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Cited by 127SARS-CoV-2 and COVID-19 Researchvaccines and immunoinformatics approachesSARS-CoV-2 detection and testing

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine

Open access
Gebre, Makda S., Rauch, Susanne, Roth, Nicole, Yu, Jingyou, Chandrashekar, Abishek, Mercado, Noe B., He, Xuan, Liu, Jinyan +26 more·2022·Nature
DOI 10.1038/s41586-021-04231-6PMC PMC8770133
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Abstract

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans 1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 μg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

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Written from the paper’s full text.

TL;DR

In a head-to-head non-human primate study, a second-generation non-modified mRNA SARS-CoV-2 vaccine (CV2CoV) with optimized non-coding regions induced substantially higher antibody, memory B cell, and T cell responses and more robust protection against SARS-CoV-2 challenge than CVnCoV. CV2CoV was comparably immunogenic to BNT162b2 in macaques, and binding/neutralizing antibody titres correlated with protection.

Key findings

  • CV2CoV induced higher binding and neutralizing antibodies than CVnCoV. At week 8, median RBD-binding titres were 14,827 (range 2,133–37,079) vs 214 (range 47–1,238) (P = 0.0043), and median pseudovirus neutralizing titres were 4,752 (range 414–6,793) vs 196 (range 20–405) (P = 0.0022).
  • CV2CoV elicited higher pseudovirus neutralizing titres against variants including D614G, Alpha, Beta, Lambda, Kappa, and Delta (e.g., Delta median 568 vs 36; Beta 755 vs 189; Alpha 1,813 vs 101; P values as reported). At week 5, CV2CoV neutralizing antibody responses were comparable to BNT162b2.
  • CV2CoV gave lower peak viral loads after challenge: median BAL sgRNA 2.90 vs CVnCoV 4.92 vs sham 6.02 log10 copies per ml; nasal swab 3.17 vs 6.42 vs 7.35 log10 copies per swab. CV2CoV was lower than CVnCoV in BAL (P = 0.0411) and nasal swab (P = 0.0087); no detectable infectious virus by TCID50 in five of six CV2CoV animals on day 2.
  • Binding and neutralizing antibody titres inversely correlated with peak viral loads (BAL: P = 0.0008, R = −0.7148 and P = 0.0015, R = −0.6912; nasal: P < 0.0001, R = −0.8346 and P < 0.0001, R = −0.8766). CV2CoV also increased memory B cells (P = 0.022 and P = 0.0152) and IFNγ T cell responses (P = 0.0065); IL-4 was not detectable.

Methods

  • Head-to-head non-human primate study: 18 cynomolgus macaques (both sexes, 3–20 years) randomized to 12 µg CVnCoV (n = 6), 12 µg CV2CoV (n = 6), or sham (n = 6); intramuscular prime on day 0, boost at week 4, challenge at week 8 with 1.0 × 10^5 TCID50 SARS-CoV-2 USA-WA1/2020 by intranasal and intratracheal routes; necropsy 10 days after challenge.
  • Immunological and virological assays included RBD ELISA, pseudovirus and live-virus neutralization, B cell flow cytometry, IFNγ and IL-4 ELISPOT, sgRNA RT–PCR, TCID50, and histopathology; assays were performed with blinding.
  • Statistics: two-tailed non-parametric Mann–Whitney U test for group comparisons; two-sided Spearman rank-correlation for correlates; P < 0.05 considered significant; GraphPad Prism v9.0.

Limitations

  • Stated: the study was not specifically designed as a safety study; no adverse effects or unexpected/enhanced pathology were observed. Future studies are required to directly assess protective efficacy of CV2CoV against SARS-CoV-2 variants of concern in non-human primates. B cell germinal centre responses and durability of protective efficacy following CV2CoV vaccination remain to be determined.
  • Stated: the challenge virus was SARS-CoV-2 WA1/2020, not a variant of concern.
  • Evident from text: small group sizes (n = 6 per group), non-human primate model rather than humans, and the BNT162b2 comparison used leftover clinical product and only pseudovirus neutralizing titres at week 5; no human efficacy data for CV2CoV are reported.

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Integrity signals

Registry

Crossref · 10.1038/s41586-021-04231-6

Cites

40 of 40 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

39 of 48 cited sources checked · 14 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Previous studies with rodents and non-human primates have demonstrated protection by CVnCoV.

    “Previous studies with rodents and non-human primates have demonstrated protection by CVnCoV2,23,24.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [2] Hoffmann, D. et al. CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2. Nat. Commun. 12, 4048 (2021). source
    • cites [23] Rauch, S. et al. mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents. NPJ Vaccines 6, 57 (2021). source

      The Discussion reports that CVnCoV protected hamsters against SARS-CoV-2 challenge, supporting the rodent portion of the claim. It does not report protection by CVnCoV in non-human primates; its comparison with primate challenge models concerns other vaccine studies. No page numbers are available in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [24] Rauch, S. et al. mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection. Preprint at https://doi.org/10.1101/2021.03.22.435960 (2020). source
  • Premisecitation

    CV2CoV has previously been shown to lead to higher antigen expression than CVnCoV in cell culture.

