CoChat
Sign Up
PreprintCited by 636SARS-CoV-2 and COVID-19 ResearchCircular RNAs in diseasesAnimal Virus Infections Studies

Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

Open access
Qu, Liang, Yi, Zongyi, Shen, Yong, Lin, Liangru, Chen, Feng, Xu, Yiyuan, Wu, Zeguang, Tang, Huixian +22 more·2021·Cell
DOI 10.1101/2021.03.16.435594PMC PMC8971115
Open this paper in CoChat — free account

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants of concern (VOC), such as Delta (B.1.617.2) and Omicron (B.1.1.529), has continued to drive the worldwide pandemic. Therefore, there is a high demand for vaccines with enhanced efficacy, high thermostability, superior design flexibility, and fast manufacturing speed. Here, we report a circular RNA (circRNA) vaccine that encodes the trimeric RBD of SARS-CoV-2 Spike protein. Without the need of nucleotide modification, 5’-capping or 3’-polyadenylation, circRNA could be rapidly produced via in vitro transcription and is highly thermostable whether stored in naked or lipid-nanoparticle (LNP)-encapsulated format. LNP-encapsulated circRNA RBD elicited potent neutralizing antibodies and T cell responses, providing robust protection against Beta (B.1.351) and native viruses in mice and rhesus macaques, respectively. Notably, circRNA vaccine enabled higher and more durable antigen production than 1mΨ-modified mRNA vaccine, eliciting a higher proportion of neutralizing antibodies and stronger Th1-biased immune responses. Importantly, we found that circRNA RBD-Omicron vaccine induced effective neutralizing antibodies against only Omicron but not Delta variant. By contrast, circRNA RBD-Delta could elicit high level of neutralizing antibodies against both Delta and Omicron. Following two doses of either native- or Delta-specific vaccination, circRNA RBD-Delta , but not Omicron or Beta vaccines, could effectively boost the neutralizing antibodies against both Delta and Omicron variants. These results suggest that circRNA RBD-Delta is a favorable choice for vaccination to provide a broad-spectrum protection against the current variants of concern of SARS-CoV-2.

AI snapshot

Written from the paper’s full text.

TL;DR

A circular RNA vaccine expressing trimeric SARS-CoV-2 RBD elicited neutralizing antibodies and T cell responses in mice and rhesus macaques, with higher and more durable antigen expression than 1mΨ-modified mRNA. The circRNARBD-Delta vaccine showed broad neutralization against Delta and Omicron and was an effective booster, whereas the Omicron-specific vaccine neutralized Omicron but not Delta.

Key findings

  • LNP-circRNARBD induced RBD-specific IgG endpoint GMTs of ∼1.9 × 10⁴ (10 μg) and ∼5.7 × 10⁵ (50 μg) in mice, with pseudovirus NT50 ∼4.5 × 10³ and authentic SARS-CoV-2 NT50 ∼7.0 × 10⁴.
  • circRNARBD-Delta elicited an NT50 of ∼1.4 × 10⁵ against Delta pseudovirus at 10 μg and maintained Omicron neutralization with NT50 ∼4.7 × 10³; as a third booster after two-dose circRNARBD, it raised Omicron geometric mean NT50 from ∼4 × 10² to ∼3.2 × 10⁴.
  • circRNARBD-Omicron induced Omicron-specific IgG GMTs of ∼4.7 × 10⁴ (5 μg) and ∼2.2 × 10⁵ (10 μg), with Omicron NT50s of ∼2.5 × 10³ and ∼8.6 × 10³, but neutralizing activity was hardly detected against native strain or Delta.
  • In rhesus macaques, circRNARBD IgG GMTs were ∼2.1 × 10⁴ (20 μg), ∼1.6 × 10⁴ (100 μg), and ∼7 × 10³ (500 μg); authentic virus NT50s were ∼80, ∼120, and ∼50, respectively. Vaccinated macaques showed nearly 1,000-fold reduced lung genomic RNA at 100/500 μg and nearly no detectable subgenomic RNA at all doses, with no severe clinical adverse effects.

