Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jul:286:121570.
doi: 10.1016/j.biomaterials.2022.121570. Epub 2022 May 7.

An imidazole modified lipid confers enhanced mRNA-LNP stability and strong immunization properties in mice and non-human primates

Manon Ripoll  1 , Marie-Clotilde Bernard  2 , Céline Vaure  3 , Emilie Bazin  4 , Sylvie Commandeur  5 , Vladimir Perkov  6 , Katia Lemdani  7 , Marie-Claire Nicolaï  8 , Patrick Bonifassi  9 , Antoine Kichler  10 , Benoit Frisch  11 , Jean Haensler  12
Affiliations

An imidazole modified lipid confers enhanced mRNA-LNP stability and strong immunization properties in mice and non-human primates

Manon Ripoll et al. Biomaterials. 2022 Jul.

Abstract

The mRNA vaccine technology has promising applications to fight infectious diseases as demonstrated by the licensing of two mRNA-based vaccines, Comirnaty® (Pfizer/BioNtech) and Spikevax® (Moderna), in the context of the Covid-19 crisis. Safe and effective delivery systems are essential to the performance of these vaccines and lipid nanoparticles (LNPs) able to entrap, protect and deliver the mRNA in vivo are considered by many as the current "best in class". Nevertheless, current mRNA/LNP vaccine technology has still some limitations, one of them being thermostability, as evidenced by the ultracold distribution chain required for the licensed vaccines. We found that the thermostability of mRNA/LNP, could be improved by a novel imidazole modified lipid, DOG-IM4, in combination with standard helper lipids. DOG-IM4 comprises an ionizable head group consisting of imidazole, a dioleoyl lipid tail and a short flexible polyoxyethylene spacer between the head and tail. Here we describe the synthesis of DOG-IM4 and show that DOG-IM4 LNPs confer strong immunization properties to influenza HA mRNA in mice and macaques and a remarkable stability to the encapsulated mRNA when stored liquid in phosphate buffered saline at 4 °C. We speculate the increased stability to result from some specific attributes of the lipid's imidazole head group.

