). anti-rabbit IgGsuggested: NoneA11037suggested: (Thermo Fisher Scientific Cat# A-11037, RRID:AB_2534095)1.1.529 S 15-mer (overlapping by 11 amino acids) peptide pool (GenScript) in plates pre-coated with anti-IFN-γ or anti-IL-4 antibodies. anti-IFN-γsuggested: Noneanti-IL-4suggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293T-hACE2 cells were cultured with 1 μg/ml puromycin to maintain stable expression of hACE2. HEK293T-hACE2suggested: RRID:CVCL_A7UK)Immunofluorescence assay: Transfected HEK293T cells were fixed with 4% paraformaldehyde (PFA). HEK293Tsuggested: RRID:CVCL_HA71)Briefly, serially diluted heat-inactivated sera (starting with a dilution of 1:20) were pre-incubated with equal volumes of 100 TCID50 of SARS-CoV-2 for one hour at 37°C. 100 ml of the virus-serum mixtures were added to pre-seeded Vero E6/TMPRSS2 cell monolayers (1×104 cells/well) in duplicate on a 96-well microtiter plate. Vero E6/TMPRSS2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources In vivo bioluminescence imaging: For detection of in vivo expression of FLuc-encoding circRNA-LNP, 7-week-old female BALB/c mice were inoculated with 1 μg of the circRNA-LNP via intramuscular route. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)Recombinant DNA Sentences Resources Briefly, the SARS-CoV-2 pseudovirus were produced by co-transfection of plasmids pHAGE-CMV-Luc2-IRES-ZsGreen-W, HDM-Hgpm2, HDM-tat1b, pRC-CMV-Rev1b, and SARS-CoV-2 spike expressing plasmid into HEK293T cells using jetPRIME transfection reagent. pHAGE-CMV-Luc2-IRES-ZsGreen-Wsuggested: RRID:Addgene_164432)pRC-CMV-Rev1bsuggested: RRID:Addgene_164443)Software and Algorithms Sentences Resources An unrooted phylogenetic tree comparing amino acid sequences derived from wildtype spike, SARS-CoV-2 spike variants of concern (VOCs) and variants of interest (VOIs), and wildtype-X spike (wildtype spike with the six rationally substituted amino acids) was constructed by maximum likelihood using IQ-TREE software. IQ-TREEsuggested: (IQ-TREE, RRID:SCR_017254)Cells were then acquired on a BD Accuri C6 plus and analyzed by FlowJo software version 10.6.2. FlowJosuggested: (FlowJo, RRID:SCR_008520)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.
Results from scite Reference Check: We found no unreliable references.
-