Methods

  • circRNA vaccines encoded trimeric SARS-CoV-2 RBD with a human tPA signal peptide and T4 fibritin foldon, produced by group I intron autocatalysis or T4 RNA ligase, and encapsulated in LNPs; comparisons used 1mΨ-modified and unmodified mRNA encoding the same RBD sequence.
  • Female BALB/c mice (6–8 weeks) were immunized intramuscularly with varying circRNA doses; 2–4-year-old male rhesus macaques received 20, 100, or 500 μg circRNARBD, circRNACtrl, or PBS on days 0 and 21. Mice were challenged with Beta variant; macaques were challenged with native SARS-CoV-2.
  • Analyses included ELISA endpoint GMTs, pseudovirus and authentic virus NT50 assays, RT-qPCR viral loads, ELISpot and flow cytometry for T cell cytokines, histopathology, and unpaired or paired two-sided Student’s t tests.

Limitations

  • Stated by the paper: small rhesus macaque numbers caused high variation and large error bars; immunogenicity of IVT-produced circRNAs remains a potential concern; safety awaits further NHP and clinical trials; the mRNA comparator encoded trimeric RBD and differed from mRNA-1273 and BNT162b2, which encode full-length spike and use different manufacturing processes.
  • Evident from the text: no human clinical data are reported; protection against Omicron and Delta was assessed mainly by neutralization assays, while mouse challenge used Beta and macaque challenge used native SARS-CoV-2.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1101/2021.03.16.435594

Cites

109 of 109 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 54 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Abundant RBD antigens were detected in the supernatant of both human HEK293T and murine NIH3T3 cells transfected with purified circRNARBD.

    “Abundant RBD antigens in the supernatant of both human and murine cells were detected by western blot, indicating the high compatibility of circRNAs (Figure 1B).”
    Quote in the paperSubstantial claim
  • Findingown data

    The circRNARBD vaccine elicited a high level of RBD-specific IgG endpoint GMTs, reaching ∼1.9 × 10^4 for the 10-μg dose and ∼5.7 × 10^5 for the 50-μg dose in BALB/c mice.

    “The circRNARBD elicited a high level of RBD-specific IgG endpoint geometric mean titers (GMTs), reaching ∼1.9 × 104 for the 10-μg dose and ∼5.7 × 105 for the 50-μg dose (Figure 1I).”
    Quote in the paperSubstantial claim
  • Findingown data

    The RBD-Beta-specific IgG endpoint GMT elicited by circRNARBD-Beta was ∼1.6 × 10^4 at 1 week after the boost.

    “ELISA showed that the RBD-Beta-specific IgG endpoint GMT was ∼1.6 × 104 at 1 week after the boost (Figure 1L).”
    Quote in the paperNot a substantial claim
  • Findingown data

    The concentration of RBD antigens produced by circRNARBD reached ∼1,400 ng/mL, 600-fold higher than those produced by its linear precursor RNA.

    “The concentration of RBD antigens produced by circRNARBD reached ∼1,400 ng/mL, 600-fold higher than those produced by its linear precursor RNA (Figure 1D).”
    Quote in the paperSubstantial claim
  • Findingown data

    Sera from circRNARBD-vaccinated mice neutralized authentic SARS-CoV-2 virus with an NT50 of ∼7.0 × 10^4.

    “and authentic SARS-CoV-2 virus with an NT50 of ∼7.0 × 104 (Figure 1K).”
    Quote in the paperSubstantial claim
  • May combine two claims. 41
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 12

  • May combine two claims.

    Findingcitation

    The Beta variant could infect wild-type BALB/c mice and replicate in their lungs.

    “Consistent with a recent report (Gu et al., 2020; Chen et al., 2021; Montagutelli et al., 2021), the Beta variant could infect wild-type BALB/c mice and replicate in their lungs (Figure 1Q), likely due to mutations in the spike such as K417N, E484K, and N501Y.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Gu et al., 2020

      Not checked in the free run.

    • cites Chen et al., 2021

      Not checked in the free run.

