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Cited by 257RNA Interference and Gene DeliveryImmunotherapy and Immune ResponsesImmune responses and vaccinations

Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines

Open access
Lutz, Johannes, Lazzaro, Sandra, Habbeddine, Mohamed, Schmidt, Kim Ellen, Baumhof, Patrick, Mui, Barbara L., Tam, Ying K., Madden, Thomas D. +3 more·2017·npj Vaccines
DOI 10.1038/s41541-017-0032-6PMC PMC5648897
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Abstract

mRNA represents a promising new vaccine technology platform with high flexibility in regard to development and production. Here, we demonstrate that vaccines based on sequence optimized, chemically unmodified mRNA formulated in optimized lipid nanoparticles (LNPs) are highly immunogenic and well tolerated in non-human primates (NHPs). Single intramuscular vaccination of NHPs with LNP-formulated mRNAs encoding rabies or influenza antigens induced protective antibody titers, which could be boosted and remained stable during an observation period of up to 1 year. First mechanistic insights into the mode of action of the LNP-formulated mRNA vaccines demonstrated a strong activation of the innate immune response at the injection site and in the draining lymph nodes (dLNs). Activation of the innate immune system was reflected by a transient induction of pro-inflammatory cytokines and chemokines and activation of the majority of immune cells in the dLNs. Notably, our data demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses. Importantly, we show that the developed LNP-formulated mRNA vaccines can be used as a vaccination platform allowing multiple, sequential vaccinations against different pathogens. These results provide strong evidence that the mRNA technology is a valid approach for the development of effective prophylactic vaccines to prevent infectious diseases.

AI snapshot

Written from the paper’s full text.

TL;DR

Sequence-optimized, chemically unmodified mRNA formulated in lipid nanoparticles (LNPs) induced protective antibody titers and durable immune responses in non-human primates after intramuscular vaccination. The platform was well tolerated, boostable, usable for sequential vaccination against different pathogens, and competitive with or superior to licensed inactivated-virus vaccines in humoral and T cell responses.

Key findings

  • A single intramuscular dose of LNP-formulated RABV-G mRNA induced rabies virus neutralization titers at or above the WHO protective threshold of 0.5 IU/ml in all non-human primates at day 28; a 10-fold higher mRNA dose yielded 10-fold higher titers. A single 10 µg dose of H1N1-HA mRNA/LNP induced H1N1 hemagglutination inhibition titers at or above the protective 1:40 threshold.
  • Boosting increased rabies VNTs 20-fold; titers stabilized at about 40 IU/ml for the 10 µg dose and about 4 IU/ml for the 1 µg dose. Recall vaccination 5 months later produced a rapid 10-fold VNT increase. H1N1-HI titers stabilized at about 640 and remained above the protective limit for 5 months, with stable titers until the end of a 1-year observation period.
  • Compared with licensed vaccines, RABV-G mRNA/LNP after one shot gave median VNTs of 4.9 IU/ml (10 µg) and 71.2 IU/ml (100 µg) versus 1.8 IU/ml for Rabipur®; after boost, 100 µg mRNA reached median 842 IU/ml versus 31.3 IU/ml for Rabipur®. H3N2-HA mRNA/LNP was comparable to or higher than Fluad®, and mRNA/LNP induced antigen-specific CD4+ and CD8+ T cell responses that were absent in Rabipur®- or Fluad®-vaccinated animals.
  • LNP-formulated mRNA induced transient local pro-inflammatory cytokines and chemokines, with TNF and IL-6 peaking at 14 h; more than 90% of T and B cells in draining lymph nodes expressed CD69 at 24 h, and CD11b+ Gr1+ cells increased 10-fold within 4 h. Injection-site reactions were limited to slight erythema and/or edema resolving in 1–6 days, with no pain on palpation.

