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LASSA VIRUS VACCINE

20190351044 · 2019-11-21

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention provides mRNAs usable as vaccines against lassa virus (LASV) infections. Further, the invention relates to (pharmaceutical) compositions and vaccines comprising said mRNAs and their use for treatment or prophylaxis of a lassa virus infection. The present invention further features a kit comprising the mRNAs, (pharmaceutical) compositions or vaccines and a method for treatment or prophylaxis of lassa virus infections using said mRNAs, pharmaceutical) compositions or vaccines.

    Claims

    1. mRNA comprising at least one coding region, said coding region encoding at least one antigenic peptide or protein derived from a protein of a lassa virus, or a variant or fragment thereof.

    2. The mRNA according to claim 1 usable as a vaccine.

    3. The mRNA according to claim 1 or 2, wherein the lassa virus is selected from lassa virus clade I, II, III and/or IV or from lassa virus lineage I, II, III and/or IV.

    4. The mRNA according to any one of claims 1 to 3, wherein said coding region encodes at least one antigenic peptide or protein derived from glycoprotein precursor (GPC) of a lassa virus, or a variant or fragment thereof, and/or at least one antigenic peptide or protein derived from nucleoprotein (NP) of a lassa virus, or a variant or fragment thereof, and/or at least one antigenic peptide or protein derived from zinc-binding matrix protein (Z) of a lassa virus, or a variant or fragment thereof, or a combination thereof.

    5. The mRNA according to any one of claims 1 to 4, wherein the mRNA is an artificial mRNA.

    6. The mRNA according to any one of claims 1 to 5, wherein the mRNA is a modified mRNA, preferably a stabilized mRNA.

    7. The mRNA according to any one of claims 1 to 6, wherein the at least one coding region encodes at least one full-length glycoprotein precursor (GPC) of a lassa virus, or a variant or fragment thereof, and/or at least one full-length nucleoprotein (NP) of a lassa virus, and/or at least one full-length zinc-binding matrix protein (Z) of a lassa virus, or a variant or fragment thereof.

    8. The mRNA according to any one of claims 1 to 7, wherein the mRNA comprises at least one coding region encoding at least one antigenic peptide or protein derived from glycoprotein precursor (GPC) of a lassa virus comprising or consisting of an amino acid sequence as defined by SEQ ID Nos: 1-186, or a variant or fragment of any of these sequences.

    9. The mRNA according to claim 8, wherein the coding region comprises an RNA sequence as defined by any of SEQ ID NOs: 376-561, or a variant or fragment of any of these sequences.

    10. The mRNA according to claim 8 or 9, wherein said the coding region comprises an RNA sequence selected from RNA sequences as defined by any of SEQ ID NOs: 751-936, 1126-1311, 1501-1686, 1876-2061, 2251-2436, 2626-2811 or 3001-3186, or a variant or fragment of any of these sequences.

    11. The mRNA according to any one of claims 1 to 10, wherein the mRNA comprises at least one coding region encoding at least one antigenic peptide or protein derived from nucleoprotein (NP) of a lassa virus comprising or consisting of an amino acid sequence as defined by any one of SEQ ID NOs. 187-375 or a variant or fragment of any of these sequences.

    12. The mRNA according to claim 11, wherein the coding region comprises an RNA sequence as defined by any of SEQ ID NOs: 562-750, or a variant or fragment of any of these sequences.

    13. The mRNA according to claim 10 or 12, wherein said the coding region comprises an RNA sequence selected from RNA sequences as defined by any of SEQ ID NOs: 937-1125, 1312-1500, 1687-1875, 2062-2250, 2437-2625, 2812-3000 or 3187-3375, or a variant or fragment of any of these sequences.

    14. The mRNA according to any one of claims 1 to 13, wherein the mRNA comprises at least one coding region encoding at least one antigenic peptide or protein derived from zinc-binding matrix protein (Z) of a lassa virus comprising or consisting of an amino acid sequence as defined by any one of SEQ ID NOs. 3451-3603 or a variant or fragment of any of these sequences.

    15. The mRNA according to claim 14, wherein the coding region comprises an RNA sequence as defined by any of SEQ ID NOs: 3604-3756, or a variant or fragment of any of these sequences.

    16. The mRNA according to claim 14 or 15, wherein said the coding region comprises an RNA sequence selected from RNA sequences as defined by any of SEQ ID NOs: 3757-3909, 3910-4062, 4063-4215, 4216-4368, 4369-4521, 4522-4674, or 4675-4827, or a variant or fragment of any of these sequences.

    17. The mRNA according to any one of claims 1 to 16, wherein the G/C content of the coding region of the mRNA is increased compared to the G/C content of the corresponding coding sequence of the wild type mRNA, or wherein the C content of the coding region of the mRNA is increased compared to the C content of the corresponding coding sequence of the wild type mRNA, or wherein the codon usage in the coding region of the mRNA is adapted to the human codon usage, or wherein the codon adaptation index (CAI) is increased or maximised in the coding region of the mRNA, wherein the encoded amino acid sequence of the mRNA is preferably not being modified compared to the encoded amino acid sequence of the wild type mRNA.

    18. The mRNA according to any of claims 1 to 17 comprising additionally a) a 5-cap structure, b) a poly(A) sequence, c) and optionally a poly (C) sequence.

    19. The mRNA according to claim 18, wherein the poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 50 to about 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably a sequence of about 60 to about 250 adenosine nucleotides.

