WO2024185696A1 - アレナウイルスワクチン - Google Patents

アレナウイルスワクチン Download PDF

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WO2024185696A1
WO2024185696A1 PCT/JP2024/007794 JP2024007794W WO2024185696A1 WO 2024185696 A1 WO2024185696 A1 WO 2024185696A1 JP 2024007794 W JP2024007794 W JP 2024007794W WO 2024185696 A1 WO2024185696 A1 WO 2024185696A1
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mrna
vaccine
lasgpc
lnp
mice
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正治 岩▲崎▼
芽衣 橋爪
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University of Osaka NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10071—Demonstrated in vivo effect

Definitions

  • the present invention relates to an arenavirus vaccine.
  • Lassa virus which causes Lassa fever
  • Lassa virus has the greatest impact on humans, infecting an estimated 900,000 people in West Africa every year, with a very high mortality rate of about 15% for patients who require hospitalization.
  • virus-specific cellular immunity plays an important role in controlling Lassa virus, and so far, live attenuated vaccines that can confer long-term cellular and humoral immunity with a single vaccination have been the focus of Lassa virus vaccine development.
  • SARS2-S-mRNA-LNP SARS-CoV-2 spike protein
  • SARS2-S SARS-CoV-2 spike protein
  • Patent Documents 1 and 2 it has been revealed that SARS2-S-mRNA-LNP induces not only neutralizing antibodies but also SARS2-S-specific CD8 + T cell responses, and it is believed that in addition to neutralizing antibodies, virus-specific cellular immunity also contributes to the prevention of onset and aggravation of the disease.
  • the objective of the present invention is to provide a new vaccine against mammalian arenavirus infections.
  • the present inventors conducted intensive research to solve the above problems and found that an mRNA vaccine expressing the glycoprotein precursor (GPC) or nucleoprotein (NP) of an arenavirus can prevent and/or treat mammalian arenavirus infections.
  • the present invention was completed through further research based on this finding, and includes the following aspects.
  • Item 1 A vaccine comprising lipid nanoparticles encapsulating mRNA encoding an arenavirus glycoprotein precursor (GPC) or nucleoprotein (NP).
  • Item 2. The vaccine according to Item 1, wherein the GPC and NP are the same or different and are derived from Lassa virus or lymphocytic choriomeningitis virus.
  • Item 3. Item 3. The vaccine according to Item 1 or 2, wherein the GPC and NP are naturally occurring.
  • Item 4. Item 4. The vaccine according to any one of Items 1 to 3, comprising lipid nanoparticles encapsulating both or each of mRNA encoding the GPC and NP of an arenavirus.
  • Item 5. Item 5.
  • Item 6. The vaccine of Item 5, wherein the 5'UTR and 3'UTR are derived from ⁇ -globin or ⁇ -globin.
  • Item 7. The vaccine of Item 5 or 6, wherein the 3' polyadenylation strand is 200 nucleotides or less in length.
  • Item 9. Item 9.
  • Item 10. The vaccine according to any one of Items 1 to 9, for intravenous or intramuscular administration.
  • Item 11. The vaccine according to any one of Items 1 to 10, which is administered to a subject at risk of infection with an arenavirus.
  • Item 12. The vaccine of clause 11, wherein the Arenavirus is Lassa virus and/or Lymphocytic Choriomeningitis virus.
  • Item 13 Item 13.
  • a method for preventing arenavirus infection comprising the step of administering a vaccine comprising lipid nanoparticles encapsulating mRNA encoding the GPC or NP of an arenavirus.
  • a vaccine comprising lipid nanoparticles encapsulating mRNA encoding the GPC or NP of an arenavirus.
  • the present invention provides a novel vaccine against mammalian arenavirus infections.
  • FIG. 1 Diagram showing the synthesis of IVT-mRNA expressing LASgpc or LCMnp.