Methods

  • Mice (BALB/c, 7–9 weeks) were vaccinated intramuscularly into the tibialis anterior with 25 µl at days 0 and 21; cynomolgus monkeys were vaccinated intramuscularly into the biceps femoris with 500 µl at days 0 and 28, with NHP groups of n = 2 male and 2 female per group.
  • Vaccines used sequence-optimized, chemically unmodified mRNA in LNPs composed of an ionizable amino lipid, phospholipid, cholesterol, and PEGylated lipid, encoding RABV-G, H1N1-HA, H3N2-HA, luciferase, or non-translatable fluorescent mRNA; licensed comparators were Rabipur® and Fluad®.
  • Analyses included in vivo luciferase imaging, cytokine/chemokine measurement by cytometric bead array or Luminex, flow cytometry of draining lymph nodes, rabies VNT by FAVN, influenza HI assays, intracellular cytokine staining, safety observations, and Mann–Whitney statistical tests.

Limitations

  • The paper states that further investigations are required to determine whether monocytes and granulocytes contribute to antigen transport to draining lymph nodes. It also notes that the vaccinated non-human primates were naïve to the influenza strains used, unlike most adult humans who have prior exposure or vaccination, so human responses may differ.
  • Evident from the text: NHP immunogenicity groups were small (n = 2 male, 2 female per group), and protection in NHPs was inferred from antibody correlates rather than live-virus challenge. The paper also states that the technology warrants clinical testing, so no human efficacy data are reported.

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

Integrity signals

Registry

Crossref · 10.1038/s41541-017-0032-6

Cites

35 of 35 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

22 of 35 cited sources checked · 9 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Protective efficacy against Zika virus challenge infections was demonstrated in non-human primates after intradermal vaccination.

    “Importantly, protective efficacy against Zika virus challenge infections was also demonstrated in non-human primates (NHPs) after i.d. vaccination.14”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [14] Pardi, N. et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature https://doi.org/10.1038/nature21428 (2017). source
  • Premisecitation

    Chemically modified nucleosides have been used in recent publications to reach sufficient antigen expression.

    “In recent publications chemically modified nucleosides were used to reach sufficient antigen expression,12–15 which is in contrast to our proprietary mRNA technology, which employs sequence optimization and selected untranslated regions (UTRs) to achieve high antigen expression.16”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [12] Richner, J. M. et al. Modified mRNA vaccines protect against zika virus infection. Cell. https://doi.org/10.1016/j.cell.2017.02.017 (2017). source
    • cites [13] Richner, J. M. et al. Vaccine mediated protection against zika virus-induced congenital disease. Cell 170, 273–283.e12 (2017). source

      The Method Details section (page unavailable) confirms that the vaccine mRNA used a chemically modified nucleoside, substituting 1-methylpseudoUTP for UTP. It attributes increased translation efficiency to the cap structure, while the Discussion (page unavailable) says modified nucleosides were used to minimize innate immune activation; the paper does not establish that the nucleoside modification was used to reach sufficient antigen expression. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [14] Pardi, N. et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature https://doi.org/10.1038/nature21428 (2017). source
    • cites [15] Bahl, K. et al. Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 Influenza Viruses. Mol. Ther. J. Am. Soc. Gene Ther. 25, 1316–1327 (2017). source
  • Premisecitation

    In prior studies, mice vaccinated intramuscularly with LNP-formulated mRNA vaccines were protected against placental damage and fetal demise in challenged pregnant mice.

    “In these studies, mice were vaccinated i.m. with LNP-formulated mRNA vaccines inducing protection against Zika virus challenge infections,12 and protection against placental damage and fetal demise in challenged pregnant mice.13”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [13] Richner, J. M. et al. Vaccine mediated protection against zika virus-induced congenital disease. Cell 170, 273–283.e12 (2017). source
  • Premisecitation

    Non-replicating mRNA is able to induce antibody titers in non-human primates.

    “The study by Pardi et al. demonstrates that non-replicating mRNA is able to induce antibody titers in NHPs.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [14] Pardi, N. et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature https://doi.org/10.1038/nature21428 (2017). source
  • Premisecitation

    Our proprietary mRNA technology employs sequence optimization and selected untranslated regions (UTRs) to achieve high antigen expression.