    • cites Montagutelli et al., 2021

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Purified circRNARBD fractions were mostly resistant to exonuclease-RNase R, whereas nicked RNARBD were almost completely degraded, indicating that purified circRNARBD were mostly in circular format.

    “We found that the majority of the purified circRNARBD fractions were resistant to exonuclease-RNase R, whereas the nicked RNARBD were almost completely degraded, indicating that purified circRNARBD were mostly in circular format (Figure S1F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The circRNARBD encapsulation efficiency was greater than 93%, with an average diameter of 100 nm.

    “The circRNARBD encapsulation efficiency was greater than 93%, with an average diameter of 100 nm (Figure 1G).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    CircRNARBD-Beta-elicited antibodies could neutralize all three pseudoviruses (D614G, Alpha, Beta), with the highest activity against the Beta variant.

    “The circRNARBD-Beta-elicited antibodies could also neutralize all three pseudoviruses, with the highest activity against its corresponding Beta variant (Figure 1N).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CircRNARBD-elicited antibodies could effectively neutralize all three pseudoviruses (D614G, Alpha, Beta), with the highest neutralizing activity against the native (D614G) strain.

    “Pseudovirus neutralization assays revealed that circRNARBD-elicited antibodies could effectively neutralize all three pseudoviruses, with the highest neutralizing activity against the native (D614G) strain (Figure 1M).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    With the help of foldon, circRNARBD encoded stable homogeneous RBD trimers in the supernatant, which dissociated into monomers under reducing conditions.

    “With the help of foldon, the circRNARBD encoded stable homogeneous RBD trimers in the supernatant, which were dissociated into monomers under reducing conditions (Figure 1C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The innate immune responses elicited by LNP-encapsulated circRNARBD were comparable with those by LNP-encapsulated 1mΨ-mRNARBD and significantly lower than those by the transfected RNAs.

    “Importantly, we found that the innate immune responses elicited by LNP-encapsulated circRNARBD were comparable with those by LNP-encapsulated 1mΨ-mRNARBD and significantly lower than those by the transfected RNAs (Figure 3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sera from circRNARBD-Delta-vaccinated mice effectively neutralized the Delta pseudovirus in a dose-dependent manner, with an NT50 of ∼1.4 × 10^5 for the 10-μg dose.

    “The sera from circRNARBD-Delta-vaccinated mice effectively neutralized the Delta pseudovirus in a dose-dependent manner, with an NT50 of ∼1.4 × 105 for the 10-μg dose (Figure 2C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The circRNA produced much higher levels of RBD antigens at all time points than both 1mΨ-mRNA and unmodified mRNA, and they were maintained for a longer period.

    “The circRNA produced much higher levels of RBD antigens at all time points than both 1mΨ-mRNA and unmodified mRNA, and they were maintained for a longer period (Figure 3 A).”
    • Also stated in: § Abstract · plain
      “Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sera from circRNARBD-Delta-immunized mice exhibited the highest neutralizing activity against Delta and the lowest against Beta.

    “The sera from circRNARBD-Delta-immunized mice exhibited the highest neutralizing activity against Delta and the lowest against Beta (Figures 2D and 2E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant.

    “Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In rhesus macaques immunized with circRNARBD, IgG endpoint GMTs reached approximately 2.1 × 10^4 for 20 μg, approximately 1.6 × 10^4 for 100 μg, and approximately 7 × 10^3 for 500 μg, whereas circRNACtrl or PBS failed to induce RBD-specific antibodies.

    “The IgG endpoint GMTs reached ∼2.1 × 104 (20-μg dose), ∼1.6 × 104 (100-μg dose), and ∼7 × 103 (500-μg dose) for circRNARBD vaccines, whereas circRNACtrl- or PBS-immunized rhesus macaques failed to induce RBD-specific antibodies (Figure 6B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Authentic SARS-CoV-2 neutralization assay showed NT50 values of approximately 80 for the 20-μg dose, approximately 120 for the 100-μg dose, and approximately 50 for the 500-μg dose in circRNARBD-immunized rhesus macaques.