  • A Schematic diagram of IVT-mRNA synthesis encoding the ORF of a viral protein. Naked RNA containing the 5'-UTR of human ⁇ -globin, the ORF of a viral protein and the 3'-UTR of human ⁇ -globin were transcribed in vitro using a PCR-amplified DNA fragment as a template. A 5' cap structure was added to the naked RNA, followed by polyadenylation at the 3' end.
  • B IVT-mRNA with or without a polyA tail (w/o) was subjected to agarose gel electrophoresis.
  • C 200 ng of LCMnp-mRNA (C) or LASgpc-mRNA (D) was introduced into HEK293T cells, and Opti-MEM was used as mock for HEK293T cells. Twenty-four hours after transfection, LCMnp expression in fixed cells was examined by indirect immunofluorescence using a monoclonal antibody against LCMnp (C), and LASgpc in cell lysates after removal of non-soluble components was examined by Western blotting using a polyclonal antibody against LASV GP2 (D).
  • Figure 1 Protective immunity confers by intravenous administration of mRNA-LNP vaccine in C57BL/6 mice.
  • A Schematic of rLCMV/LASgpc 2m genome structure.
  • B Plasma anti-LASgpc IgG was measured 14 days after the second administration.
  • C C57BL/6 mice were inoculated intravenously with rLCMV/LASgpc 2m and plasma virus titers were measured 28 days after the second administration. Data are shown as mean ⁇ SD. LoD indicates low limit of detection. ** p ⁇ 0.01.
  • Figure 1. Virus-specific immunity and protective immunity against lethal rLCMV/LASgpc 2m challenge by intramuscular administration of LASgpc-mRNA-LNP vaccine in CBA mice.
  • FIG. 1 shows the virus-specific immunity conferred by intramuscular administration of LASgpc-mRNA-LNP vaccine in CBA mice and the protective immunity against lethal rLCMV/LASgpc 2m challenge.
  • FIG. 1 Virus-specific immunity conferred by intramuscular administration of LCMnp-mRNA-LNP vaccine in FVB mice and protective immunity against viral challenge leading to lethal hemorrhagic pathology.
  • FIG. 1 Schematic diagram of the experiments in (B) and (C).
  • C FVB mice were inoculated intravenously with wild-type rLCMV 28 days after the second administration and survival was monitored daily.
  • D Schematic diagram of the experiment in (E).
  • the vaccine of the present invention comprises lipid nanoparticles encapsulating mRNA encoding glycoprotein precursor (GPC) or nucleoprotein (NP) of arenavirus.
  • the vaccine of the present invention may comprise lipid nanoparticles encapsulating both or each of mRNA encoding GPC and mRNA encoding NP.
  • the degree of immune induction is synergistically improved compared to the case of containing lipid nanoparticles encapsulating only mRNA encoding GPC or containing lipid nanoparticles encapsulating only mRNA encoding NP.
  • the GPC or NP encoded by the mRNA encapsulated in the lipid nanoparticles contained in the vaccine of the present invention is a protein derived from an arenavirus or a partial protein thereof.
  • the mRNAs encoding the GPC or NP are preferably derived from Lassa virus or lymphocytic choriomeningitis virus, whether identical or different, and more preferably are mRNA encoding the GPC derived from Lassa virus or mRNA encoding the NP derived from lymphocytic choriomeningitis virus.
  • a structure similar to the ORF of natural mRNA includes those in which the uridines that make up the mRNA have been completely replaced with chemically modified uridines.
  • the mRNA encoding GPC or NP may further contain, in addition to the above structure, a 5' cap structure, a 5' untranslated region (UTR), a 3' UTR, and a 3' polyadenylation region.
  • the 5' cap structure is preferably a cap 1 structure.
  • the 5'UTR and 3'UTR are preferably derived from human ⁇ -globin or human ⁇ -globin.
  • the 3' polyadenylation strand is preferably 200 nucleotides or less in length, more preferably 150 nucleotides or less in length.
  • uridine is completely replaced with chemically modified uridine.
  • chemically modified uridine include 5-methoxyuridine, pseudouridine, N1-methylpseudouridine, 2-thiouridine, etc. Among these, complete replacement with 5-methoxyuridine is preferable.