    “In recent publications chemically modified nucleosides were used to reach sufficient antigen expression,12–15 which is in contrast to our proprietary mRNA technology, which employs sequence optimization and selected untranslated regions (UTRs) to achieve high antigen expression.16”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [16] Thess, A. et al. Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals. Mol. Ther. J. Am. Soc. Gene Ther. 23, 1456–1464 (2015). source

      The Materials and Methods describe codon optimization and selection of 5′ and 3′ UTRs, and the Results report high expression from sequence-engineered mRNA. However, the expression experiments described in the Results use luciferase and erythropoietin, not an antigen; the source does not establish high antigen expression specifically. Page numbers were not provided in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • May combine two claims. 32
  • Classified as probably not a substantial claim. 1

  • May combine two claims.

    Premisecitation

    Vaccines based on mRNA offer multifaceted advantages including flexible antigen design, a cost-effective manufacturing process allowing for parallel production of multiple mRNA vaccines, and rapid manufacturing.

    “Vaccines based on mRNA could meet these requirements because they offer multifaceted advantages including flexible antigen design, a cost-effective manufacturing process allowing for parallel production of multiple mRNA vaccines1–5 and rapid manufacturing, which could be of crucial importance during pandemics.6”
    Quote in the paperSubstantial claim4 of 6 sources checked
    • cites [1] 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 [2] Kallen, K.-J. et al. A novel, disruptive vaccination technology: self-adjuvanted RNActive(®) vaccines. Hum. Vaccines Immunother. 9, 2263–2276 (2013). source

      The Introduction describes nucleotide-based vaccines as a simple, inexpensive platform compatible with protein engineering, and the Conclusion describes RNA production as flexible and scalable with modest investment (page numbers unavailable). The paper does not establish parallel production of multiple mRNA vaccines. Rapid manufacturing is attached to a separate citation in the citing sentence, not specifically to this source. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [3] Sullenger, B. A. & Nair, S. From the RNA world to the clinic. Science 352, 1417–1420 (2016). source

      The “Therapeutic coding RNAs: mRNA-based immunotherapy” section describes mRNA vaccines encoding different tumor or viral antigens and says direct-injection approaches could replace more complicated and costly cell-based therapies. It does not establish a cost-effective manufacturing process or parallel production of multiple vaccines. Rapid manufacturing is attached to a separate citation in the citing sentence. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [4] Ulmer, J. B. & Geall, A. J. Recent innovations in mRNA vaccines. Curr. Opin. Immunol. 41, 18–22 (2016). source
    • cites [5] Nabel, G. J. Designing tomorrow’s vaccines. N. Engl. J. Med 368, 551–560 (2013). source

      The opening section says older vaccine technologies were ill-suited to a rapid response to the 2009 H1N1 pandemic, but it does not say mRNA vaccines can be manufactured rapidly. The passages on manufacturing and gene-based delivery likewise do not address mRNA vaccine production, its cost, or parallel production (searched: mRNA vaccines, messenger RNA, rapid manufacturing). (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [6] Koff, W. C. et al. Accelerating next-generation vaccine development for global disease prevention. Science 340, 1232910 (2013). source

      The Introduction and Antigen Discovery Technologies sections discuss advances in vaccine design generally, but do not address the claimed advantages of mRNA vaccines. Searches for mRNA, manufacturing, and parallel production found no passage in our copy that substantiates those advantages; the citing sentence assigns rapid manufacturing to a separate citation. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The prior unmodified mRNA vaccine approach was specifically optimized for intradermal administration and showed lower efficacy when given by the intramuscular route.