    “CV2CoV has previously been shown to lead to higher antigen expression than CVnCoV in cell culture3.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [3] Roth, N. et al. CV2CoV, an enhanced mRNA-based SARS-CoV-2 vaccine candidate, supports higher protein expression and improved immunogenicity in rats. Preprint at https://doi.org/10.1101/2021.05.13.443734 (2021). source
  • Premisecitation

    Protection in macaques was primarily observed in the lower respiratory tract.

    “However, protection in macaques was primarily observed in the lower respiratory tract23,24.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [23] Rauch, S. et al. mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents. NPJ Vaccines 6, 57 (2021). source
    • cites [24] Rauch, S. et al. mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection. Preprint at https://doi.org/10.1101/2021.03.22.435960 (2020). source

      The Results (page unavailable) report near-complete protection in the lower respiratory tract but residual viral replication in the upper respiratory tract of vaccinated mice. The paper mentions protection in non-human primates in a previous study, but does not say that protection in macaques was primarily in the lower respiratory tract. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    Neutralizing antibody titres induced by CV2CoV in this study appear similar to those reported for other mRNA vaccines in macaques.

    “The neutralizing antibody titres induced by CV2CoV in this study also appear to be similar to those reported for other mRNA vaccines in macaques27,28.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [27] Corbett, K. S. et al. Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates. N. Engl. J. Med. 383, 1544–1555 (2020). source
    • cites [28] Vogel, A. B. et al. BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature 592, 283–289 (2021). source
  • Interpretationown data

    Binding and neutralizing antibody titres are important correlates of protection for these SARS-CoV-2 vaccines in non-human primates.

    “Consistent with prior observations from our laboratory and others15,16,22, these findings suggest that binding and neutralizing antibody titres are important correlates of protection for these SARS-CoV-2 vaccines in non-human primates.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [15] Yu, J. et al. DNA vaccine protection against SARS-CoV-2 in rhesus macaques. Science 369, 806–811 (2020). source
    • cites [16] Mercado, N. B. et al. Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Nature 586, 583–588 (2020). source
    • cites [22] McMahan, K. et al. Correlates of protection against SARS-CoV-2 in rhesus macaques. Nature 590, 630–634 (2021). source

      The IgG Adoptive Transfer Protection Study reports protection thresholds for neutralizing titres and binding-antibody ELISA titres in rhesus macaques. The Discussion also says neutralizing titres correlated with protection in earlier DNA and Ad26 vaccine studies. This paper’s direct binding-titre evidence comes from transfer of convalescent IgG, however, rather than from vaccinated animals, so it does not directly establish both as correlates for the vaccines. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • May combine two claims. 29
  • Classified as probably not a substantial claim. 10
  • The quote only partly states this claim. 1

  • May combine two claims.

    Premisecitation

    CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression.

    “CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression.”
    • Also stated in: § Main · plain
      “CV2CoV is a second-generation mRNA vaccine that incorporates modifications of non-coding regions that were selected by empiric screening for improved antigen expression2,3.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [2] Hoffmann, D. et al. CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2. Nat. Commun. 12, 4048 (2021). source

      Methods > Vaccination (page unavailable) describes CV2CoV as an mRNA vaccine without chemically modified nucleosides and specifies its untranslated regions. Results (page unavailable) says CV2CoV and CVnCoV encode the same protein but differ in their non-coding regions. Our copy does not establish that those regions are optimized or that they enhance antigen expression; its reported antibody responses do not directly measure antigen expression. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [3] Roth, N. et al. CV2CoV, an enhanced mRNA-based SARS-CoV-2 vaccine candidate, supports higher protein expression and improved immunogenicity in rats. Preprint at https://doi.org/10.1101/2021.05.13.443734 (2021). source

  • May combine two claims.

    Premisecitation

    Both vaccines encode the same full-length, pre-fusion stabilized SARS-CoV-2 spike protein and are encapsulated in lipid nanoparticles with identical composition.