  • the mRNA encapsulated in the lipid nanoparticles contained in the vaccine of the present invention can be designed by standard methods using commonly used molecular and cellular tools, and can be produced by various known methods.
  • the number-average particle diameter of the lipid nanoparticles contained in the vaccine of the present invention is preferably 70 nm to 130 nm, more preferably 80 nm to 120 nm, and even more preferably 90 nm to 110 nm.
  • the average particle size of lipid nanoparticles refers to the number-average particle size measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be performed, for example, using a commercially available DLS device.
  • DLS dynamic light scattering
  • the lipid nanoparticles contained in the vaccine of the present invention are composed of, for example, 30-50 mol% ionizable cationic lipids (pH-sensitive lipids), 35-50 mol% sterols, 0.5-3 mol% PEG-modified lipids, and 10 mol% or less neutral phospholipids, relative to 100 mol% lipid nanoparticles.
  • ionizable cationic lipids examples include dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 1,2-dioleyl-3-dimethylaminopropane (DODMA), [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoic acid ester) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl
  • Sterols include, for example, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, etc.
  • PEG-modified lipids include 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxylpropyl-3-amine (PEG-c-DMA), etc.
  • PEG-DMG 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • PEG-DSPE 1,2-distearoyl-sn-glycero
  • Neutral phospholipids include, for example, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-
  • the method for producing the lipid nanoparticles contained in the vaccine of the present invention is not particularly limited, and any method available to those skilled in the art can be used. For example, a commercially available device for producing nanoparticles may be used.
  • the form of the lipid nanoparticles contained in the vaccine of the present invention is not particularly limited, and examples include unilamellar liposomes, multilayer liposomes, spherical micelles, irregular layered structures, etc.
  • adjuvants include aluminum salts, and examples of aluminum salts include aluminum hydroxide and aluminum phosphide.
  • stabilizers include refined white sugar, sodium chloride, potassium chloride, magnesium chloride, sodium hydrogen phosphate dihydrate, potassium dihydrogen phosphate, sodium acetate hydrate, glacial acetic acid, tris(hydroxymethyl)aminomethane (also known as trometamol), trometamol hydrochloride, L-histidine, L-histidine hydrochloride hydrate, absolute ethanol, polyoxyethylene sorbitan oleate (also known as polysorbate 80), etc.
  • preservatives examples include sodium ethylmercurithiosalicylate (also known as thimerosal) and phenoxyethanol.
  • the vaccine of the present invention is administered to a subject at risk of infection with an arenavirus.
  • it is preferably administered to a subject at risk of infection with Lassa virus and/or lymphocytic choriomeningitis virus.
  • Subject refers to a human or non-human animal.
  • Animals include mammals, rodents, birds, reptiles, amphibians, fish, and insects.
  • Non-human animals are mammals such as mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and pigs.
  • the method of administration of the vaccine of the present invention can be appropriately selected depending on the target disease, the condition of the subject, and other conditions.
  • Examples of the administration method include intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, nasal administration, oral administration, intralymph node administration, and mucosal administration. Among these, intravenous administration or intramuscular administration is preferred, and intramuscular administration is more preferred.
  • the number of doses and the interval between doses of the vaccine of the present invention can be appropriately selected depending on the target disease, the condition of the subject, the route of administration, and other conditions.
  • the number of doses of the vaccine of the present invention is preferably at least twice.
  • the interval between doses of the vaccine of the present invention is preferably at least every three weeks.
  • the dosage of the vaccine of the present invention can be appropriately selected depending on the target disease, the condition of the subject, the route of administration, and other conditions.
  • the dosage of the vaccine of the present invention per administration can be appropriately determined by methods known to those skilled in the art depending on the age, sex, condition, and other factors of the subject.
  • the vaccine is administered so that the amount of mRNA contained in each administration is 0.1 ⁇ g to 200 ⁇ g, and it is more preferable that the vaccine is administered so that the amount is 5 ⁇ g to 50 ⁇ g.