    “This approach was specifically optimized for i.d. administration and showed lower efficacy when given by the intramuscular route (i.m.) as preferred route for prophylactic vaccination, highlighting the important role of formulation in vaccine design.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [7] 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 vaccine contained no chemically modified nucleotides. The Immunizations procedure (page unavailable) says the mRNA vaccine was given intradermally, but describes no intramuscular mRNA group. The copy describes optimization of the mRNA and its formulation, not optimization specifically for intradermal delivery, and does not establish lower efficacy by the intramuscular route. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [8] 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

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

    Premisecitation

    Induction of protective immune responses against two different influenza strains could be demonstrated after intramuscular administration in non-human primates and humans using LNP-formulated mRNA vaccines based on chemically modified nucleosides.

    “Meanwhile, induction of protective immune responses against two different influenza strains could be demonstrated also after i.m. administration in NHPs and humans, again using LNP-formulated mRNA vaccines based on chemically modified nucleosides.15”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [15] Bahl, K. et al. Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 Influenza Viruses. Mol. Ther. J. Am. Soc. Gene Ther. 25, 1316–1327 (2017). source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Self-amplifying mRNA vaccines benefited from formulation with lipid nanoparticles or cationic nanoemulsion and were able to induce protective antibody titers.

    “Studies with self-amplifying mRNA vaccines investigated the effect of different formulations such as lipid-nanoparticles (LNP)10 or cationic nanoemulsion (CNE)11 on the immunogenicity of mRNA vaccines. Self-amplifying vaccines benefited from formulation with LNP or CNE and were able to induce protective antibody titers.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [10] Geall, A. J. et al. Nonviral delivery of self-amplifying RNA vaccines. Proc. Natl. Acad. Sci. USA 109, 14604–14609 (2012). source
    • cites [11] Brito, L. A. et al. A cationic nanoemulsion for the delivery of next-generation RNA vaccines. Mol. Ther. J. Am. Soc. Gene Ther. 22, 2118–2129 (2014). source

  • May combine two claims.

    Premisecitation

    Non-replicating mRNA vaccines using exclusively unmodified nucleosides are immunogenic and capable of inducing protection against lethal rabies and influenza virus infections after intradermal vaccination in mice, rats, ferrets, and pigs.

    “Using exclusively unmodified nucleosides, we have demonstrated that non-replicating mRNA vaccines are immunogenic and capable of inducing protection against lethal rabies and influenza virus infections after intradermal vaccination (i.d.) in mice, rats, ferrets, and pigs.7,8”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [7] 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 states that the rabies mRNA contained no chemically modified nucleotides and that mice and pigs received intradermal injections. The Abstract reports protection against lethal rabies challenge in mice, while the Results report protective-correlate antibody titers—not challenge-proven protection—in pigs. This paper does not establish the sentence’s influenza or rat and ferret claims. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [8] 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

  • May combine two claims.

    Premisecitation

    Recent studies using chemically modified mRNA revealed protective efficacy of LNP-formulated mRNA vaccines for Zika virus after i.m. administration in mice or i.d. administration in NHPs, and immunogenicity for influenza HA strains H10N8 and H7N9 after i.m. administration in NHPs and humans.

    “Recent studies using chemically modified mRNA revealed protective efficacy of LNP-formulated mRNA vaccines for Zika virus after i.m. administration in mice or i.d. administration in NHPs, as well as immunogenicity for influenza HA of the strains H10N8 and H7N9 after i.m. administration in NHPs and humans.12–15”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [12] Richner, J. M. et al. Modified mRNA vaccines protect against zika virus infection. Cell. https://doi.org/10.1016/j.cell.2017.02.017 (2017). source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [13] Richner, J. M. et al. Vaccine mediated protection against zika virus-induced congenital disease. Cell 170, 273–283.e12 (2017). source

      Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [14] Pardi, N. et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature https://doi.org/10.1038/nature21428 (2017). source

      Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [15] Bahl, K. et al. Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 Influenza Viruses. Mol. Ther. J. Am. Soc. Gene Ther. 25, 1316–1327 (2017). source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Previously, i.d. administration of protamine-formulated prophylactic mRNA vaccines based on unmodified nucleosides protected mice against influenza or rabies challenge infections and induced strong humoral responses in ferrets and pigs.