    20. The mRNA according to any of claims 1 to 19 comprising additionally at least one histone stem-loop.

    21. The mRNA according to claim 20, wherein the at least one histone stem-loop comprises a nucleic acid sequence according to the following formulae (I) or (II): formula (I) (stem-loop sequence without stem bordering elements): ##STR00011## formula (II) (stem-loop sequence with stem bordering elements): ##STR00012## wherein: TABLE-US-00018 stem1 or stem2 bordering elements N1-6 is a consecutive sequence of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more preferably of 3 to 5, most preferably of 4 to 5 or 5N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C, or a nucleotide analogue thereof; stem1 [N.sub.0-2GN.sub.3-5] is reverse complementary or partially reverse complementary with element stem2, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N.sub.3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof, and wherein G is guanosine or an analogue thereof, and may be optionally replaced by a cytidine or an analogue thereof, provided that its complementary nucleotide cytidine in stem2 is replaced by guanosine; loop sequence [N.sub.0-4(U/T)N.sub.0-4] is located between elements stem1 and stem2, and is a consecutive sequence of 3 to 5 nucleotides, more preferably of 4 nucleotides; wherein each N.sub.0-4 is independent from another a consecutive sequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein U/T represents uridine, or optionally thymidine; stem2 [N.sub.3-5CN.sub.0-2] is reverse complementary or partially reverse complementary with element stem1, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N.sub.0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein C is cytidine or an analogue thereof, and may be optionally replaced by a guanosine or an analogue thereof provided that its complementary nucleotide guanosine in stem1 is replaced by cytidine; wherein stem1 and stem2 are capable of base pairing with each other forming a reverse complementary sequence, wherein base pairing may occur between stem1 and stem2, or forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stem1 and stem2.

    22. The mRNA according to claim 20 or 21, wherein the at least one histone stem-loop comprises a nucleic acid sequence according to the following formulae (Ia) or (IIa): formula (Ia) (stem-loop sequence without stem bordering elements): ##STR00013## formula (IIa) (stem-loop sequence with stem bordering elements): ##STR00014##

    23. The mRNA according to any one of claims 20 to 22, wherein the at least one histone stem loop comprises a nucleic acid sequence according to SEQ ID NO: 3394 and most preferably a RNA sequence according to SEQ ID NO: 3395.

    24. The mRNA according to any one of claims 1 to 23, wherein the mRNA comprises a poly(A) sequence, preferably comprising 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, and/or a poly(C) sequence, preferably comprising 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides.

    25. The mRNA according to any one of claims 1 to 24, wherein the mRNA comprises, preferably in 5 to 3 direction, the following elements: a.) a 5-cap structure, preferably m7GpppN, b.) at least one coding region encoding at least one antigenic peptide or protein derived from a protein of a lassa virus or a variant or fragment thereof, c.) a poly(A) tail, preferably consisting of 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides, d.) optionally a poly(C) tail, preferably consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and e.) optionally a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 3395.

    26. The mRNA according to any one of claims 1 to 25 comprising additionally a 3-UTR element.

    27. The mRNA according to claim 26, wherein the at least one 3-UTR element comprises or consists of a sequence element according to SEQ ID NO: 6412, 6413, 6314 or 6415 or a homolog, a fragment or a variant thereof.

    28. The mRNA according to claims 1 to 27, wherein the RNA comprises or consists of an RNA sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5884-6069, 6070-6259 or 6259-6411 (column 7 F mRNA design 3, Table 1-3) or a fragment or variant of any of these sequences.

    29. The mRNA according to claim 26 or 27, wherein the at least one 3-UTR element comprises or consists of a nucleic acid sequence which is derived from a 3-UTR of a gene providing a stable mRNA or from a homolog, a fragment or a variant thereof.

    30. The mRNA according to claim 29, wherein the 3-UTR element comprises or consists of a nucleic acid sequence derived from a 3-UTR of a gene selected from the group consisting of an albumin gene, an alpha-globin gene, a beta-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, or from a homolog, a fragment or a variant thereof.

    31. The mRNA according to any one of claims 29 to 30, wherein the 3-UTR element comprises a nucleic acid sequence derived from a 3-UTR of an alpha-globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 3386, a homolog, a fragment, or a variant thereof.

    32. The mRNA according to any of claims 1 to 31, wherein the mRNA comprises, preferably in 5- to 3-direction: a.) a 5-cap structure, preferably m7GpppN; b.) at least one coding region encoding at least one antigenic peptide or protein derived from a protein of a lassa virus or a variant or fragment thereof as defined in any one of the preceding claims, c.) a 3-UTR element comprising or consisting of a nucleic acid sequence which is derived from an alpha globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 3386, a homolog, a fragment or a variant thereof; d.) optionally, a poly(A) sequence, preferably comprising 64 adenosines; e.) optionally, a poly(C) sequence, preferably comprising 30 cytosines; and f.) optionally, a histone stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 3395.

    33. The mRNA according to claims 1 to 32, wherein the RNA comprises or consists of an RNA sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4828-5013, 5014-5202 or 5203-5355 (column 5 D mRNA design 1, Table 1-3) or a fragment or variant of any of these sequences.

    34. The mRNA according to claim 26, wherein the at least one 3-UTR element comprises a nucleic acid sequence, which is derived from the 3-UTR of a vertebrate albumin gene or from a variant thereof, preferably from the 3-UTR of a mammalian albumin gene or from a variant thereof, more preferably from the 3-UTR of a human albumin gene or from a variant thereof, even more preferably from the 3 UTR of the human albumin gene according to GenebankGenebank Accession number NM_000477.5, or from a variant or fragment thereof.

    35. The mRNA according to claim 32, wherein the 3-UTR element is derived from a nucleic acid sequence according to SEQ ID NO: 3390 or 3392, preferably from a corresponding RNA sequence, or a homolog, a fragment or a variant thereof.