    “The authentic SARS-CoV-2 neutralization assay showed NT50 values of ∼80 for the 20-μg dose, ∼120 for the 100-μg dose, and ∼50 for the 500-μg dose (Figures 6D and 6E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ELISpot assay showed evident IFN-γ and IL-2 responses but nearly undetectable IL-4 in circRNARBD-immunized rhesus macaques.

    “The ELISpot assay showed evident IFN-γ and IL-2 responses but nearly undetectable IL-4 in circRNARBD-immunized rhesus macaques (Figure 6F)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    circRNARBD-immunized rhesus macaques of all doses were well protected, with only very mild pneumonia observed.

    “circRNARBD-immunized rhesus macaques of all doses were well protected because only very mild pneumonia was observed (Figure 6H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The circRNA vaccine expressing the trimeric RBD of the spike protein elicited potent neutralizing antibodies and T cell responses.

    “Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein,”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mice previously immunized with two-dose circRNARBD, circRNARBD-Delta was a much better booster than circRNARBD against Delta and Omicron, elevating the geometric mean NT50 against Omicron from approximately 4 × 10^2 to approximately 3.2 × 10^4.

    “CircRNARBD-Delta appeared to be a much better booster than circRNARBD against both Delta and Omicron variants, which elevated the geometric mean NT50 from ∼4 × 102 to ∼3.2 × 104 against the Omicron (Figures 5K and 5L).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The circRNARBD-Delta vaccine protected against both Delta and Omicron.

    “the circRNARBD-Delta vaccine protected against both Delta and Omicron”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Neutralizing antibody titers against a SARS-CoV-2 variant in vitro predict protection against that variant in vivo.

  • May combine two claims.

    Findingown data

    In mice previously immunized with two-dose circRNARBD, both circRNARBD and circRNARBD-Delta boosters effectively boosted neutralizing antibodies against Delta and Omicron.

    “Both vaccines effectively boosted neutralizing antibodies against both Delta (Figure 5K) and Omicron (Figure 5L).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The neutralizing activity against Omicron elicited by each of the three circRNA vaccines dropped 74-fold (native), 15-fold (Beta), and 44-fold (Delta) in comparison with the neutralizing activity against their corresponding variants.

    “The neutralizing activity against Omicron elicited by each one of the three circRNA vaccines dropped 74-fold (native), 15-fold (Beta), and 44-fold (Delta) in comparison with the neutralizing activity against their corresponding variants (Figure 5 A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pseudovirus neutralization assay showed NT50 values of approximately 180 for the 20-μg dose, approximately 520 for the 100-μg dose, and approximately 390 for the 500-μg dose in circRNARBD-immunized rhesus macaques.

    “The pseudovirus neutralization assay showed NT50 values of ∼180 for the 20-μg dose, ∼520 for the 100-μg dose, and ∼390 for the 500-μg dose (Figure 6C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CircRNARBD vaccines induced evident IL-6 and MCP-1, whereas TNF-α, IL-1β, and IFN-α were nearly undetectable in rhesus macaques.

    “CircRNARBD vaccines induced evident IL-6 and monocyte chemoattractant protein-1 (MCP-1) (Figures S7 A and S7B), whereas TNF-α, IL-1β, and IFN-α were nearly undetectable (Figures S7C–S7E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    circRNARBD vaccine-immunized rhesus macaque plasma effectively inhibited native SARS-CoV-2, and also inhibited Alpha, Delta, and Beta variants with reduced activity, especially against Beta.

    “Both the pseudotyped and authentic SARS-CoV-2 neutralization assays showed that the circRNARBD vaccine-immunized rhesus macaque plasma could effectively inhibit the corresponding native strain, whereas the Alpha, Delta, and Beta variants could also be inhibited, but with reduced activity, especially against the Beta variant (Figures 6D and 6E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Omicron-specific vaccine failed to cross-protect against the Delta variant or boost the two-dose Delta vaccine.