    “We have previously demonstrated that i.d. administration of protamine-formulated prophylactic mRNA vaccines based on unmodified nucleosides can protect mice against influenza or rabies challenge infections and induces also in ferrets and pigs strong humoral responses.7,8”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [7] 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
    • cites [8] 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

  • May combine two claims.

    Findingown data

    The strongest elevation in cell numbers in draining lymph nodes was observed 24 h after injection, except for NK cells which increased over time.

    “The strongest elevation in cell numbers was observed 24 h after injection, except for NK cells which increased over time.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The increase in cellularity in draining lymph nodes was accompanied by a strong activation of both adaptive and innate immune cells, which peaked at 24 h after injection, when more than 90% of the T and B cells expressed the activation marker CD69.

    “The increase in cellularity in dLNs was accompanied by a strong activation of both adaptive and innate immune cells, which peaked at 24 h after injection, when more than 90% of the T and B cells expressed the activation marker CD69 (Fig. 2h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-6 concentrations remained 10-fold lower in the serum compared to the injection site and returned to baseline at 96 h after injection.

    “The IL-6 concentrations remained 10-fold lower in the serum compared to the injection site and returned to baseline at 96 h after injection (Fig. 2c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The increase in cytokine concentrations after LNP-formulated mRNA injection is predominantly local, with systemic TNF concentrations below detection limit and only a transient increase in IL-6 concentrations.

    “Importantly, the increase in cytokine concentrations was predominantly local with systemic TNF concentrations below detection limit and only a transient increase in IL-6 concentrations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The immune responses induced by the mRNA vaccines could be boosted and stayed stable during an observation period of up to 1 year.

    “The immune responses could be boosted and stayed stable during an observation period of up to 1 year.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The non-LNP formulation did not induce antigen-specific humoral responses and no H3N2-HI titers could be detected after prime or boost vaccination.

    “In contrast to the LNP formulation, the non-LNP formulation did not induce antigen-specific humoral responses and no H3N2-HI titers could be detected after prime or boost vaccination (Supplementary Fig. 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Antibody titers of all groups benefited from a second vaccination given at day 28, and VNTs (measured on day 49) induced by 100 μg of mRNA vaccine reached a median of 842 IU/ml, outperforming Rabipur® more than 20-fold (median 31.3 IU/ml).

    “Antibody titers of all groups benefited from a second vaccination given at day 28, and VNTs (measured on day 49) induced by 100 µg of mRNA vaccine reached a median of 842 IU/ml, outperforming Rabipur® more than 20-fold (median 31.3 IU/ml).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A second dose of both vaccines further increased the titers, to yet higher titers for the mRNA/LNP vaccine.

    “A second dose of both vaccines further increased the titers, to yet higher titers for the mRNA/LNP vaccine.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The RABV-G mRNA/LNP vaccine induced after a single vaccination neutralizing antibody titers above 0.5 IU/ml, which were comparable (for the 10 μg mRNA dose) or higher (for the 100 μg mRNA dose) than the VNTs induced by a full human dose of Rabipur®.

    “The RABV-G mRNA/LNP vaccine induced after a single vaccination neutralizing antibody titers above 0.5 IU/ml, which were comparable (for the 10 µg mRNA dose) or higher (for the 100 µg mRNA dose) than the VNTs induced by a full human dose of Rabipur® (Fig. 5a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    i.m. application of unmodified mRNA in optimized LNPs led to high expression of the encoded protein in mice, which translated into strong humoral and cellular immune responses.

    “We observed that i.m. application of unmodified mRNA in optimized LNPs led to high expression of the encoded protein in mice, which translated into strong humoral and cellular immune responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All vaccinated animals maintained H1N1-HI titers clearly above the protective limit for 5 months starting after prime vaccination, pointing to longevity of the mRNA-mediated humoral response.