    36. The mRNA according to any one of claims 1 to 35, wherein the mRNA sequence comprises a 5-UTR element.

    37. The mRNA according to claim 36, wherein the 5-UTR element comprises or consists of a nucleic acid sequence which is derived from the 5-UTR of a TOP gene preferably from a corresponding RNA sequence, a homolog, a fragment, or a variant thereof, preferably lacking the 5TOP motif.

    38. The mRNA according to claim 37, wherein the 5-UTR element comprises or consists of a nucleic acid sequence which is derived from a 5-UTR of a TOP gene encoding a ribosomal protein, preferably from a corresponding RNA sequence or from a homolog, a fragment or a variant thereof, preferably lacking the 5TOP motif.

    39. The mRNA according to claim 38, wherein the 5-UTR element comprises or consists of a nucleic acid sequence which is derived from a 5-UTR of a TOP gene encoding a ribosomal Large protein (RPL) or from a homolog, a variant or fragment thereof, preferably lacking the 5TOP motif and more preferably comprising or consisting of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 3376.

    40. The mRNA according to claim 39, wherein the 5-UTR element which is derived from a 5-UTR of a TOP gene comprises or consists of a corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO: 3377.

    41. The mRNA according to any one of claims 1 to 26 and 34 to 40, wherein the mRNA comprises, preferably in 5- to 3-direction: a.) a 5-cap structure, preferably m7GpppN; b.) a 5-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5-UTR of a TOP gene, preferably comprising or consisting of the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO: 3376 or 3378, a homolog, a fragment or a variant thereof; c.) at least one coding region encoding at least one antigenic peptide or protein derived from a protein of a lassa virus or a variant or fragment thereof as defined in any one of the preceding claims, d.) a 3-UTR element comprising or consisting of a nucleic acid sequence which is derived from a gene providing a stable mRNA, preferably comprising or consisting of the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO: 3390 or 3392, a homolog, a fragment or a variant thereof; e.) a poly(A) sequence preferably comprising 64 adenosines; f.) optionally a poly(C) sequence, preferably comprising 30 cytosines; and g.) optionally a histone-stem-loop, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 3395.

    42. The mRNA according to any one of claims 1 to 26 and 34 to 41, wherein the RNA comprises or consists of an RNA sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5356-5541, 5542-5730 or 5731-5883 (column 5 E mRNA design 2, Table 1-3) or a fragment or variant of any of these sequences.

    43. Composition comprising at least one mRNA according to any one of claims 1 to 42 and a pharmaceutically acceptable carrier.

    44. The composition according to claim 43, wherein the at least one mRNA is complexed with one or more cationic or polycationic compounds, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins, e.g. protamine, cationic or polycationic polysaccharides and/or cationic or polycationic lipids.

    45. The composition according to claim 44, wherein the N/P ratio of the at least one mRNA to the one or more cationic or polycationic compounds is in the range of about 0.1 to 20, including a range of about 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and of about 0.7 to 1.5.

    46. The composition according to claim 44 or 45, wherein the at least one mRNA is complexed with one or more cationic or polycationic compounds in a weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w) of mRNA to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate ratio of mRNA to cationic or polycationic compound and/or polymeric carrier in the range of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and most preferably in a range of about 0.5-1 or 0.7-1, and even most preferably in a range of about 0.3-0.9 or 0.5-0.9.

    47. The composition according to any one of claims 43 to 46 comprising the at least one mRNA, which is complexed with one or more cationic or polycationic compounds, and at least one free (m)RNA.

    48. The composition according to claim 47, wherein the at least one complexed mRNA is identical to the at least one free (m)RNA.

    49. The composition according to claim 47 or 48, wherein the molar ratio of the complexed mRNA to the free (m)RNA is selected from a molar ratio of about 0.001:1 to about 1:0.001, including a ratio of about 1:1.

    50. The composition according to any one of claims 47 to 49, wherein the ratio of the complexed mRNA to the free (m)RNA is selected from a range of about 5:1 (w/w) to about 1:10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of the complexed mRNA to the free (m)RNA is selected from a ratio of 1:1 (w/w).

    51. The composition according to any one of claims 43 to 50, wherein the mRNA is complexed or associated with one or more lipids, thereby forming liposomes, lipid nanoparticles and/or lipoplexes.