    “Our result argues against this possibility because our Omicron-specific vaccine failed to cross-protect against the Delta variant (Figures 5D–5F) or boost the two-dose Delta vaccine (Figures 5H and 5I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In comparison with the mRNA vaccine, the circRNA vaccine elicited higher and more durable immunogens.

    “In comparison with the mRNA vaccine, the circRNA vaccine elicited higher and more durable immunogens, leading to distinct Th1-biased T cell immune responses from the mRNA vaccine (Figures 3 and S3).”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    circRNA vaccine did not cause clinical signs of illness or enhanced pathology in vaccinated NHPs, even at high doses of 500 μg per rhesus macaque.

    “circRNA vaccine did not cause clinical signs of illness or enhanced pathology in vaccinated NHPs”
    • Also stated in: § Limitations of the study · plain
      “our study showed that circRNA vaccines did not cause any clinical signs of illness in rhesus macaques, even at high doses (500 μg per rhesus macaque) (Figure 6)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Omicron carries over 30 mutations on the spike protein, 15 of which are located in the receptor-binding domain (RBD), resulting in a significant decrease in the effectiveness of prior neutralizing antibodies.

    “Omicron carries over 30 mutations on the spike protein, 15 of which are located in the receptor-binding domain (RBD) (Dejnirattisai et al., 2022), resulting in a significant decrease in the effectiveness of prior neutralizing antibodies (Cameroni et al., 2022; Cao et al., 2021; Cele et al., 2022; Liu et al., 2022a; Planas et al., 2022).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Dejnirattisai et al., 2022

      Not checked in the free run.

    • cites Cameroni et al., 2022

      Not checked in the free run.

    • cites Cao et al., 2021

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The S protein, S1 subunit, or RBD antigen of SARS-CoV-2 can induce both B cell and T cell responses, generating highly potent neutralizing antibodies against SARS-CoV-2.

    “The S protein, S1 subunit, or RBD antigen of SARS-CoV-2 can induce both B cell and T cell responses, generating highly potent neutralizing antibodies against SARS-CoV-2 (Bangaru et al., 2020; Hsieh et al., 2020; Walls et al., 2020).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Bangaru et al., 2020

      Not checked in the free run.

    • cites Hsieh et al., 2020

      Not checked in the free run.

    • cites Walls et al., 2020

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    An additional boost with original SARS-CoV-2 vaccines after a prior two-dose vaccination regimen could partly elevate neutralizing capability, but neutralization of Omicron pseudovirus was 4- to 13-fold lower than that of the wild type.

    “Although it has recently been reported that an additional boost with original SARS-CoV-2 vaccines after receiving a prior two-dose vaccination regimen could partly elevate the neutralizing capability, the neutralization of Omicron pseudovirus was 4- to 13-fold lower than that of the wild type (Garcia-Beltran et al., 2022).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Garcia-Beltran et al., 2022

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The current mRNA vaccine has limitations including inherent instability and suboptimal thermostability after LNP encapsulation for in vivo administration due to its susceptibility to exonuclease digestion.

    “However, due to its susceptibility to exonuclease digestion, the current mRNA vaccine still has certain limitations including inherent instability and suboptimal thermostability after LNP encapsulation for in vivo administration (Durymanov and Reineke, 2018; Fenton et al., 2016; Jackson et al., 2020).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Durymanov and Reineke, 2018

      Not checked in the free run.

    • cites Fenton et al., 2016

      Not checked in the free run.

    • cites Jackson et al., 2020

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    The T4 RNA ligase-based method produced circRNARBD with precise circularization confirmed by Sanger sequencing.

    “Sanger sequencing confirmed the precise circularization of circRNARBD by this approach (Figure S2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    CircRNAs are covalently closed ring RNA molecules that are highly stable due to their covalently closed ring structure, which protects them from exonuclease-mediated degradation.

    “Compared with the linear mRNA, circRNA is highly stable due to its covalently closed ring structure, which protects it from exonuclease-mediated degradation (Enuka et al., 2016; Kristensen et al., 2019; Memczak et al., 2013).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Enuka et al., 2016

      Not checked in the free run.