    “Importantly, all vaccinated animals maintained H1N1-HI titers clearly above the protective limit for 5 months starting after prime vaccination pointing to longevity of the mRNA-mediated humoral response.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Systemic cytokine concentrations after vaccination stayed for most animals and time points below the detection limit for IL-1β, IL-4, IL-5, IFN-γ and TNF, or did not increase significantly above pre-dose concentrations for IL-2, IL-8 and G-CSF.

    “Systemic cytokine concentrations after vaccination stayed for most animals and time points below the detection limit (IL-1β, IL-4, IL-5, IFN-γ and TNF) or did not increase significantly above pre-dose concentrations (IL-2, IL-8 and G-CSF)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After an initial decline, neutralizing titers stabilized at a level of about 40 IU/ml for the 10 μg mRNA dose and about 4 IU/ml for the 1 μg mRNA dose.

    “Monitoring the antibody titers for 6 months we could demonstrate that after an initial decline neutralizing titers stabilized at a level of about 40 IU/ml for the 10 µg mRNA dose and about 4 IU/ml for the 1 µg mRNA dose (Fig. 4b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A single dose of the H3N2-HA mRNA/LNP vaccine was sufficient to induce H3N2-HI titers above 1:40 and comparable to titers induced by a full human dose of Fluad®.

    “A single dose of the H3N2-HA mRNA/LNP vaccine was sufficient to induce H3N2-HI titers, which were above the titer of 1:40 and comparable to titers induced by a full human dose of Fluad®.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A second dose of the H1N1-HA mRNA/LNP vaccine strongly increased the H1N1-HI titers, which after an initial decline stabilized at a titer of about 640.

    “A second dose of the H1N1-HA mRNA/LNP vaccine strongly increased the H1N1-HI titers, which again after an initial decline stabilized at a titer of about 640 (Fig. 4e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only limited reactogenicity at the injection site and minor changes in systemic cytokine and chemokine concentrations were observed.

    “We observed only limited reactogenicity at the injection site and minor changes in systemic cytokine and chemokine concentrations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The mRNA/LNP vaccines induced CD4+ and CD8+ T cell responses against RABV-G and CD4+ T cell responses against H3N2-HA, which were absent in NHPs that received the licensed vaccines Rabipur or Fluad.

    “The mRNA/LNP vaccines induced even CD4+ and CD8+ T cell responses against RABV-G and CD4+ T cell responses against H3N2-HA, which were absent in NHPs that had received the licensed vaccines Rabipur® or Fluad®”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses.

    “Notably, our data demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Intramuscular injection of LNP-formulated mRNA vaccines induces a broad but transient local immunostimulatory milieu, which is relevant for the induction of strong adaptive immune responses.

    “Taken together, these results suggest that i.m. injection of LNP-formulated mRNA vaccines induces a broad but transient local immunostimulatory milieu, which is relevant for the induction of strong adaptive immune responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    1-methylpseudouridine (m1Ψ) has been demonstrated to reduce immunostimulation and increase protein expression.

    “the Zika studies used mRNA harboring 1-methylpseudouridine (m1Ψ) which has been demonstrated to reduce immunostimulation and increase protein expression.26”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Andries, O. et al. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J. Control. Release 217, 337–344 (2015). source

  • May combine two claims.

    Interpretationown data

    The specific approach described yields strong immunogenicity in NHPs, superior to licensed vaccines based on inactivated virus.

    “The specific approach we describe here yields strong immunogenicity in NHPs, which is even superior to licensed vaccines based on inactivated virus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    LNP-formulated mRNA can be used as a platform technology, which does not induce anti-vector immunity that can be observed with viral vectors such as adenoviruses.

    “An important finding of our study is that LNP-formulated mRNA can be used as a platform technology, which does not induce anti-vector immunity that can be observed with viral vectors such as adenoviruses.29”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Lopez-Gordo, E., Podgorski, I. I., Downes, N. & Alemany, R. Circumventing antivector immunity: potential use of nonhuman adenoviral vectors. Hum. Gene Ther. 25, 285–300 (2014). source

  • May combine two claims.

