NOVEL LASSA VIRUS RNA MOLECULES AND COMPOSITIONS FOR VACCINATION
20210260178 · 2021-08-26
Assignee
Inventors
Cpc classification
C12N2760/10034
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
A61K2039/55555
HUMAN NECESSITIES
A61K2039/545
HUMAN NECESSITIES
International classification
Abstract
The present invention is directed to RNA suitable for use in treatment and/or prophylaxis of an infection with Lassa virus (LASV) or of a disorder related to such an infection. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the RNA, compositions, and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the RNA, compositions and vaccines.
Claims
1. RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from a Lassa virus (LASV) protein or a fragment or variant thereof, wherein said coding sequence is operably linked to a 5′-UTR derived from a HSD17B4 gene, a NDUFA4 gene, or a RPL32 gene and/or a 3′-UTR derived from a PSMB3 gene, a CASP1 gene, an ALB7 gene, or an alpha-globin gene.
2. RNA according to claim 1, wherein said coding sequence is operably linked to a 5′-UTR and/or 3′-UTR, comprising a-1. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or a-2. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or b-4. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or i-2. at least one 5′-UTR derived from a 5′-UTR of a RPL32 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a ALB7 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or i-3. at least one 3′-UTR derived from a 3′-UTR of a alpha-globin gene gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof.
3. RNA according to claim 1 or 2, wherein said 5′-UTR derived from a HSD17B4 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13804 or 13805 or a fragment or a variant thereof; said 5′-UTR derived from a NDUFA4 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13812 or 13813 or a fragment or a variant thereof; said 5′-UTR derived from a RPL32 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13824 or 13825 or a fragment or a variant thereof; said 3′-UTR derived from a PSMB3 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13826 or 13827 or a fragment or a variant thereof; said 3′-UTR derived from a CASP1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13828 or 13829 or a fragment or a variant thereof; said 3′-UTR derived from a ALB7 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13838 or 13839 or a fragment or a variant thereof; said 3′-UTR derived from a alpha-globin gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to EQ ID NO: 13840 or 13841 or a fragment or a variant thereof.
4. RNA according to any one of the preceding claims, wherein the LASV protein is derived from glycoprotein precursor (GPC), a prefusion-stabilized GPC, nucleoprotein (NP), zinc-binding matrix protein (Z), or a variant, fragment, or combination thereof, wherein GPC, prefusion-stabilized GPC, NP, Z are preferably full-length proteins.
5. RNA according claim 4, wherein the prefusion-stabilized GPC comprises at least one of the following mutations A, preferably of A1, A2, A3 and B, and C: A amino acid substitutions allowing a covalent link of different structural elements of GPC, preferably by introduction of two additional cysteine residues, wherein preferably A1 the amino acid substitutions allowing a covalent link of GP1 and GP2, preferably by introduction of two additional cysteine residues, preferably at position 207 or 206 and 360 or 359, preferably by amino acid substitutions R2070 or R2060 and G3600 or G359C, or A2 the amino acid substitutions allowing a covalent link of GP1 to HR1 of GP2, preferably by introduction of two additional cysteine residues, preferably at position 81 or 80 and 319 or 318, preferably by amino acid substitutions T81 C or T800 and N3190 or N3180, or A3 the amino acid substitutions allowing a covalent link of T-loop in GP2 to adjacent loop in GP2, preferably by introduction of two additional cysteine residues, preferably at positon 370 or 369 and 386 or 385, preferably by amino acid substitutions W3700 or W369C and W386C or W385C, B amino acid substitutions allowing stabilization of GP2, preferably by introduction of a helix-breaking point mutation in the metastable region of HR1 of GP2 subunit, more preferably by introducing E329P or E328P, C replacing the native GP1-GP2 cleavage site (S1 P) with a furin cleavage site, e.g. RRLL to RRRR to facilitate efficient processing of the mRNA encoded antigenic protein in target cells, preferably by amino acid substitutions L258R, L259R or L257R, L258R, wherein prefusion-stabilized GPC preferably comprises at least one mutation A selected from A1, A2 and A3.
6. RNA according to any one of the preceding claims, wherein said at least one coding sequence encodes at least one antigenic peptide or protein derived from LASV GPC comprising or consisting of an amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 1-254 or a fragment or a variant thereof; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut1” or “GPCstabilized”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3567-3820 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut2”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 22949-22980 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut3”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 24645-24676 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut4”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 26341-26372 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut5”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 28037-28068 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut6”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 29733-29764 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut7”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 31429-31460 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut8”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 33125-33156 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut9”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 34821-34852 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut10”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 36517-36548 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut11”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 38213-38244 or a fragment or variant of any of these sequences, said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV prefusion-stabilized GPC (“GPCmut12”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 39909-39940 or a fragment or variant of any of these sequences, said at least one coding sequence encoding at least one antigenic peptide or protein derived from LASV GPC lacking the cytoplasmic tail (“GPCmut13”) comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 41605- 41636 or a fragment or variant of any of these sequences, said at least one coding sequence encodes at least one antigenic peptide or protein derived from LASV NP comprising or consisting of an amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 7547-7797 or a fragment or a variant thereof; said at least one coding sequence encoding at least one antigenic peptide or protein derived from HsPLAT_NP comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 17968-18001 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from HsALB_NP comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 19328-19361 or a fragment or variant of any of these sequences; said at least one coding sequence encoding at least one antigenic peptide or protein derived from IgE_NP comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 20689-20722 or a fragment or variant of any of these sequences; said at least one coding sequence encodes at least one antigenic peptide or protein derived from LASV Z comprising or consisting of an amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 11166-11347 or a fragment or a variant thereof.