    “Both vaccines encode the same full-length, pre-fusion stabilized severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein13,14 and are encapsulated in lipid nanoparticles (LNPs) with identical composition.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [13] Pallesen, J. et al. Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc. Natl Acad. Sci. USA 114, E7348–E7357 (2017). source

      The Results (p. 3) describe a two-proline design that stabilizes MERS-CoV spike in its prefusion form and report analogous effects for SARS-CoV spike. The paper does not establish that the two vaccines encode the same full-length SARS-CoV-2 spike. The citation is attached to the spike-protein description, not the subsequent LNP-composition statement. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [14] Kirchdoerfer, R. N. et al. Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis. Sci. Rep. 8, 15701 (2018). source

      The Introduction (page unavailable) describes prefusion stabilization of coronavirus spike proteins using two proline mutations, and the Results examine a stabilized SARS-CoV spike ectodomain. The cited paper does not establish that both vaccines encode the same full-length SARS-CoV-2 spike protein. The LNP-composition clause follows the citations and is not judged as attributed to this source. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Both CVnCoV and CV2CoV are based on RNActive technology and consist of non-chemically modified sequence-engineered mRNA without pseudouridine.

    “Both CVnCoV and CV2CoV are based on RNActive technology4–7 and consist of non-chemically modified sequence-engineered mRNA without pseudouridine6–12.”
    • Also stated in: § Discussion · plain
      “Both CVnCoV and CV2CoV contain only non-modified nucleosides with no pseudouridine or derivates, and CV2CoV has previously been shown to lead to higher antigen expression than CVnCoV in cell culture3.”
    Quote in the paperSubstantial claim6 of 9 sources checked
    • cites [4] Hoerr, I., Obst, R., Rammensee, H. G. & Jung, G. In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies. Eur. J. Immunol. 30, 1–7 (2000). source
    • cites [5] Fotin-Mleczek, M. et al. Highly potent mRNA based cancer vaccines represent an attractive platform for combination therapies supporting an improved therapeutic effect. J. Gene Med. 14, 428–439 (2012). source

      The RNActive technology section (page unavailable) describes CureVac’s RNActive mRNA vaccines, and Results and Discussion describes sequence engineering of their coding and noncoding regions. The paper does not identify CVnCoV or CV2CoV or establish that either uses this technology; it also does not state that their mRNA is chemically unmodified or lacks pseudouridine. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [6] Fotin-Mleczek, M. et al. Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity. J. Immunother. 34, 1–15 (2011). source
    • cites [7] Kubler, H. et al. Self-adjuvanted mRNA vaccination in advanced prostate cancer patients: a first-in-man phase I/IIa study. J. Immunother. Cancer 3, 26 (2015). source

      The Background and Methods (RNActive® technology; page not provided) describe RNActive as an mRNA-vaccine platform using free and protamine-complexed mRNA. Our copy studies CV9103, however, and does not identify CVnCoV or CV2CoV or establish that their mRNA is sequence-engineered, non-chemically modified, or free of pseudouridine. (LLM, conf=0.79)

      Experimental — accuracy not yet measured
    • cites [8] Lutz, J. et al. Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines. NPJ Vaccines 2, 29 (2017). source

      The Materials and methods section (‘mRNA vaccines’; page not provided) says the vaccines studied used the RNActive platform, lacked chemically modified nucleosides, and underwent sequence optimization. That supports the description of the technology, including the absence of pseudouridine, but the paper discusses rabies and influenza vaccines and does not identify CVnCoV or CV2CoV or establish their composition. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [9] Stitz, L. et al. A thermostable messenger RNA based vaccine against rabies. PLoS Negl. Trop. Dis. 11, e0006108 (2017). source

      The Introduction and Methods describe a rabies vaccine based on RNActive technology, including optimization of its mRNA sequence. They do not identify CVnCoV or CV2CoV or establish that either uses this technology. The passages also do not say whether their mRNA is chemically unmodified or lacks pseudouridine. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [10] Schnee, M. et al. An mRNA vaccine encoding rabies virus glycoprotein induces protection against lethal infection in mice and correlates of protection in adult and newborn pigs. PLoS Negl. Trop. Dis. 10, e0004746 (2016). source

      Materials and Methods (mRNA- and protein-based vaccines; page unavailable) says the rabies mRNA vaccines used the RNActive platform, had GC-enriched coding sequences and enhanced UTRs, and contained no chemically modified nucleotides. That supports the technology description, including the absence of pseudouridine, for the vaccines studied here. The paper does not identify CVnCoV or CV2CoV or establish that either has those properties. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [11] Petsch, B. et al. Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection. Nat. Biotechnol. 30, 1210–1216 (2012). source
    • cites [12] Aldrich, C. et al. Proof-of-concept of a low-dose unmodified mRNA-based rabies vaccine formulated with lipid nanoparticles in human volunteers: a phase 1 trial. Vaccine 39, 1310–1318 (2021). source

      The Introduction (page unavailable) identifies RNActive® as CureVac’s mRNA platform and says the vaccine approach is being applied to SARS-CoV-2. It does not identify CVnCoV or CV2CoV or establish that either uses sequence-engineered mRNA without pseudouridine. Searches for those vaccine names and the claimed mRNA features found no supporting passage. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    CV2CoV has different non-coding regions flanking the open reading frame, which have previously been shown to improve transgene expression and protection against SARS-CoV-2 in ACE2-transgenic mice.