    52. The composition according to any one of claims 4336 to 51, wherein the composition comprises at least one adjuvant.

    53. The composition according to claim 52, wherein the adjuvant is selected from the group consisting of: cationic or polycationic compounds, comprising cationic or polycationic peptides or proteins, including protamine, nucleoline, spermin or spermidine, poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, pIsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, protamine, spermine, spermidine, or histones, cationic polysaccharides, including chitosan, polybrene, cationic polymers, including polyethyleneimine (PEI), cationic lipids, including DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, including modified polyaminoacids, including -aminoacid-polymers or reversed polyamides, modified polyethylenes, including PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, including pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified Amidoamines including pAMAM (poly(amidoamine)), modified polybetaaminoester (PBAE), including diamine end modified 1,4 butanediol diacrylate-co-5-amino-1-pentanol polymers, dendrimers, including polypropylamine dendrimers or pAMAM based dendrimers, polyimine(s), including PEI: poly(ethyleneimine), poly(propyleneimine), polyallylamine, sugar backbone based polymers, including cyclodextrin based polymers, dextran based polymers, Chitosan, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., block polymers consisting of a combination of one or more cationic blocks selected from a cationic polymer as mentioned before, and of one or more hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); or cationic or polycationic proteins or peptides, selected from the following proteins or peptides having the following total formula (III): (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x, wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except from Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50% of all amino acids of the oligopeptide; or nucleic acids having the formula (V): G.sub.lX.sub.mG.sub.n, wherein: G is guanosine (guanine), uridine (uracil) or an analogue of guanosine (guanine) or uridine (uracil); X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of the above-mentioned nucleotides (nucleosides); l is an integer from 1 to 40, wherein, when l=1 G is guanosine (guanine) or an analogue thereof, when l>1 at least 50% of the nucleotides (nucleosides) are guanosine (guanine) or an analogue thereof; m is an integer and is at least 3; wherein when m=3 X is uridine (uracil) or an analogue thereof, when m>3 at least 3 successive uridines (uracils) or analogues of uridine (uracil) occur; n is an integer from 1 to 40, wherein when n=1 G is guanosine (guanine) or an analogue thereof, when n>1 at least 50% of the nucleotides (nucleosides) are guanosine (guanine) or an analogue thereof; or nucleic acids having the formula (VI): C.sub.lX.sub.mC.sub.n, wherein: C is cytidine (cytosine), uridine (uracil) or an analogue of cytidine (cytosine) or uridine (uracil); X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of the above-mentioned nucleotides (nucleosides); I is an integer from 1 to 40, wherein when l=1 C is cytidine (cytosine) or an analogue thereof, when l>1 at least 50% of the nucleotides (nucleosides) are cytidine (cytosine) or an analogue thereof; m is an integer and is at least 3; wherein when m=3 X is uridine (uracil) or an analogue thereof, when m>3 at least 3 successive uridine (uracils) or analogues of uridine (uracil) occur; n is an integer from 1 to 40, wherein when n=1 C is cytidine (cytosine) or an analogue thereof, when n>1 at least 50% of the nucleotides (nucleosides) are cytidine (cytosine) or an analogue thereof; or adjuvants selected from the group consisting of: TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMER (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE (propanediamine); BAY R1005 ((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate); CALCITRIOL (1-alpha,25-dihydroxy-vitamin D3); calcium phosphate gel; CAP (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein, sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine); imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine); ImmTher (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRVs (immunoliposomes prepared from dehydration-rehydration vesicles); interferon-gamma; interleukin-ibeta; interleukin-2; interleukin-7; interleukin-12; ISCOMS; ISCOPREP 7.0.3.; liposomes; LOXORIBINE (7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MF59; (squalene-water emulsion); MONTANIDE ISA 51 (purified incomplete Freund's adjuvant); MONTANIDE ISA 720 (metabolisable oil adjuvant); MPL (3-Q-desacyl-4-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt); MURAMETIDE (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE and D-MURAPALMITINE (Nac-Mur-L-Thr-D-isoGIn-sn-glyceroldipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISVs (non-ionic surfactant vesicles); PLEURAN (-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolic acid; microspheres/nanospheres); PLURONIC L121; PMMA (polymethyl methacrylate); PODDS (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5 c]quinoline-1-ethanol); SAF-1 (Syntex adjuvant formulation); Sendai proteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane (2,6,10,15,19,23-hexamethyltetracosan and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyltyrosine (octadecyltyrosine hydrochloride); Theramid (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); Theronyl-MDP (Termurtide or [thr 1]-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys, in particular aluminium salts, such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, including Stealth, cochleates, including BIORAL; plant derived adjuvants, including QS21, Quil A, Iscomatrix, ISCOM; adjuvants suitable for costimulation including Tomatine, biopolymers, including PLG, PMM, Inulin; microbe derived adjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid sequences, CpG7909, ligands of human TLR 1-10, ligands of murine TLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists including CGRP neuropeptide.

    54. The composition according to any one of claims 43 to 53, wherein the composition comprises a plurality or more than one of the mRNAs each defined in any one of claims 1 to 42.

    55. The composition according to claim 54, wherein each of the mRNAs comprises a coding region encoding at least one different antigenic peptide or protein derived from proteins of the same lassa virus.

    56. The composition according to claim 55, wherein each of the mRNAs comprises a coding region which encodes at least one different antigenic peptide or protein derived from different proteins of the same lassa virus.

    57. The composition according to claim 55, wherein each of the mRNAs comprises a coding region which encodes at least one different antigenic peptide or protein derived from different proteins of different lassa viruses.

    58. The composition according to any one of claims 54 to 57, wherein each of the mRNAs comprises a coding region which encodes at least one antigenic peptide or protein derived from glycoprotein precursor (GPC) or nucleoprotein (NP) or zinc-binding matrix protein (Z) of a lassa virus or a variant or fragment thereof.

    59. The composition according to any one of claims 54 to 57, wherein each of the mRNAs comprises a coding region which encodes at least one antigenic peptide or protein derived from glycoprotein precursor (GPC) and nucleoprotein (NP) of a lassa virus or a variant or fragment thereof.

    60. The composition according to any one of claims 54 to 57, wherein each of the mRNAs comprises a coding region which encodes at least one antigenic peptide or protein derived from glycoprotein precursor (GPC) and zinc-binding matrix protein (Z) of a lassa virus or a variant or fragment thereof.

    61. The composition according to any one of claims 54 to 57, wherein each of the mRNAs comprises a coding region which encodes at least one antigenic peptide or protein derived from glycoprotein precursor (GPC) and nucleoprotein (NP) and zinc-binding matrix protein (Z) of a lassa virus or a variant or fragment thereof.

    62. The composition according to claims 54 to 61, wherein at least one antigenic peptide or protein derived from glycoprotein precursor (GPC) and/or nucleoprotein (NP) and/or zinc-binding matrix protein (Z) of 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 100 different lassa viruses are encoded by the plurality of mRNAs.