    Interpretationown data

    Intramuscular application of LNP-formulated prophylactic mRNA vaccines based on sequence-optimized, unmodified nucleosides leads to strong induction of local innate immune responses and systemic adaptive immune responses.

    “In summary, we demonstrated that intramuscular application of LNP-formulated prophylactic mRNA vaccines based on sequence-optimized, unmodified nucleosides leads to a strong induction of local innate immune responses and systemic adaptive immune responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    mRNA vaccines can compete with or even outperform licensed vaccines.

    “Here, we demonstrate that mRNA vaccines can compete with or even outperform licensed vaccines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The humoral and cellular immune responses in NHPs induced by mRNA/LNP vaccines against rabies and influenza H3N2 were superior to the licensed vaccines Rabipur and Fluad, respectively.

    “Notably, the humoral and cellular immune responses in NHP induced by mRNA/LNP vaccines against rabies and influenza H3N2 were superior to the licensed vaccines Rabipur® and Fluad®, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The maintenance of H1N1-HI titers during RABV-G mRNA vaccinations and induction of high rabies neutralizing antibodies in H1N1-HA-vaccinated animals using the same vaccine format suggest that LNP-formulated mRNA enables multiple prophylactic vaccinations.

    “The maintenance of the H1N1-HI titers during the RABV-G mRNA vaccinations and the induction of high titers of rabies neutralizing antibodies in H1N1-HA-vaccinated animals using the same vaccine format suggest that LNP-formulated mRNA enables multiple prophylactic vaccinations.”
    Quote in the paperSubstantial claim
33 of 33

  • The paper relies on this without support.

    Muscle tissue has low infiltration by immune cells and lacks costimulatory molecules and optimal antigen presentation on muscle cells.

    “The induction of strong immune responses after intramuscular injection of mRNA represents a very high hurdle due to a low infiltration of muscle tissue by immune cells and the lack of costimulatory molecules and optimal antigen presentation on muscle cells.”

  • The paper relies on this without support.

    Potent intramuscular mRNA vaccines must allow high antigen expression and presentation and induce strong immunostimulatory signals to improve immune cell infiltration.

    “Thus, potent intramuscular mRNA vaccines must allow high antigen expression and presentation and induce strong immunostimulatory signals to improve immune cell infiltration.”

  • The paper relies on this without support.

    Immunogenicity and protection data in non-human primates predict efficacy of prophylactic vaccines in humans.

    “In the present study, we compare mRNA vaccines to licensed vaccines based on inactivated virus and demonstrate that unmodified mRNA, formulated in optimized LNPs, fulfills all the key requirements to be a viable vaccine platform for human prophylaxis.”

  • The paper relies on this without support.

    Fluad® contains the surface antigens HA and neuraminidase of the influenza strains H1N1, H3N2 and B/Brisbane, as well as the adjuvant MF59C.1.

    “Fluad® contains the surface antigens HA and neuraminidase of the influenza strains H1N1, H3N2 and B/Brisbane, as well as the adjuvant MF59C.1.”

  • The paper relies on this without support.

    Nonhuman primates mimic the human situation most closely.

    “we tested whether this also translates to NHPs, which mimic the human situation most closely.”

  • The paper relies on this without support.

    The antibody titers measured serve as correlates of protection for rabies and influenza.

    “were maintained during this period well above the titers that serve as correlate of protection.”
6 of 6

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?

  • LNP-formulated mRNAs with unmodified nucleotides are a potent modality to deliver antigens for presentation on HLA class I and II molecules due to their intrinsic immunostimulatory capacities.

    “Due to their intrinsic immunostimulatory capacities, LNP-formulated mRNAs with unmodified nucleotides are a potent modality to deliver antigens for presentation on HLA class I and II molecules.”