    • cites Kristensen et al., 2019

      Not checked in the free run.

    • cites Memczak et al., 2013

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The S protein of SARS-CoV-2 mediates viral entry into host cells by binding to ACE2 through the RBD at the C terminus of the S1 subunit.

    “The S protein mediates viral entry into host cells by binding to its receptor, angiotensin-converting enzyme 2 (ACE2), through the RBD at the C terminus of the S1 subunit.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Hoffmann et al., 2020

      Not checked in the free run.

    • cites Shang et al., 2020

      Not checked in the free run.

    • cites Wrapp et al., 2020

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    circRNA holds the potential to become an effective and safe platform for vaccination against viral infection, including SARS-CoV-2 emerging variants, as well as possibly becoming a therapeutic platform, owing to its specific properties.

    “In summary, circRNA holds the potential to become an effective and safe platform for vaccination against viral infection—including SARS-CoV-2 emerging variants—as well as possibly becoming a therapeutic platform, owing to its specific properties.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Results in mice and rhesus macaques predict immunogenicity, protection, and safety of circRNA vaccines in humans.

  • May combine two claims.

    Interpretationown data

    The circRNARBD-Delta vaccine appeared to produce antigens possessing high immunogenicity and consequently elicit a high level of neutralizing antibodies against Delta.

    “By contrast, the circRNARBD-Delta vaccine appeared to produce antigens possessing high immunogenicity and consequently elicit a high level of neutralizing antibodies against Delta (Figures 2 and 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    circRNAs encode therapeutic antibodies in the cytoplasm, allowing them to target intracellular targets such as TP53 and KRAS, bypassing the cytomembrane barrier.

    “Unlike antibodies and protein drugs, circRNAs encode therapeutic antibodies in the cytoplasm, allowing them to target intracellular targets such as TP53 (Sabapathy and Lane, 2018) and KRAS (Mukhopadhyay et al., 2021), bypassing the cytomembrane barrier.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Sabapathy and Lane, 2018

      Not checked in the free run.

    • cites Mukhopadhyay et al., 2021

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The circRNA-encoded RBD trimers were functional and successfully induced potent neutralizing antibodies and specific T cell responses against SARS-CoV-2 in both mice and rhesus macaques.

    “The circRNA-encoded RBD trimers were functional and successfully induced potent neutralizing antibodies and specific T cell responses against SARS-CoV-2 in both mice and rhesus macaques (Figures 2, 4, and 6).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All animal experiments with SARS-CoV-2 challenge were conducted under animal biosafety level 3 facilities at the Institute of Pathogen Biology, Chinese Academy of Medical Sciences.

    “All animal experiments with SARS-CoV-2 challenge were conducted under animal biosafety level 3 (ABSL3) facilities at the Institute of Pathogen Biology, Chinese Academy of Medical Sciences.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All animal experiments with SARS-CoV-2 challenge were reviewed and approved by the Committee on the Ethics of Animal Experiments of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences.

    “All the animal experiments with SARS-CoV-2 challenge were reviewed and approved by the Committee on the Ethics of Animal Experiments of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Delta-specific vaccination could cross-protect against all other variants, including Omicron, and could also be used as an effective booster following two-dose original SARS-CoV-2 vaccines.

    “Our Delta-specific vaccination could cross-protect against all other variants, including Omicron (Figures 2E, 5A, 5B, and 5C), and could also be used as an effective booster following two-dose original SARS-CoV-2 vaccines (Figures 5K and 5L).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Results in mice and rhesus macaques predict immunogenicity, protection, and safety of circRNA vaccines in humans.
    • Rests on an assumption: Neutralizing antibody titers are a correlate of protection against SARS-CoV-2 in humans.

  • May combine two claims.

    Study design and conductcitation

    The mRNA vaccine used for comparison is different from mRNA-1273 and BNT162b2, which encode full-length spike antigens and were produced by different manufacturing processes, whereas the mRNA vaccine in this study encoded the trimeric RBD of spike.