    “CV2CoV has been engineered with different non-coding regions flanking the open reading frame, which have previously been shown to improve transgene expression3 and protection against SARS-CoV-2 in ACE2-transgenic mice2.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [2] Hoffmann, D. et al. CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2. Nat. Commun. 12, 4048 (2021). source

      The Methods (Vaccination; page unavailable) describe CV2CoV’s different non-coding regions, and the Discussion reports that CV2CoV protected hACE2-transgenic mice against disease caused by both tested SARS-CoV-2 variants. The paper does not show that those regions improved protection relative to CVnCoV: the Discussion reports full protection with both vaccines. The transgene-expression claim is attached to a different citation and is not assessed here. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [3] Roth, N. et al. CV2CoV, an enhanced mRNA-based SARS-CoV-2 vaccine candidate, supports higher protein expression and improved immunogenicity in rats. Preprint at https://doi.org/10.1101/2021.05.13.443734 (2021). source

      The citation to this paper is attached to the claim of protection against SARS-CoV-2 in ACE2-transgenic mice. Sections 2.3–2.4 describe experiments in Wistar rats, and the Introduction mentions protective efficacy in non-human primates; no passage found reports protection in ACE2-transgenic mice. The paper does describe different non-coding regions and increased protein expression, but those are not the protection claim attached to this citation. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    Following challenge, anamnestic binding and neutralizing antibody responses were observed in all CVnCoV-vaccinated animals and in a subset of CV2CoV-vaccinated animals.

    “Following challenge, we observed anamnestic binding and neutralizing antibody responses in all CVnCoV-vaccinated animals and in a subset of the CV2CoV-vaccinated animals16 (Extended Data Fig. 6).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [16] Mercado, N. B. et al. Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Nature 586, 583–588 (2020). source

      The Methods (Animals and study design) list Ad26 vaccine groups, not CVnCoV or CV2CoV. The Immune Responses in Vaccinated Animals Following Challenge section reports increased neutralizing antibodies in most Ad26-vaccinated animals but no anamnestic neutralizing response in the Ad26-S.PP group. Searches for CVnCoV and CV2CoV found no passage addressing responses in those groups; the Ad26 findings do not establish or contradict the claim about different vaccines. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Previous mRNA vaccine clinical trials have demonstrated onset of protective efficacy after the first dose with improved protection after the boost immunization.

    “Previous mRNA vaccine clinical trials have demonstrated onset of protective efficacy after the first dose with improved protection after the boost immunization25,26.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [25] Polack, F. P. et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N. Engl. J. Med. 383, 2603–2615 (2020). source
    • cites [26] Baden, L. R. et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 384, 403–416 (2021). source

      The Discussion (p. 11) reports fewer symptomatic COVID-19 cases after one dose of mRNA-1273, but cautions that the trial was not designed to evaluate single-dose efficacy. The Efficacy section (p. 9) establishes strong efficacy for the two-dose regimen, but does not demonstrate that protection improved specifically because of the second dose. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Depletion of CD8+ T cells partially abrogated protective efficacy against SARS-CoV-2 re-challenge in convalescent macaques.

    “We previously reported that depletion of CD8+ T cells partially abrogated protective efficacy against SARS-CoV-2 re-challenge in convalescent macaques22.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] McMahan, K. et al. Correlates of protection against SARS-CoV-2 in rhesus macaques. Nature 590, 630–634 (2021). source

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

    Premisecitation

    Neutralizing antibody titres against certain SARS-CoV-2 variants, such as B.1.351 (Beta) and B.1.617.2 (Delta), were lower than those against the parental strain WA1/2020.

    “As previously reported for other vaccines29–33, the neutralizing antibody titres against certain SARS-CoV-2 variants, such as the B.1.351 (Beta) and B.1.617.2 (Delta) variants, were lower than those against the parental strain WA1/2020.”
    Quote in the paperNot a substantial claim5 of 5 sources checked
    • cites [29] Liu, C. et al. Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum. Cell https://doi.org/10.1016/j.cell.2021.06.020 (2021). source

      The Results section on vaccine serum reports lower neutralizing titres against B.1.617.2 than against Victoria, and the Introduction reports reduced titres against B.1.351. The paper uses Victoria, an early Wuhan-related strain, as its comparator—not the specifically named WA1/2020 strain. (LLM, conf=0.87)

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    • cites [30] Haas, E. J. et al. Impact and effectiveness of mRNA BNT162b2 vaccine against SARS-CoV-2 infections and COVID-19 cases, hospitalisations, and deaths following a nationwide vaccination campaign in Israel: an observational study using national surveillance data. Lancet 397, 1819–1829 (2021). source