    63. The composition according to claims 57 to 62, wherein the different lassa viruses are belonging to different lassa virus clades or different lassa virus lineages, preferably to the lassa virus clades I, II, III and IV or to the lassa virus lineages I, II, III and IV.

    64. The composition according to claim 62 or 63, wherein the composition comprises at least one mRNA encoding at least one antigenic peptide or protein derived from glycoprotein precursor (GPC) of a lassa virus and at least one mRNAs encoding at least one antigenic peptide or protein derived from nucleoprotein (NP) of the same lassa virus.

    65. Vaccine comprising the mRNA according to any one of claims 1 to 42 and a pharmaceutically acceptable carrier, or comprising the composition according to any one of claims 43 to 64.

    66. The vaccine according to claim 65, wherein the mRNA according to any one of claims 1 to 42 or the composition according to any one of claims 43 to 65 elicits an adaptive immune response.

    67. The vaccine according to claim 65 or 66 comprising an adjuvant.

    68. Kit or kit of parts comprising the components of the mRNA according to any one of claims 1 to 42, the composition according to any one of claims 43 to 64, the vaccine according to any one of claims 65 to 67 and optionally technical instructions with information on the administration and dosage of the components.

    69. mRNA as defined according to any one of claims 1 to 42, the composition according to any one of claims 43 to 64, the vaccine according to any one of claims 65 to 67, the kit or kit of parts according to claim 68 for use as a medicament.

    70. mRNA as defined according to any one of claims 1 to 42, the composition according to any one of claims 43 to 64, the vaccine according to any one of claims 65 to 67, the kit or kit of parts according to claim 68 for use in the treatment or prophylaxis of lassa infections or disorders related thereto.

    71. mRNA, composition, vaccine or kit or kit of parts for use according to claim 69 or 70, wherein the mRNA, the composition, the vaccine or the kit or kit of parts is administered by subcutaneous, intramuscular or intradermal injection, preferably by intramuscular or intradermal injection, more preferably by intradermal injection.

    72. mRNA, composition, vaccine and kit or kit of parts for use according to claim 71, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

    73. A method of treatment or prophylaxis of lassa infections comprising the steps: a) providing the mRNA as defined according to any one of claims 1 to 42, the composition according to any one of claims 43 to 65, the vaccine according to any one of claims 65 to 67, the kit or kit of parts according to claim 68; b) applying or administering the mRNA, the composition, the vaccine or the kit or kit of parts to a tissue or an organism.

    74. The method according to claim 73, wherein the mRNA, the composition, the vaccine or the kit or kit of parts is administered to the tissue or to the organism by subcutaneous, intramuscular or intradermal injection, preferably by intramuscular or intradermal injection, more preferably by intradermal injection.

    75. The method according to claim 74, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

    Description

    BRIEF DECRYPTION OF THE DRAWINGS

    [0662] FIG. 1 shows that mRNA constructs encoding Lassa virus glycoprotein precursor (GPC) of different lassa virus clades (clade I, II, III and IV) were expressed and processed in vitro. Further details are provided in Example 3.

    EXAMPLES

    [0663] The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

    Example 1: Preparation of mRNA Constructs for In Vitro and In Vivo Experiments

    [0664] 1.1. Preparation of DNA and mRNA Constructs:

    [0665] For the present Examples, DNA sequences encoding Lassa virus proteins are prepared and used for subsequent RNA in vitro transcription reactions. The generated RNA constructs (RNA sequences) are provided in the sequence listing (Lassa virus sequences: SEQ ID NOs: 5541, 5729, 5731, 5529, 5356, 5540, and 5913) and in Table 6 with the encoded proteins and virus origin and virus clade indicated.

    TABLE-US-00013 TABLE 6 mRNA constructs used in the present expamles Virus SEQ ID mRNA RNA ID strain Virus clade Construct description SEQ ID NO RNA Protein design R6023/ Josiah clade IV LASV(Josiah)-GPC(GC) SEQ ID NO: SEQ ID NO: design 2 R6630 5541 186 R6024 Josiah clade IV LASV(Josiah)-NP(GC) SEQ ID NO: SEQ ID NO: design 2 5729 374 R6025 Josiah clade IV LASV(Josiah)-Z(GC) SEQ ID NO: SEQ ID NO: design 2 5731 3451 R6026/ LP clade I LASV(LP)-GPC(GC) SEQ ID NO: SEQ ID NO: design 2 R6631 5529 174 R6027/ 803213 clade II LASV(803213)- SEQ ID NO: SEQ ID NO: 1 design 2 R6632 GPC(GC) 5356 R6028/ GA391 clade III LASV(GA391)- SEQ ID NO: SEQ ID NO: design 2 R6633 GPC(GC) 5540 185 R6717 Josiah clade IV LASV(Josiah)-GPC(GC) SEQ ID NO: SEQ ID NO: design 1 5013 186

    [0666] DNA sequences are prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in silico algorithms that increase the GC content of the respective coding sequence. Moreover, sequences are introduced into a pUC19 derived vector and modified to comprise stabilizing sequences derived from alpha-globin-3-UTR, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3-terminal end (poly-A-tail) (indicated as mRNA design 1 in Table 1-3 and 6). Other sequences were introduced into a pUC19 derived vector to comprise stabilizing sequences derived from 32L4 5-UTR ribosomal 5 TOP UTR and 3-UTR derived from albumin 7, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3-terminal end (poly-A-tail) (indicated as mRNA design 2 in Table 1-3 and 6). Generated mRNA constructs are provided in Table 6

    [0667] The obtained plasmid DNA constructs are transformed and propagated in bacteria (Escherichia coli) using common protocols known in the art.