  • Example 1 Preparation of in vitro transcribed (IVT) mRNA and confirmation of expression in cultured cells mRNA expressing the glycoprotein precursor (GPC) (hereinafter referred to as "LASgpc”) of Lassa virus (LASV) or the nucleoprotein (NP) (hereinafter referred to as "LCMnp”) of Lymphocytic choriomeningitis virus (LCMV) (hereinafter referred to as "LASgpc-mRNA” and “LCMnp-mRNA”) was synthesized in vitro (Figs. 1A and 1B), and the expression in cultured cells was confirmed (Figs. 1C and 1D).
  • GPC glycoprotein precursor
  • NP nucleoprotein
  • LCMV Lymphocytic choriomeningitis virus
  • LASgpc NCBI Accession ID, NC_004296.1
  • LCMnp NCBI Accession ID, KY514256.
  • mRNAs were synthesized in which a cap structure was added to the 5' end and a polyadenylation structure was added to the 3' end, and uridine 5' triphosphate was replaced with 5-methoxyuridine 5' triphosphate (TriLink BioTechnologies) using the T7 mScript standard mRNA Production System (Cell Script).
  • IVT-mRNA was purified using the Monarch RNA clean up kit (New England Biolabs), and the quality of the purified IVT-mRNA was examined by non-denaturing agarose gel electrophoresis using RNA High for Easy Electrophoresis (DynaMarker Laboratory) ( Figure 1B).
  • Example 2 Preparation of mRNA-LNP
  • LNPs lipid nanoparticles
  • LNP formulations (IVT-mRNA-LNPs) were prepared from GenVoy-ILM (Precision Nanosystems) and IVT-mRNA using a microfluidic mixer NanoAssemblr® Ignite (Precision Nanosystems).
  • the N/P ratio (molar ratio of cationic amine of ionic lipid to negatively charged phosphate of mRNA) was set to 4.
  • the obtained samples were diluted with 1 ⁇ Formulation Buffer 2 (Precision Nanosystems), concentrated with an Amicon Ultra centrifugal filter (Millipore), and then passed through a 0.2 ⁇ m filter according to the manufacturer's recommendations (Precision Nanosystems). Sterile-filtered mRNA-LNPs were stored at 4°C until use.
  • the concentration of IVT-mRNA incorporated into LNPs was determined using the Quant-iT Ribogreen Assay Kit (ThermoFisher Scientific).
  • Example 3 Construction of recombinant LCMV (rLCMV/LASgpc 2m ) expressing LASV-GPC
  • rLCMV/LASgpc 2m recombinant LCMV
  • Example 4 Verification of the possibility of inducing protective immunity by intravenous administration of mRNA-LNP vaccine
  • C57BL/6 mice were intravenously administered with LASgpc-mRNA-LNP or LCMnp-mRNA-LNP containing 10 ⁇ g of IVT-mRNA, or saline twice at an interval of 3 weeks (FIG. 2-1B), and the levels of LASgpc antibody and LCMnp antibody in plasma 14 days after the second administration were measured by enzyme-linked immunosorbent assays (ELISAs).
  • ELISAs enzyme-linked immunosorbent assays
  • Example 5 Verification of protective immune effect of mRNA-LNP vaccine against lethal virus exposure
  • C57BL/6 mice were intravenously administered the above-mentioned LASgpc-mRNA-LNP or LCMnp-mRNA-LNP, or saline twice at an interval of 3 weeks, and then intracerebrally inoculated with a lethal dose of rLCMV/LASgpc 2m on the 28th day after the second administration.
  • Example 6 Verification of the possibility of LCMnp-mRNA-LNP vaccine inducing virus-specific T cell responses
  • erythrocyte-free spleen cells obtained from C57BL/6 mice that had been intravenously administered LCMnp-mRNA-LNP or saline twice ( Figure 2-2E) were cultured in the presence of H-2d-restricted LCMnp T cell epitope peptide (NP396), and intracellular cytokine expression was examined by flow cytometry.