    “It is worth noting that the mRNA vaccine we used for the comparison study is different from the two widely administered vaccines, mRNA-1273 and BNT162b2, both of which encode the full-length spike antigens and were produced by different manufacturing processes, whereas the mRNA vaccine in this study encoded the trimeric RBD of spike (Corbett et al., 2020a, 2020b; Vogel et al., 2021).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Corbett et al., 2020a, 2020b

      Not checked in the free run.

    • cites Vogel et al., 2021

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The animal experiments were approved by Peking University Laboratory Animal Center and undertaken in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals.

    “The animal experiments were approved by Peking University Laboratory Animal Center (Beijing) and undertaken in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All rhesus macaque procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Science.

    “All animal procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Science.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The rhesus macaque experiments were performed in the animal biosafety level 4 facility of the National Kunming High-level Biosafety Primate Research Center, Yunnan, China.

    “The 2- to 4-year-old male rhesus macaque experiments were performed in the animal biosafety level 4 (ABSL-4) facility of the National Kunming High-level Biosafety Primate Research Center, Yunnan, China.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    RBD trimers bind to hACE2 better than their monomeric counterparts and have been shown to enhance the humoral immune response.

    “Given that RBD trimers bind to hACE2 better than their monomeric counterparts and have been shown to enhance the humoral immune response (Bouwman et al., 2021; Routhu et al., 2021; Sahin et al., 2020)”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Bouwman et al., 2021

      Not checked in the free run.

    • cites Routhu et al., 2021

      Not checked in the free run.

    • cites Sahin et al., 2020

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The mouse model for the SARS-CoV-2 Beta variant challenge has previously been characterized.

    “The mouse model for the SARS-CoV-2 Beta variant challenge has previously been characterized (Montagutelli et al., 2021).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Montagutelli et al., 2021

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Amplified SARS-CoV-2 was confirmed via RT-PCR, sequencing and transmission electron microscopy, and titrated via plaque assay.

    “Amplified SARS-CoV-2 was confirmed via RT–PCR, sequencing and transmission electronic microscopy, and titrated via plaque assay (106 PFU/ml).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Phytohemagglutinin (PHA-P) and CELL STIMULATION COCKTAIL were used as positive controls in the ELISpot assay.

    “Dimethyl sulfoxide (DMSO) served as an unstimulating control, and phytohemagglutinin (PHA-P, Sigma) and CELL STIMULATION COCKTAIL (Thermo Fisher) were used as positive controls.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Dimethyl sulfoxide (DMSO) served as an unstimulating control in the ELISpot assay.

    “Dimethyl sulfoxide (DMSO) served as an unstimulating control, and phytohemagglutinin (PHA-P, Sigma) and CELL STIMULATION COCKTAIL (Thermo Fisher) were used as positive controls.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Histopathological analysis of tissue slides was scored by 3 independent pathologists blinded to the groups of animals.

    “Histopathological analysis of tissue slides was scored by 3 independent pathologists blinded to the groups of animals.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The ELISpot assay for each rhesus macaque was performed in two or three technical repetitions.

    “The test for each rhesus macaque was performed in two or three technical repetitions.”
    Quote in the paperNot a substantial claim
51 of 51

  • The paper relies on this without support.

    Neutralizing antibody titers against a SARS-CoV-2 variant in vitro predict protection against that variant in vivo.

    “Among all three, the circRNARBD-Delta vaccine maintained sufficient neutralizing activity against Omicron (Figure 5A), with an NT50 of ∼4.7 × 103,”
    “the circRNARBD-Delta vaccine protected against both Delta and Omicron”

  • The paper relies on this without support.

    Results in mice and rhesus macaques predict immunogenicity, protection, and safety of circRNA vaccines in humans.

    “In summary, circRNA holds the potential to become an effective and safe platform for vaccination against viral infection—including SARS-CoV-2 emerging variants—as well as possibly becoming a therapeutic platform, owing to its specific properties.”

  • The paper relies on this without support.

    Neutralizing antibody titers are a correlate of protection against SARS-CoV-2 in humans.