    [0668] 1.2. RNA In Vitro Transcription on Linearized pDNA:

    [0669] The DNA plasmids prepared according to paragraph 1 are enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG) under suitable buffer conditions. RNA production is performed under current good manufacturing practice according to WO2016/180430. The obtained mRNAs are purified using PureMessenger (CureVac, Tbingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. The generated mRNA constructs are indicated as mRNA design 1 and as mRNA design 2 in Table 1-3.

    [0670] 1.3. RNA In Vitro Transcription on PCR Amplified DNA Templates:

    [0671] DNA plasmids prepared according to paragraph 1, or synthic DNA constructs are used for PCR-amplification. The generated PCR templates are used for subsequent RNA in vitro transcription using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG) under suitable buffer conditions. The obtained mRNA constructs are purified using PureMessenger (CureVac, Tubingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. The generated mRNA constructs are indicated as mRNA design 3 in Table 1-3.

    Example 2: Expression of Lassa Virus Proteins in HeLa Cells and Analysis by FACS

    [0672] To determine in vitro protein expression of the constructs, HeLa cells are transiently transfected with mRNA encoding Lassa virus antigens and stained using suitable customized anti-LASV antibodies (raised in rabbits), counterstained with a FITC-coupled secondary antibody.

    [0673] HeLa cells are seeded in a 6-well plate at a density of 400,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24 h prior to transfection. HeLa cells are transfected with 1 and 2 g unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs according to Example 1 are used in the experiment, including a negative control encoding an irrelevant protein.

    [0674] 24 hours post transfection, HeLa cells are stained with suitable anti anti-LASV antibodies (raised in rabbits; 1:200) and anti-rabbits FITC labelled secondary antibody (1:500) and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal is performed using the FlowJo software package (Tree Star, Inc.).

    Example 3: Expression of Lassa Virus Proteins Using Western Blot

    [0675] The results of the present Example show that mRNA encoding Lassa Virus GPC protein are expressed and processed in Hela cells after transfection.

    [0676] For the analysis of Lassa virus protein expression, HeLa cells are transfected with 1 g and 2 g unformulated mRNA using Lipofectamine as the transfection agent. Supernatants, harvested 24 hours post transfection, are filtered through a 0.2 m filter. Clarified supernatants are applied on top of 1 ml 20% sucrose cushion (in PBS) and centrifuged at 14000 rcf (relative centrifugal force) for 2 hours at 4 C. Protein content is analyzed by Western Blot using suitable customized polyclonal anti-LASV antibodies (raised in rabbits) (1:1000) as primary antibody in combination with secondary anti rabbit antibody coupled to IRDye 800CW (Licor Biosciences). The presence of -tubulin is also analyzed as control for cellular contamination (-tubulin; Cell Signalling Technology; 1:1000 diluted) in combination with secondary anti mouse antibody coupled to IRDye 680RD (Licor Biosciences).

    [0677] For the analysis LASV proteins in cell lysates, HeLa cells were transfected with 2 g unformulated mRNAs (generated according to Example 1, see Table 6) using Lipofectamine as the transfection agent 24 hours post transfection, HeLa cells are detached by trypsin-free/EDTA buffer, harvested, and cell lysates are prepared. Cell lysates are subjected to SDS-PAGE under denaturating and reducing conditions followed by western blot detection. Western Blot analysis is performed using suitable customized polyclonal rabbit anti-LASV GP antibodies (diluted 1:200) as primary antibody in combination with secondary anti anti-rabbit antibody coupled to IRDye 800CW (Licor Biosciences). The presence of -tubulin was analyzed (-tubulin; Cell Signalling Technology; 1:1000 diluted) in combination with secondary anti rabbit antibody coupled to IRDye 680RD (Licor Biosciences). Inactivated Lassa virus (Josiah) was used as positive control for the western blot. The outline of the experiment is shown in Table 7. The result of the experiment is shown in FIG. 1.

    TABLE-US-00014 TABLE 7 Expression analysis experiment (Example 3): Lassa virus Lane SEQ ID No R# encoded antigen clade 1 SEQ ID NO: 5541 R6023 LASV(Josiah)-GP clade IV 2 SEQ ID NO: 5529 R6026 LASV(LP)-GP clade I 3 SEQ ID NO: 5356 R6027 LASV(803213)-GP clade II 4 SEQ ID NO: 5540 R6028 LASV(GA391)-GP clade III 5 SEQ ID NO: 5729 R6024 LASV(Josiah)-NP clade IV 6 WFI 7 Inactivated clade IV LASV Josiah

    [0678] Results:

    [0679] As shown in FIG. 1, mRNA constructs encoding lassa virus glycoprotein precursors (GPC) were expressed in vitro. Moreover, the expressed encoding lassa virus glycoprotein precursors were properly processed, thus cotranslationally cleaved by signal peptidases. The subunit protein GP2 was detected as expected.

    [0680] The results exemplify that the inventive mRNA encoding Lassa virus GPC protein is translated in cells and that alternative mRNA constructs according to the invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

    Example 4: Vaccination of Mice with mRNA Encoding Lassa Virus Proteins

    [0681] 4.1. Preparation of Protamine Complexed mRNA (Vaccine Composition 1):

    [0682] Lassa virus mRNA constructs are complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consists of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA is complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes are stably generated, free mRNA is added, and the final concentration of the vaccine is adjusted with Ringer's lactate solution.

    [0683] 4.2. Immunization:

    [0684] Female BALB/c mice are injected intradermally (i.d.) with mRNA vaccine compositions with doses, application routes and vaccination schedules as indicated in Table 8. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 0, 21 and 42. Blood samples are collected on day 21, 35, and 49 for the determination of antibody titers. Splenocytes are isolated on day 49 for T-cell analysis.