  • Example 7 Verification of the possibility of inducing virus-specific immunity by intramuscular administration of LASgpc-mRNA-LNP vaccine in C57BL/6 mice and the protective immunity effect against lethal virus challenge
  • LASgpc-mRNA-LNP containing 2 ⁇ g of IVT-mRNA or saline was administered intramuscularly to C57BL/6 mice twice at an interval of 3 weeks, and blood was collected from the mice to examine the possibility of inducing protective immunity and the protective immune effect against virus challenge (Figure 3A).
  • Example 8 Verification of the possibility of inducing virus-specific immunity by intramuscular administration of LASgpc-mRNA-LNP vaccine in CBA mice and the protective immunity effect against lethal virus challenge CBA mice are known to be more susceptible to LCMV than C57BL/6 mice.
  • CBA mice were inoculated intravenously with various amounts ( 102 to 105 FFU per mouse) of rLCMV/LASgpc 2m and monitored for clinical signs, body weight changes, and survival.
  • LASgpc-mRNA-LNP containing 2 ⁇ g of IVT-mRNA or saline was administered intramuscularly to CBA mice twice at an interval of 3 weeks (Fig. 4-1C), and the amount of LASgpc-specific antibodies in the plasma collected from the CBA mice 14 days after the second administration was examined (4-1D). Almost no LASgpc-specific antibodies were detected in the plasma of CBA mice.
  • Intramuscular administration of the LASgpc-mRNA-LNP vaccine in CBA mice demonstrated protective immune effects against lethal virus challenge, whereas no LASgpc-specific antibody responses were observed, suggesting that viral antigen-specific T cell responses are involved in the protective immune effects of LASgpc-mRNA-LNP against rLCMV/LASgpc 2m challenge.
  • Example 9 Verification of the ability of LASgpc-mRNA-LNP vaccine to induce viral antigen-specific T cell responses in CBA mice
  • erythrocyte-free spleen cells obtained from CBA mice that had been intramuscularly administered LASgpc-mRNA-LNP or saline twice ( Figure 4-2H) were stimulated with an LASgpc peptide cocktail, and intracellular cytokine expression was examined by flow cytometry.
  • Example 10 Induction of humoral and cellular immunity by intramuscular administration of LCMnp-mRNA-LNP vaccine in FVB mice and verification of protective effect against viral challenge causing fatal hemorrhagic symptoms
  • FVB mice LCMV-infected mouse model
  • FVB mice were intramuscularly administered 2 ⁇ g of IVT-mRNA-containing LCMnp-mRNA-LNP or saline twice at an interval of 3 weeks, and blood was collected from the mice 14 days after the second administration (Figure 5A).
  • FVB mice administered LCMnp-mRNA-LNP produced high levels of LCMnp-specific antibodies ( Figure 5B). This result demonstrated that intramuscular administration of the LCMnp-mRNA-LNP vaccine induced humoral immunity.
  • mice were intravenously inoculated with a normally lethal dose of WT rLCMV (Fig. 5A) and monitored for clinical signs and survival.
  • FVB mice administered saline died within 8 days after inoculation with WT rLCMV, whereas all FVB mice administered LCMnp-mRNA-LNP survived without obvious clinical signs (Fig. 5C).
  • erythrocyte-free spleen cells obtained from FVB mice that had been intramuscularly administered LCMnp-mRNA-LNP or saline twice (Fig. 5D) were stimulated with an H-2q-restricted LCMnp-specific T cell epitope peptide (NP118) and the expression of intracellular cytokines was examined by flow cytometry.
  • Example 11 Verification of protective effect by intramuscular administration of LASnp-mRNA-LNP vaccine in CBA mice We verified whether protective immunity was induced in CBA mice by intramuscular administration of an mRNA-LNP vaccine (LASnp-mRNA-LNP) using LASgpc-mRNA or mRNA (LASnp-mRNA) expressing Lassa virus NP (hereinafter referred to as "LASnp").
  • LASnp mRNA-LNP vaccine
  • the ORF of LASgpc or LASnp was cloned into the Linearized Template Vector of the Cloning Kit for mRNA Template (TaKaRa, product number 6143) to prepare a plasmid.