    “Our Delta-specific vaccination could cross-protect against all other variants, including Omicron (Figures 2E, 5A, 5B, and 5C), and could also be used as an effective booster following two-dose original SARS-CoV-2 vaccines (Figures 5K and 5L).”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • circRNA has recently been developed for COVID-19 mRNA vaccines due to its natural high stability.

    “In addition, circRNA has recently been developed for COVID-19 mRNA vaccines due to its natural high stability.112”

    From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [112]

  • CircRNA prepared with sequences of Delta strains resulted in broad-spectrum protection and production of neutralizing antibodies against both Delta and Omicron.

    “the results revealed that circRNA prepared with sequences of Delta strains resulted in broad-spectrum protection and production of neutralizing antibodies against both Delta and Omicron.”

    From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [227]

  • Vaccination with circRNA prepared with the original SARS-CoV-2 strain sequence followed by a booster dose of the vaccine containing circRNA prepared with Delta sequences conferred good protection against Delta and Omicron.

    “In addition, vaccination with circRNA prepared with the original SARS-CoV-2 strain sequence followed by a booster dose of the vaccine containing circRNA prepared with Delta sequences conferred good protection against Delta and Omicron.”

    From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [227]

  • The circRNA vaccine showed neutralizing activity against the Beta variant (B.1.351).

    “Moreover, the vaccine also showed neutralizing activity against the Beta variant (B.1.351).”

    From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [112]

  • After immunizing mice with the circRNA vaccine, long-lasting neutralizing antibodies and Th1-biased cellular immune responses were produced.

    “After immunizing mice with the obtained vaccine, long-lasting neutralizing antibodies and Th1-biased cellular immune responses were produced.”

    From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [112]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • IL-4 and TNF-α antibody identifiers are reversed

    Minor

    The key resources table identifies #506324 as the TNF-α antibody and #504104 as the IL-4 antibody, but the flow-cytometry method assigns each identifier to the other cytokine.

1 of 1

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Qu, L., Yi, Z., Shen, Y., Lin, L., Chen, F., Xu, Y., Wu, Z., Tang, H., Zhang, X., Tian, F., Wang, C., Xiao, X., Dong, X., Guo, L., Lu, S., Yang, C., Tang, C., Yang, Y., Yu, W., Wang, J., Zhou, Y., Huang, Q., Yisimayi, A., Cao, Y., Wang, Y., Zhou, Z., Peng, X., Wang, J., Xie, X. S., & Wei, W.
Affiliations
  • Peking University (CN)
  • Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
  • National Institutes for Food and Drug Control (CN)
Corresponding author
Wei, Wensheng
ORCID
Venue
Cell
Publisher
openRxiv
Published
2021-03-16
Volume
185
Issue
10
Pages
1728-1744.e16
ISSN
0092-8674 (ISSN-L)
DOI
10.1101/2021.03.16.435594
PMC
PMC8971115
OpenAlex
W4226027283
Cited by
636
References
107
Citations by year
2026: 107 · 2025: 170 · 2024: 151 · 2023: 129 · 2022: 73 · 2021: 4 · 2013: 1
FWCI
64.04
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Natural Science Foundation of China (2018ZX10301401, 2020YFA0707800, 31870893, 31930016)
  • Chinese Academy of Sciences
  • Ministry of Science and Technology of the People's Republic of China
  • China Postdoctoral Science Foundation
  • Tsinghua University
  • Peking University
  • Beijing Municipal Science and Technology Commission
  • National Postdoctoral Program for Innovative Talents
  • Institute of Microbiology, Chinese Academy of Sciences
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Circular RNAs in diseases
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
Keywords
SARS-CoV-2, COVID-19 vaccines, SARS-CoV-2 variants of concern, Omicron variant, Delta variant, booster vaccination, vaccine protection
MeSH terms
RNA, Circular, COVID-19, SARS-CoV-2, COVID-19 Vaccines, mRNA Vaccines, Animals, Antibodies, Viral, Humans, Macaca mulatta, Vaccines, Synthetic, Mice, Antibodies, Neutralizing

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review