    TABLE-US-00015 TABLE 8 Vaccination regimen (Example 4): Number Vaccine Group of mice composition SEQ ID NO Dose Route/Volume 1 6 R6630 SEQ ID NO: 80 g i.d. 2 50 l (Lassa GPC 5541 Josiah strain) 2 6 R6631 SEQ ID NO: 80 g i.d. 2 50 l (Lassa GPC 5529 LP strain) 3 6 RiLa buffer

    [0685] 4.3. Detection of Specific Humoral Immune Responses:

    [0686] Hela cells are transfected with 2 g of either R6630 or R6631 mRNA using lipofectamine. The cells are harvested 20 h post transfection, and seeded at 110.sup.5 per well into an 96 well plate. The cells are incubated with sera of the vaccinated mice (diluted 1:50) followed by a FITC-conjugated anti-mouse IgG antibody. Cells are acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo.

    [0687] 4.4. Determination of Anti Lassa Virus Protein Antibodies by ELISA:

    [0688] ELISA is performed using recombinant Lassa Glycoproteins for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Lassa virus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies directed against the Lassa virus antigens are measured by ELISA on day 49 after three vaccinations.

    [0689] 4.5. Intracellular Cytokine Staining:

    [0690] Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art. Briefly, isolated spleens are grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS splenocytes are seeded into 96-well plates (2106 cells per well). The cells are stimulated with recombinant LASV virus in the presence of 2.5 g/ml of an anti-CD28 antibody (BD Biosciences) and -CD107a-PE-Cy7 (1:100) for 24 hours at 37 C. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: anti-CD8-APC-H7 (1:100), anti-CD4-BD-Horizon V450 (1:200), anti-CD3-Thy1.2-FITC (1:200) and incubated with Fc-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

    Example 5: Preparation of Lassa Virus Vaccine Compositions

    [0691] For further in vivo vaccination experiments, different compositions of Lassa virus mRNA vaccine are prepared using constructs obtained in Example 1. One composition comprises protamine-complexed mRNA, one composition comprises mRNA that is formulated without protamine (naked), one composition comprises mRNA that is encapsulated in lipid nanoparticles (LNPs), and one composition comprises polymer-lipidoid complexed mRNA.

    [0692] 5.1. Preparation of Protamine Complexed mRNA (Vaccine Composition 1):

    [0693] Lassa virus mRNA constructs are complexed as described in Example 4.

    [0694] 5.2. Preparation of Naked mRNA (Vaccine Composition 2):

    [0695] Lassa virus mRNA constructs are formulated without protamine. The final concentration of the vaccine is adjusted with Ringer's lactate solution.

    [0696] 5.3. Preparation of LNP Encapsulated mRNA (Vaccine Composition 3):

    [0697] A lipid nanoparticle (LNP)-encapsulated mRNA mixture is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. LNPs are prepared as follows. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol. Briefly, mRNA mixture is diluted to a total concentration of 0.05 mg/mL in 50 mM citrate buffer, pH 4. Syringe pumps are used to mix the ethanolic lipid solution with the mRNA mixture at a ratio of about 1:6 to 1:2 (vol/vol). The ethanol is then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 m pore sterile filter. Lipid nanoparticle particle diameter size is determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

    [0698] 5.4. Preparation of Polymer-Lipidoid Complexed mRNA (Vaccine Composition 4):

    [0699] 20 mg peptide (CHHHHHHRRRRHHHHHHCNH2; SEQ ID NO: 3450) TFA salt was dissolved in 2 mL borate buffer pH 8.5 and stirred at room temperature for approximately 18 h. Then, 12.6 mg PEG-SH 5000 (Sunbright) dissolved in N-methylpyrrolidone was added to the peptide solution and filled up to 3 mL with borate buffer pH 8.5. After 18 h incubation at room temperature, the reaction mixture was purified and concentrated by centricon procedure (MWCO 10 kDa), washed against water, and lyophilized. The obtained lyophilisate was dissolved in ELGA water and the concentration of the polymer was adjusted to 10 mg/mL. The obtained polyethylene glycol/peptide polymers (HO-PEG 5000-S(SCHHHHHHRRRRHHHHHHCS-)7-S-PEG 5000-OHpeptide component: SEQ ID NO: 3450) were used for further formulation and are hereinafter referred to as PB83.

    [0700] Preparation of 3-C12-OH Lipidoid:

    [0701] First, lipidoid 3-C12 was obtained by acylation of tris(2-aminoethyl)amine with an activated lauric (C12) acid derivative, followed by reduction of the amide. Alternatively, it may be prepared by reductive amination with the corresponding aldehyde. Lipidoid 3-C12-OH was prepared by addition of the terminal C12 alkyl epoxide with the same oligoamine according to Love et al., pp. 1864-1869, PNAS, vol. 107 (2010), no. 5.

    [0702] Preparation of Compositions with Nanoparticles of Polymer-Lipidoid Complexed mRNA:

    [0703] First, ringer lactate buffer (RiLa; alternatively e.g. saline (NaCl) or PBS buffer may be used), respective amounts of lipidoid, and respective amounts of a polymer (PB83) were mixed to prepare compositions comprising a lipidoid and a peptide or polymer. Then, the carrier compositions were used to assemble nanoparticles with the mRNA by mixing the mRNA with respective amounts of polymer-lipidoid carrier and allowing an incubation period of 10 minutes at room temperature such as to enable the formation of a complex between the lipidoid, polymer and mRNA. In order to characterize the integrity of the obtained polymer-lipidoid complexed mRNA particles, RNA agarose gel shift assays were performed. In addition, size measurements were performed (gel shift assay, Zetasizer) to evaluate whether the obtained nanoparticles have a uniform size profile.