      The Discussion reports effectiveness against B.1.1.7 and says effectiveness against B.1.351 could not be estimated. Its mention of lower neutralising antibodies compares one vaccine dose with two, not variants with WA1/2020. Searches for Delta, WA1/2020, and variant-specific neutralising titres found no passage addressing the claimed comparison; page numbers were not available in our copy. (LLM, conf=0.76)

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    • cites [31] Wu, K. et al. Serum neutralizing activity elicited by mRNA-1273 vaccine. N. Engl. J. Med. 384, 1468–1470 (2021). source

      In the main text (page not specified), the paper reports lower neutralizing titers against B.1.351 in serum from mRNA-1273 recipients, using the original Wuhan-Hu-1 isolate as a reference. It does not report results for B.1.617.2 (Delta) or name WA1/2020 as the comparator. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [32] Wibmer, C. K. et al. SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma. Nat. Med. 27, 622–625 (2021). source

      The Figure 2 passage reports reduced neutralization of 501Y.V2 (Beta) relative to the original Wuhan-1 D614G lineage using convalescent plasma; the vaccine discussion also reports reduced neutralization of Beta by vaccinee sera. The paper does not address Delta, and its tested comparator is Wuhan-1 D614G, not the specified WA1/2020 strain. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [33] Wall, E. C. et al. Neutralising antibody activity against SARS-CoV-2 VOCs B.1.617.2 and B.1.351 by BNT162b2 vaccination. Lancet 397, 2331–2333 (2021). source

      The main text reports neutralising antibody titres 5.8-fold lower against B.1.617.2 and 4.9-fold lower against B.1.351 than against its Wild-type strain. It describes that comparator as having the original spike sequence but does not identify it as WA1/2020, so the named comparator is not established by our copy. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

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

    Interpretationcitation

    Memory B cells might contribute to the durability of antibody responses.

    “Memory B cells might contribute to the durability of antibody responses38,39; B cell germinal centre responses and the durability of protective efficacy following CV2CoV vaccination remain to be determined.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [38] Abayasingam, A. et al. Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection. Cell Rep. Med. 2, 100228 (2021). source
    • cites [39] Dan, J. M. et al. Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science 371, https://doi.org/10.1126/science.abf4063 (2021). source

      The Concluding remarks report durable Spike IgG titers and persistent memory B cells 5–8 months after infection. They do not establish that memory B cells contribute to the durability of circulating antibody responses; the paper instead describes antibody levels as reflecting production by short- and then long-lived plasma cells. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Sham controls had peak medians of 6.02 log10 sgRNA copies per ml in BAL and 7.35 log10 sgRNA copies per swab in nasal swab samples on day 2.

    “The sham controls had peak medians of 6.02 (range, 4.62–6.81) log10-transformed sgRNA copies per ml in the BAL and 7.35 (range, 5.84–8.09) log10-transformed sgRNA copies per swab in the nasal swab samples on day 2 (Fig. 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At week 2, binding antibody titres were detected only with CV2CoV and not with CVnCoV, with median values of 25 for CVnCoV and 799 for CV2CoV.

    “At week 2, binding antibody titres were detected only with CV2CoV and not with CVnCoV, with median values of 25 (range, 25–25) and 799 (range, 82–2,010) for CVnCoV and CV2CoV, respectively (Fig. 2a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At week 2, neutralizing antibodies were detected only with CV2CoV and not with CVnCoV, with median values of 20 for CVnCoV and 131 for CV2CoV.

    “At week 2, neutralizing antibodies were detected only with CV2CoV and not with CVnCoV, with median values of 20 (range, 20–20) and 131 (range, 62–578) for CVnCoV and CV2CoV, respectively (Fig. 2b).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    At peak immunity at week 5, neutralizing antibody responses induced by two immunizations with 12 μg CV2CoV were comparable to those induced by two immunizations with 30 μg BNT162b2.

    “At peak immunity at week 5, the neutralizing antibody responses induced by CV2CoV were comparable to those induced by BNT162b2 (Fig. 2e).”
    • Also stated in: § Abstract · plain
      “Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The BNT162b2 clinical vaccine obtained as leftover product from pharmacies retained immunogenicity comparable to the clinical vaccine.

  • May combine two claims.

    Findingown data

    Live-virus neutralizing antibody responses elicited by CV2CoV were higher than those elicited by CVnCoV against WA1/2020 and B.1.617.2 (Delta) strains (P = 0.0466 and 0.0152), with similar trends for Alpha and Beta.

    “The live-virus neutralizing antibody responses elicited by CV2CoV were higher than those elicited by CVnCoV against the WA1/2020 and B.1.617.2 (Delta) strains (P = 0.0466 and 0.0152, respectively), with similar trends for B.1.1.7 (Alpha) and B.1.351 (Beta) (P = 0.0628 and 0.1450, respectively) (Fig. 2d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    By week 8, binding antibody titres had increased in the CVnCoV group but were still >50 times lower than those in the CV2CoV group (P = 0.0043), with medians of 214 and 14,827.