Keywords: Imidazole lipid; Ionizable lipid; Lipid nanoparticles; Storage stability; mRNA vaccine.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Comparison of DOG-IM4, DOG-IM2 and L319 LNPs characteristics.DOG-IM4, DOG-IM2 and L319 were synthesized (A) and combined with standard helper lipids for the preparation of mRNA-containing LNPs as described in Materials and Methods. The LNPs were characterized for pKa by TNS dye binding assay (B), particle size and surface charge by using a Zetasizer (Malvern instrument), and mRNA entrapment by Ribogreen accessibility assay (C). FI stands for fluorescence intensity, EE for encapsulation efficiency and PDI for polydispersity index.
Fig. 2
Fig. 2
Cryo EM micrograph for DOG-IM4 LNPs; scale = 100 nm.
Fig. 3
Fig. 3
DOG-IM4 LNPs induce higher functional antibody titers than DOG-IM2 LNPs in immunogenicity studies with mice and are stable at 4°C.PanelA: Individual and mean hemagglutination inhibiting antibody titers (HI titers) measured in sera collected at D42 from Balb/c mice (n = 8) immunized at D0 and D21 with 10 μg of a monovalent A/California/07/2009 (H1N1) split influenza vaccine (Vaxigrip®; MIV) or with DOG-IM4, DOG-IM2 and L319 LNPs loaded with indicated amounts of mRNA encoding full-length hemagglutinin (HA) of closely related influenza virus strain A/Netherlands/602/2009 (H1N1). The vaccines in PBS were injected into the mouse quadriceps under a final volume of 50 μL. PanelB: Samples from the lot of DOG-IM4 LNPs used in (A) were retested after 6 and 12 months of storage in PBS under nitrogen at 4°C. Individual and mean HI titers are shown. The study comprised also a group of mice injected with MIV (10 μg Vaxigrip®) at each timepoint to confirm the consistency of HI titration test (not shown). NS = not statistically significant.
Fig. 4
Fig. 4
DOG-IM4 LNPs induce high functional antibody titers with both UNR and MNR in immunogenicity studies with Balb/c mice.Balb/c mice (n = 8) were immunized as inFig. 3with 10 μg of a monovalent A/California/07/2009 (H1N1) split influenza vaccine (Vaxigrip®; MIV) or with DOG-IM4 LNPs loaded with 5 μg of unmodified (UNR) or 1MpU-modified mRNA (MNR) encoding full-length hemagglutinin (HA) of closely related influenza virus strain A/Netherlands/602/2009 (H1N1). Individual and mean HI titers measured three weeks after the second injection (D42) are shown. UNR was either from Trilink (as inFig. 3) or from AmpTec and MNR was from AmpTec. A group immunized with L319 LNPs loaded with 5 μg of UNR from Trilink (as inFig. 3) was included as a reference.
Fig. 5
Fig. 5
Reporter protein expression in BALB/c mice after IM injection of hEPO and FLuc mRNAs loaded in DOG-IM4 or L319 LNPs. Panel A: In vivo imaging of Balb/c mice 6h and 24h after IM administration of a 5 μg dose of Fluc mRNA in DOG-IM4 or L319 LNPs under 50 μL of PBS. One representative mouse from groups of 4 is shown for each formulation.Panel B: Luciferase expression from the injected muscles was quantified and average Radiance (total photon flux/second) (±SD) from groups of 4 injected mice is represented as a function of time.Panel C: Average (±SD) hEPO expression in the serum of Balb/c mice (4/group) determined by ELISA 6h and 24h after IM administration of 1 μg of hEPO mRNA in either DOG-IM4 or L319 LNPs under 50 μl of PBS.
Fig. 6
Fig. 6
Stability of DOG-IM4 LNPs over time at different storage temperatures.DOG-IM4 LNPs containing hEPO mRNA were stored in PBS under nitrogen at 4°C, 25°C or 37°C, and after different periods of time (indicated in weeks) the LNPs were tested for: PanelA: In vivo bioactivity via circulating hEPO determination 6h after IM injection of 1 μg mRNA into Balb/c mice (4 mice/group injected with 50 μL LNP in the quadriceps); PanelB: mRNA integrity by fragment analyzer after extraction from the DOG-IM4 LNPs; PanelC: Average particle size (z-Average and PDI in dotted lines) by dynamic light scattering (DLS); PanelD: mRNA encapsulation (Ribogreen assay). PanelE: The hEPO expression level from DOG-IM4 LNPs stored at 4°C is compared to that of L319 and MC3 LNPs stored in the same conditions. Loss of hEPO expression is expressed as a percent of hEPO measured at T0.
Fig. 7
Fig. 7
Lipid analysis of DOG-IM4 LNPs by UHPLC-CAD-MS at T0 and after 18 weeks of storage at different temperatures. The LNPs used in the stability study described inFig. 7were analyzed for lipid integrity by UHPLC-CAD-MS. PanelA: CAD chromatogram at T0; PanelB: CAD chromatograms after 18 weeks at 4°C (in red), 25°C (in blue) or 37°C (in black). The slight shift in retention times observed between T0 and T18 weeks chromatograms is due to the replacement of the HPLC column by a new one. PanelC: Total Ion Current and mass spectra after 18 weeks at 4°C (in red), 25°C (in blue) or 37°C (in black). The mass spectra correspond to the retention time of DOG-IM4 (13.1 min; indicated by the black star).
Fig. 8
Fig. 8
DOG-IM4 LNPs induce high functional antibody titers, innate cytokines and Th-1 biased T cell responses in immunogenicity studies with NHPs in a dose-dependent manner.PanelA: Time course of mean (±SD) hemagglutination inhibiting antibody titers (HI titers) measured in sera collected from cynomolgus macaques (N = 5 in DOG-IM4 groups and N = 4 in L319 group) immunized at D0 and D28 with DOG-IM4 or L319 LNPs loaded with 1MpU-modified mRNA (MNR from AmpTec) encoding full-length hemagglutinin (HA) of influenza virus strain A/Netherlands/602/2009 (H1N1). DOG-IM4 LNPs contained either 10 μg or 50 μg of MNR and L319 LNPs contained 50 μg of MNR. The LNPs in PBS were injected into the quadriceps under a final volume of 500 μL. PanelB: Twenty-four hours following the first administration, plasma samples from the immunized monkeys were tested for ten chemokines/cytokines (Eotaxin, IFN-α2a; IL-1β; IL-1RA, IL-6, IL-8, IL-17A; I-TAC, MCP-1, TNF-α) by MSD. Levels are shown in pg/mL on a spider diagram. The levels induced by an injection of 15 μg of monovalent split vaccine (Vaxigrip; MIV) come from a previous study and were plotted as a comparator. PanelC & D: Two weeks following the second administration (D42), PBMCs were collected from the immunized macaques and analyzed for IFN-γ (PanelC) and IL-4 (PanelD) secreting cells by ELISPOT assay after stimulation for 24h with recombinant influenza hemagglutinin (rHA) from A/California/07/2009 strain (H1N1). Cell counts from each individual monkey are shown after subtracting counts obtained with PBMCs collected from the same animal pre-immunization.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sahin U., Karikó K., Türeci Ö. mRNA-based therapeutics--developing a new class of drugs. Nat. Rev. Drug Discov. 2014;13:759–780. doi: 10.1038/nrd4278. - DOI - PubMed
    1. Thran M., Mukherjee J., Pönisch M., Fiedler K., Thess A., Mui B.L., Hope M.J., Tam Y.K., Horscroft N., Heidenreich R., Fotin-Mleczek M., Shoemaker C.B., Schlake T. mRNA mediates passive vaccination against infectious agents, toxins, and tumors. EMBO Mol. Med. 2017;9:1434–1447. doi: 10.15252/emmm.201707678. - DOI - PMC - PubMed
    1. Pardi N., Hogan M.J., Porter F.W., Weissman D. mRNA vaccines - a new era in vaccinology. Nat. Rev. Drug Discov. 2018;17:261–279. doi: 10.1038/nrd.2017.243. - DOI - PMC - PubMed
    1. Pardi N., Hogan M.J., Weissman D. Recent advances in mRNA vaccine technology. Curr. Opin. Immunol. 2020;65:14–20. doi: 10.1016/j.coi.2020.01.008. - DOI - PubMed
    1. Zhang C., Maruggi G., Shan H., Li J. Advances in mRNA vaccines for infectious diseases. Front. Immunol. 2019;10:594. doi: 10.3389/fimmu.2019.00594. - DOI - PMC - PubMed

Publication types