    Example 6: Vaccination of Mice and Evaluation of Lassa Virus Specific Immune Response

    [0704] 6.1. Immunization:

    [0705] Female BALB/c mice are injected intradermally (i.d.) and intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 5) with doses, application routes and vaccination schedules as indicated in Table 9. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 for the determination of binding and neutralizing antibody titers (see below). Splenocytes were isolated on day 49 for T-cell analysis.

    TABLE-US-00016 TABLE 9 Vaccination regimen - Lassa virus experiment (Example 6) Number Route/ Vaccination Group of mice Vaccine composition Volume Schedule (day) 1 10 40 g Lassa virus RNA i.d. 0/21/35 Composition 1 2 25 l 2 10 40 g Lassa virus RNA i.m. 0/21/35 Composition 1 2 25 l 3 10 20 g Lassa virus RNA i.d. 0/21/35 Composition 2 2 25 l 4 10 20 g Lassa virus RNA i.m. 0/21/35 Composition 2 2 25 l 5 10 10 g Lassa virus RNA i.d. 0/21/35 Composition 3 2 25 l 6 10 10 g Lassa virus RNA i.m. 0/21/35 Composition 3 2 25 l 7 10 40 g Lassa virus RNA i.d. 0/21/35 Composition 4 2 25 l 8 10 40 g Lassa virus RNA i.m. 0/21/35 Composition 4 2 25 l 9 10 100% RiLa i. d. 0/21/35 Control 2 25 l

    [0706] 6.2. Determination of Anti Lassa Virus Protein Antibodies by ELISA:

    [0707] ELISA is performed using inactivated Lassa virus infected cell lysate or recombinant Lassa Glycoproteins for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Lassa virus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies directed against the Lassa virus antigens are measured by ELISA on day 63 after three vaccinations.

    [0708] 6.3. Intracellular Cytokine Staining:

    [0709] Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art. Briefly, isolated spleens are grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS splenocytes are seeded into 96-well plates (2106 cells per well). The cells are stimulated with recombinant LASV virus in the presence of 2.5 g/ml of an anti-CD28 antibody (BD Biosciences) and -CD107a-PE-Cy7 (1:100) for 24 hours at 37 C. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: anti-CD8-APC-H7 (1:100), anti-CD4-BD-Horizon V450 (1:200), anti-CD3-Thy1.2-FITC (1:200) and incubated with Fc-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

    [0710] 6.4. Lassa Virus Plaque Reduction Neutralization Test (PRNT50):

    [0711] Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art. Briefly, obtained serum samples of vaccinated mice are incubated with Lassa virus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.

    Example 7: Vaccination of Mice and Evaluation of Lassa Virus Specific Immune Response

    [0712] 7.1. Immunization:

    [0713] Female BALB/c mice are injected intramuscularly (i.m.) with LNP encapsulated mRNA R6717 coding for GPC (LASV Josiah, SEQ ID NO: 5013, see Table 6, Example 1, mRNA design 1 andvaccine composition 3 prepared according to Example 5) with doses, application routes and vaccination schedules as indicated in Table 10. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 1 and 21. Blood samples are collected on day 21 and 35 for the determination of binding and neutralizing antibody titers (see below). Splenocytes were isolated on day 35 for T-cell analysis.

    TABLE-US-00017 TABLE 10 Vaccination regimen - Lassa virus experiment (Example 7) Number Route/ Vaccination Group of mice Vaccine composition Volume Schedule (day) 1 6 5 g Lassa virus RNA i.d. 0/21 GPC (Josiah; R6717) 2 25 l vaccine composition 3 2 6 2 g Lassa virus RNA i.m. 0/21 GPC (Josiah; R6717) 2 25 l vaccine composition 3 3 6 1 g Lassa virus RNA i.d. 0/21 GPC (Josiah; R6717) 2 25 l vaccine composition 3 4 6 100% RiLa i.m. 0/21 Control 2 25 l

    [0714] 7.2. Determination of Anti Lassa Virus Protein Antibodies by ELISA:

    [0715] ELISA is performed using inactivated Lassa virus infected cell lysate for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Lassa virus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies directed against the Lassa virus antigens are measured by ELISA on day 35 after two vaccinations.

    [0716] 7.3. Intracellular Cytokine Staining:

    [0717] Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art. Briefly, isolated spleens are grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS splenocytes are seeded into 96-well plates (2106 cells per well). The cells are stimulated with recombinant LASV virus in the presence of 2.5 g/ml of an anti-CD28 antibody (BD Biosciences) and -CD107a-PE-Cy7 (1:100) for 24 hours at 37 C. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: anti-CD8-APC-H7 (1:100), anti-CD4-BD-Horizon V450 (1:200), anti-CD3-Thy1.2-FITC (1:200) and incubated with Fc-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

    [0718] 7.4. Lassa Virus Plaque Reduction Neutralization Test (PRNT50):

    [0719] Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art. Briefly, obtained serum samples of vaccinated mice are incubated with Lassa virus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.

    Example 8: Clinical Development of a Lassa Virus mRNA Vaccine Composition

    [0720] To demonstrate safety and efficiency of the Lassa virus mRNA vaccine composition, a clinical trial (phase I) is initiated.

    [0721] In the clinical trial, a cohort of human volunteers is intradermally or intramuscularly injected for at least two times.

    [0722] In order to assess the safety profile of the vaccine compositions according to the invention, subjects are monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis). The efficacy of the immunization is analyzed by determination of virus neutralizing titers (VNT) in sera from vaccinated subjects. Blood samples are collected on day 0 as baseline and after completed vaccination. Sera are analyzed for virus neutralizing antibodies.