    “By week 8, the binding antibody titres had increased in the CVnCoV group but were still >50 times lower than those in the CV2CoV group (P = 0.0043), with median values of 214 (range, 47–1,238) and 14,827 (range, 2,133–37,079), respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    One week after the week-4 boost, neutralizing antibody titres increased, with medians of 55 for CVnCoV and 15,827 for CV2CoV.

    “One week after the week 4 boost, the neutralizing antibody titres were increased, with median values of 55 (range, 20–302) and 15,827 (range, 3,985–81,081) for CVnCoV and CV2CoV, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    One week after the week-4 boost, binding antibody titres increased in both groups, with medians of 48 for CVnCoV and 28,407 for CV2CoV.

    “One week after the week-4 boost, the antibody titres were increased in both groups, with medians of 48 (range, 75–710) and 28,407 (range, 2,714–86,541) for CVnCoV and CV2CoV, respectively (Fig. 2a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IFNγ responses were detected in both groups but were higher in the CV2CoV group (P = 0.0065).

    “IFNγ responses were detected in both groups but were higher in the CV2CoV group (P = 0.0065) (Extended Data Fig. 3c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    By week 8, neutralizing antibody titres had increased in the CVnCoV group but were still >20 times lower than those in the CV2CoV group (P = 0.0022), with medians of 196 and 4,752.

    “By week 8, the neutralizing antibody titres had increased in the CVnCoV group but were still >20 times lower than those in the CV2CoV group (P = 0.0022), with median values of 196 (range, 20–405) and 4,752 (range, 414–6,793), respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pathological lesions in vaccinated animals were similar to those in sham group but were overall fewer in number and more focal in distribution.

    “The pathological lesions in vaccinated animals were similar to those observed for animals in the sham group (Extended Data Fig. 7i–l) but were overall fewer in number and more focal in distribution.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sham group had more lung lobes affected and more extensive lung lesions than vaccinated animals.

    “Animals in the sham group also had more lung lobes affected (Extended Data Fig. 7b) and more extensive lung lesions, with a greater proportion of lung lobes showing evidence of interstitial inflammation, alveolar inflammatory infiltrates and type II pneumocyte hyperplasia (Extended Data Fig. 7c–h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the present study, prime immunization with CV2CoV induced binding and neutralizing antibodies in all macaques by week 2, and these responses had increased substantially by 1 week after the boost immunization.

    “In the present study, the prime immunization with CV2CoV induced binding and neutralizing antibodies in all macaques by week 2, and these responses had increased substantially by 1 week after the boost immunization.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CV2CoV-immunized animals exhibited peak medians of 2.90 log10 sgRNA copies per ml in BAL and 3.17 log10 sgRNA copies per swab in nasal swab samples.

    “The CV2CoV-immunized animals exhibited peak medians of 2.90 (range, 1.70–4.64) log10-transformed sgRNA copies per ml in the BAL and 3.17 (range, 2.59–5.63) log10-transformed sgRNA copies per swab in the nasal swab samples (Fig. 3), with resolution of sgRNA levels in the BAL samples by day 2 in most animals and by day 4 in all animals.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CVnCoV-immunized animals showed peak medians of 4.92 log10 sgRNA copies per ml in BAL and 6.42 log10 sgRNA copies per swab in nasal swab samples.

    “The CVnCoV-immunized animals showed peak medians of 4.92 (range, 2.40–6.61) log10-transformed sgRNA copies per ml in the BAL and 6.42 (range, 4.46–7.81) log10-transformed sgRNA copies per swab in the nasal swab samples (Fig. 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CV2CoV induced >10-fold-higher neutralizing antibody responses against multiple viral variants and provided >3 log reductions in sgRNA copies per ml in BAL and >4 log reductions in sgRNA copies per swab in nasal swab samples compared with sham controls.

    “In contrast to CVnCoV, CV2CoV induced >10-fold-higher neutralizing antibody responses against multiple viral variants and provided >3 log reductions in sgRNA copies per ml in BAL and >4 log reductions in sgRNA copies per swab in nasal swab samples compared with sham controls.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Sham-treated animals had higher cumulative lung pathology scores than CVnCoV animals (P = 0.0368) and CV2CoV animals (P = 0.0022).

    “Viral replication was largely resolved by day 10 in the animals vaccinated with CVnCoV and CV2CoV, and those with sham treatment had higher cumulative lung pathology scores19 (CVnCoV animals compared with sham controls, P = 0.0368; CV2CoV animals compared with sham controls, P = 0.0022) (Extended Data Fig. 7a).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] He, X. et al. Low-dose Ad26.COV2.S protection against SARS-CoV-2 challenge in rhesus macaques. Cell https://doi.org/10.1016/j.cell.2021.05.040 (2021). source

  • May combine two claims.