    From Preclinical development of an mRNA-based multiepitope immunotherapeutic for glioblastoma (2025) · cites this paper as [22]

    The Results report that unmodified, LNP-formulated mRNA produced antigen-specific CD4+ and CD8+ T-cell responses in mice, while the Discussion reports strong antigen expression and innate immune activation. These findings support its vaccine potency, but the paper does not directly demonstrate antigen presentation on HLA class I and II molecules. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • LNP-formulated mRNAs with unmodified nucleotides primarily induce CD8+ T-cell responses.

    “They primarily induce CD8+ T-cell responses [22], which can exceed those induced by LNP-formulated mRNAs with modified nucleotides [34, 35], as well as CD4+ T-cell responses.”

    From Preclinical development of an mRNA-based multiepitope immunotherapeutic for glioblastoma (2025) · cites this paper as [22]

    The Results section on mice reports antigen-specific CD8+ T-cell responses after vaccination with LNP-formulated unmodified mRNA, but also reports CD4+ responses; it does not establish that CD8+ responses predominate. In the Results section on non-human primates, CD4+ responses appeared at both rabies-vaccine doses while CD8+ responses appeared only at the higher dose, and influenza-specific CD8+ responses were absent (pages not provided). (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • 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.”

    From Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine (2022) · cites this paper as [8]

    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

  • Preclinical studies showed that protecting the mRNA by lipid encapsulation significantly enhanced the response to CV7201.

    “preclinical studies showed that protecting the mRNA by lipid encapsulation significantly enhanced the response to the CV7201 [9], [24].”

    From Proof-of-concept of a low-dose unmodified mRNA-based rabies vaccine formulated with lipid nanoparticles in human volunteers: A phase 1 trial (2021) · cites this paper as [9]

    The Results section (‘LNP-formulated mRNA induces strong humoral and cellular responses in mice’; page unavailable) reports substantially higher rabies-neutralizing antibody titers and stronger cellular responses with LNP-formulated RABV-G mRNA than with non-formulated mRNA. The Discussion says LNPs protect mRNA from RNases. Our copy does not identify the LNP-formulated vaccine as CV7201, so the CV7201-specific part of the claim is not established. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Formulation of mRNA in lipid nanoparticles (LNP) protects the mRNA and enhances the immune response in animal models.

    “Further preclinical research in animal models has found that formulation of the mRNA in lipid nanoparticles (LNP) protects the mRNA and enhances the immune response [9].”

    From Proof-of-concept of a low-dose unmodified mRNA-based rabies vaccine formulated with lipid nanoparticles in human volunteers: A phase 1 trial (2021) · cites this paper as [9]

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Authors
Lutz, J., Lazzaro, S., Habbeddine, M., Schmidt, K. E., Baumhof, P., Mui, B. L., Tam, Y. K., Madden, T. D., Hope, M. J., Heidenreich, R., & Fotin-Mleczek, M.
Affiliations
  • CureVac (Germany) (DE)
  • Acuitas Therapeutics (Canada) (CA)
ORCID
Venue
npj Vaccines
Publisher
Springer Science and Business Media LLC
Published
2017-10-19
Volume
2
Issue
1
Pages
29-29
ISSN
2059-0105 (ISSN-L)
DOI
10.1038/s41541-017-0032-6
PMC
PMC5648897
OpenAlex
W2761745911
Cited by
257
References
36
Citations by year
2026: 13 · 2025: 25 · 2024: 39 · 2023: 39 · 2022: 41 · 2021: 51 · 2020: 26 · 2019: 14 · 2018: 9
FWCI
4.77
Type
journal article
Language
en
Open access
Licence
Locations
  • npj Vaccines (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Life Sciences › Immunology and Microbiology › Immunology › Immune responses and vaccinations
Keywords
mRNA vaccines, lipid nanoparticles, rabies vaccine, influenza vaccination, prophylactic vaccination, non-human primates, intramuscular vaccination, antibody response, T cell response, innate immune activation, draining lymph nodes, cytokines and chemokines, sequential vaccination, vaccine platforms

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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