  • the prepared plasmid was digested with restriction enzymes and separated by agarose gel electrophoresis. The portion of the agarose gel containing the DNA fragment containing the T7 promoter, human ⁇ -globin 5'-UTR, viral protein ORF, human ⁇ -globin 3'-UTR, and polyA sequence was excised, and the DNA was purified.
  • mRNA was transcribed in vitro using the Takara IVTpro T7 mRNA Synthesis Kit (TaKaRa, product number 6144).
  • TriLink product number N-1093
  • CleanCap Reagent AG TriLink, product number N-7113
  • the prepared IVT-mRNA was purified using the Monarch RNA clean up kit (New England Biolabs), and the quality of the purified IVT-mRNA was examined by non-denaturing agarose gel electrophoresis using RNA High for Easy Electrophoresis (DynaMarker Laboratory).
  • the live attenuated LASV vaccine candidate strain ML29 was used for virus exposure.
  • ML29 is a genetic reassortment consisting of the S segment from LASV (containing the LASgpc and LASnp genes) and the L segment from Mopeia virus (MOPV), which is considered to be nonpathogenic to humans.
  • IVT-mRNA-LNPs LNP formulations
  • GenVoy-ILM Precision Nanosystems
  • IVT-mRNA prepared in Figure 6 using a microfluidic mixer NanoAssemblr® Ignite (Precision Nanosystems).
  • the N/P ratio molar ratio of cationic amine of ionic lipid to negatively charged phosphate of mRNA
  • the obtained sample was diluted with 1 ⁇ Formulation Buffer 2 (Precision Nanosystems), concentrated using an Amicon Ultra centrifugal filter (Millipore), and then sterile filtered through a 0.2 ⁇ m filter.
  • the purified mRNA-LNPs were stored at 4°C until use.
  • the concentration of IVT-mRNA incorporated into the LNPs was determined using the Quant-iT Ribogreen Assay Kit (ThermoFisher Scientific).
  • CBA mice were intramuscularly administered 2 ⁇ g of IVT-mRNA-LNP or LASnp-mRNA-LNP or saline twice at an interval of 3 weeks.
  • a lethal dose of ML29 was administered intracerebrally to examine the protective effect against viral challenge.
  • the results are shown in Figure 7. All mice administered saline died within 8 days. On the other hand, all mice administered LASgpc-mRNA-LNP survived, and 4 out of 5 mice administered LASnp-mRNA survived.
  • Example 12 Verification of protective effect by intramuscular administration of mRNA-LNP mixed vaccine in CBA mice We verified whether a synergistic effect could be obtained by combining the LASgpc-mRNA-LNP vaccine and the LASnp-mRNA-LNP vaccine compared to administering each vaccine alone.
  • CBA mice were intramuscularly administered with LASgpc-mRNA-LNPs containing 0.4 ⁇ g of LASgpc-mRNA, LASnp-mRNA-LNPs containing 0.4 ⁇ g of LASnp-mRNA, or a mixture of LASgpc-mRNA-LNPs containing 0.2 ⁇ g of LASgpc-mRNA and LASnp-mRNA-LNPs containing 0.2 ⁇ g of LASnp-mRNA, or saline twice at an interval of 3 weeks.
  • a lethal dose of ML29 was administered intracerebrally to examine the protective effect against viral challenge. The results are shown in Figure 8. All mice administered saline died within 8 days.
  • mice One out of five mice died in the LASgpc-mRNA-LNP group, and four out of five mice died in the LASnp-mRNA-LNP group.
  • all mice vaccinated with the mixed vaccine of LASgpc-mRNA-LNP and LASnp-mRNA-LNP survived. 100% of mice did not survive when LASgpc-mRNA-LNP or LASnp-mRNA-LNP were administered alone, but when they were mixed and administered at half the IVT-mRNA content (0.2 ⁇ g) of 0.4 ⁇ g, 100% of the mice survived, demonstrating a synergistic effect of mixing LASgpc-mRNA-LNP and LASnp-mRNA-LNP.

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