    Findingown data

    CV2CoV induced both antigen-specific memory B cell responses and T cell responses.

    “CV2CoV induced both antigen-specific memory B cell responses and T cell responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In the HERALD trial, observed vaccine efficacy against symptomatic COVID-19 was approximately 48% in the overall study population and 53% in participants 18-60 years of age.

    “In this trial, the observed vaccine efficacy against symptomatic coronavirus disease 2019 (COVID-19) was approximately 48% and 53% in the overall study population and in a subgroup of participants 18–60 years of age, respectively1.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Kremsner, P. G. et al. Efficacy and safety of the CVnCoV SARS-CoV-2 mRNA vaccine candidate: results from Herald, a phase 2b/3, randomised, observer-blinded, placebo-controlled clinical trial in ten countries in Europe and Latin America. Lancet Infect. Dis. https://doi.org/10.1016/S1473-3099(21)00677–0 (2021). source

  • May combine two claims.

    Interpretationown data

    Optimization of non-coding elements of the mRNA backbone can substantially improve the immunogenicity and protective efficacy of mRNA vaccines.

    “These data suggest that optimization of non-coding elements of the mRNA backbone can substantially improve the immunogenicity and protective efficacy of mRNA vaccines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

    “These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.”
    • Also stated in: § Discussion · plain
      “In summary, our data show that optimization of non-coding regions in a SARS-CoV-2 mRNA vaccine can substantially improve its immunogenicity against multiple viral variants and can enhance its protective efficacy against SARS-CoV-2 challenge in macaques.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    CV2CoV elicited substantially greater humoral and cellular immune responses and provided significantly improved protective efficacy against SARS-CoV-2 challenge compared with CVnCoV in macaques.

    “CV2CoV elicited substantially greater humoral and cellular immune responses and provided significantly improved protective efficacy against SARS-CoV-2 challenge as compared with CVnCoV in macaques.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All animal studies were conducted in compliance with all relevant local, state and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee.

    “All animal studies were conducted in compliance with all relevant local, state and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The immunological assays in this study were performed with blinding.

    “The immunological assays were performed with blinding.”
    • Also stated in: § B cell immunophenotyping · plain
      “The immunological assays were performed with blinding.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisenone given

    LNPs are composed of ionizable amino lipid, phospholipid and cholesterol and PEGylated lipid; the compositions for CVnCoV and CV2CoV are identical.

    “LNPs are composed of ionizable amino lipid, phospholipid and cholesterol and PEGylated lipid; the compositions for CVnCoV and CV2CoV are identical.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Blinded histopathological evaluation of tissues was performed by a board-certified veterinary pathologist.

    “Blinded histopathological evaluation was performed by a board-certified veterinary pathologist (A.J.M.).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Immunological and virological assays were performed with blinding.

    “Immunological and virological assays were performed with blinding.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Eighteen cynomolgus macaques were randomly assigned to three groups.

    “Eighteen cynomolgus macaques of both sexes between the ages of 3 and 20 years were randomly assigned to three groups.”
    Quote in the paperSubstantial claim
37 of 37

  • The paper relies on this without support.

    The BNT162b2 clinical vaccine obtained as leftover product from pharmacies retained immunogenicity comparable to the clinical vaccine.

    “We also compared the pseudovirus neutralizing antibody titres induced in macaques by two immunizations with 12 μg of CV2CoV to those induced by two immunizations with 30 μg of the Pfizer BNT162b2 clinical vaccine obtained as leftover product from pharmacies.”
1 of 1

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?

  • The optimised non-coding regions of mRNA SARS-CoV-2 vaccine CV2CoV improve homologous and heterologous neutralising antibody responses in rats and non-human primates, compared with CVnCoV and other modified mRNA vaccines.

    “The optimised non-coding regions of mRNA SARS-CoV-2 vaccine CV2CoV have previously been shown to improve homologous and heterologous neutralising antibody responses in rats and non-human primates, compared with CVnCoV and other modified mRNA vaccines10,11.”

    From Efficacy of an unmodified bivalent mRNA vaccine against SARS-CoV-2 variants in female small animal models (2023) · cites this paper as [11]

    The Vaccine immunogenicity section (page not available) reports higher neutralising antibody titres against the ancestral strain and several variants with CV2CoV than with CVnCoV in macaques. This paper does not report rat results, and it describes CV2CoV responses as comparable—not superior—to those induced by the modified mRNA vaccine BNT162b2. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Rat-derived data translated very well to improved immunogenicity and protective efficacy in a non-human primate challenge model for SARS-CoV-2.

    “The rat-derived data translated very well to improved immunogenicity and protective efficacy in a non-human primate challenge model for SARS-CoV-210,11 and our cross-neutralization results emphasize the broad antigen coverage induced by the bivalent vaccine.”

    From Efficacy of an unmodified bivalent mRNA vaccine against SARS-CoV-2 variants in female small animal models (2023) · cites this paper as [11]

    The Discussion (page not available in our copy) reports greater immune responses and improved protection against SARS-CoV-2 challenge with CV2CoV than with CVnCoV in macaques. The Main section describes earlier protection findings in ACE2-transgenic mice, not rats; our copy does not establish that rat-derived data translated to the primate results. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • mRNA vaccines are viable tools to limit the spread of COVID-19 owing to scalable production, safety and efficacy.

    “mRNA vaccines9,10 have been recognized as viable tools to limit the spread of COVID-19 owing to their scalable production, safety and efficacy1–3.”

    From Algorithm for optimized mRNA design improves stability and immunogenicity (2023) · cites this paper as [3]

  • UTR engineering has a profound effect on protein expression.

    “and UTR engineering has a profound effect on protein expression3.”

    From Algorithm for optimized mRNA design improves stability and immunogenicity (2023) · cites this paper as [3]

  • Protection induced by CV2CoV is superior to CVnCoV in a non-human primate SARS-CoV-2 challenge model.

    “Protection induced by CV2CoV was reported to be superior to CVnCoV in a non-human primate SARS-CoV-2 challenge model11.”

    From Efficacy of an unmodified bivalent mRNA vaccine against SARS-CoV-2 variants in female small animal models (2023) · cites this paper as [11]

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.

  • Figure caption names the wrong vaccine in the BNT162b2 comparison

    Minor

    The text says the BNT162b2 comparison used CV2CoV, but the caption for that comparison says it used CVnCoV. This leaves the vaccine underlying the stated comparability conclusion unclear.

  • Binding-antibody median falls below its reported range

    Minor

    The reported median of 48 cannot fall outside the stated range of 75-710 for the same group and time point.

2 of 2

Cites

Flagged first, then by citations

4 of the 44 references couldn’t be matched to a known paper.

Research works printed but not linked: 2

  1. He, X. et al. Low-dose Ad26.COV2.S protection against SARS-CoV-2 challenge in rhesus macaques. Cell 184, 3467–3473 (2021). · DOI · Paper page
  2. Rauch, S. et al. mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection. Preprint at https://doi.org/10.1101/2021.03.22.435960 (2020). · DOI · Paper page

Cited by

Sorted by citations

Related

Authors
Gebre, M. S., Rauch, S., Roth, N., Yu, J., Chandrashekar, A., Mercado, N. B., He, X., Liu, J., McMahan, K., Martinot, A., Martinez, D. R., Giffin, V., Hope, D., Patel, S., Sellers, D., Sanborn, O., Barrett, J., Liu, X., Cole, A. C., Pessaint, L., Valentin, D., Flinchbaugh, Z., Yalley-Ogunro, J., Muench, J., Brown, R., Cook, A., Teow, E., Andersen, H., Lewis, M. G., Boon, A. C. M., Baric, R. S., Mueller, S. O., Petsch, B., & Barouch, D. H.
Affiliations
  • Beth Israel Deaconess Medical Center (US)
  • CureVac (Germany) (DE)
  • Tufts University (US)
  • University of North Carolina at Chapel Hill (US)
  • Bioqual (US)
  • Washington University in St. Louis (US)
Corresponding author
  • Rauch, Susanne
  • Barouch, Dan H.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2021-11-18
Volume
601
Issue
7893
Pages
410-414
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-021-04231-6
PMC
PMC8770133
OpenAlex
W3212637969
Cited by
127
References
44
Citations by year
2026: 9 · 2025: 22 · 2024: 32 · 2023: 30 · 2022: 31 · 2021: 3
FWCI
7.17
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • Digital Commons@Becker (Washington University School of Medicine) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • ImmPort
Funders
  • Ragon Institute of MGH, MIT and Harvard
  • Massachusetts Consortium on Pathogen Readiness
  • Bundesministerium für Bildung und Forschung (01KI20703)
  • National Institutes of Health (CA260476)
  • National Cancer Institute (U01CA260476)
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 detection and testing
Keywords
mRNA vaccines, SARS-CoV-2, COVID-19 vaccines, immunogenicity, neutralizing antibodies, memory B cells, T cell response, Delta variant, viral load, protective efficacy, lipid nanoparticle formulation, non-human primates, cynomolgus macaques
MeSH terms
Immunogenicity, Vaccine, COVID-19, SARS-CoV-2, COVID-19 Vaccines, mRNA Vaccines, BNT162 Vaccine, Memory B Cells, Animals, Antibodies, Viral, Female, Macaca fascicularis, Male, Nucleosides, Respiratory System, T-Lymphocytes, Vaccines, Synthetic, Viral Load, 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 2026-10-11.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-11.

Cited sources are checked by gpt-6-sol.

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.

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