7. RNA according to any one of the preceding claims, wherein said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 255-2286 or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 3821-6106 (encoding GPCmut1) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 22981-23108 (encoding GPCmut2) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 24677-24804 (encoding GPCmut3) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 26373-26500 (encoding GPCmut4) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 28069-28196 (encoding GPCmut5) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 29765-29892 (encoding GPCmut6) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 31461-31588 (encoding GPCmut7) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 33157-33284 (encoding GPCmut8) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 34853-34980 (encoding GPCmut9) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 36549-36676 (encoding GPCmut10) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 38245-38372 (encoding GPCmut11) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 39941-40068 (encoding GPCmut12) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 41637-41764 (encoding GPCmut13) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 7798-9805 (encoding NP) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 18002-18103 (encoding SP- HsPLAT_NP) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 19362-19463 (encoding SP- HsALB_NP) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 20723-20824 (encoding SP- IgE_NP) or a fragment or a fragment or variant of any of these sequences; said the at least one coding sequence comprises or consists of at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 11348-12803 (encoding Z) or a fragment or a fragment or variant of any of these sequences.
8. RNA according to any one of the preceding claims, wherein the at least one coding sequence is a codon modified coding sequence, wherein the amino acid sequence encoded by the at least one codon modified coding sequence is preferably not being modified compared to the amino acid sequence encoded by the corresponding wild type coding sequence.
9. RNA according to claim 8, wherein the at least one codon modified coding sequence is selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof.
10. RNA according to claim 8 or 9, wherein the at least one coding sequence comprising a codon modified nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 509-2286 (encoding GPC) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 4075-6106 (encoding GPCmut1) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 22981-23108 (encoding GPCmut2) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 24677-24804 (encoding GPCmut3) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 26373-26500 (encoding GPCmut4) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 28069-28196 (encoding GPCmut5) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 29765-29892 (encoding GPCmut6) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 31461-31588 (encoding GPCmut7) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 33157-33284 (encoding GPCmut8) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 34853-34980 (encoding GPCmut9) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 36549-36676 (encoding GPCmut10) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 38245-38372 (encoding GPCmut11) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 39941-40068 (encoding GPCmut12) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 41637-41764 (encoding GPCmut13) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 8049-9805 (encoding NP) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 18002-18103, 19362-19463, or 20723-20824 (encoding SP-NP) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 11530-12803 (encoding Z) or a fragment or variant of any of these sequences.
11. RNA according to claims 8 to 10, wherein the at least one coding sequence comprising a G/C optimized or G/C content modified coding sequence (opt1, opt5, opt6, opt11) comprising a nucleic acid 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 any one of the nucleic acid sequences according to the SEQ ID NOs: 509-762, 1525-2286 (encoding GPC) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 4075-4582, 5345-6106 (encoding GPCmut1) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 22981-23044, 23077-23108 (encoding GPCmut2) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 24677-24740, 24773-24804 (encoding GPCmut3) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 26373-26436, 26469-26500 (encoding GPCmut4) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 28069-28132, 28165-28196 (encoding GPCmut5) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 29765-29828, 29861-29892 (encoding GPCmut6) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 31461-31524, 31557-31588 (encoding GPCmut7) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 33157-33220, 33253-33284 (encoding GPCmut8) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 34853-34916, 34949-34980 (encoding GPCmut9) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 36549-36612, 36645-36676 (encoding GPCmut10) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 38245-38308, 38341-38372 (encoding GPCmut11) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 39941-40004, 40037-40068 (encoding GPCmut12) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 41637-41700, 41733-41764 (encoding GPCmut13) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 8049-8299, 9053-9805 (encoding NP) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 18002-18035, 18070-18103, 19362-19395, 19430-19463, 20723-20756, 20791-20824 (encoding SP_NP) or a fragment or variant of any of these sequences, or at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 11530-11711, 12258-12803 (encoding Z) or a fragment or variant of any of these sequences.
12. RNA according to any one of the preceding claims, wherein the RNA comprises a 5′-cap structure, preferably m7G(5′), m7G(5′)ppp(5′)(2′OMeA), or m7G(5′)ppp(5′)(2′OMeG), wherein cap1 (m7G(5′)ppp(5′)(2′OMeA), or m7G(5′)ppp(5′)(2′OMeG) is particularly preferred.
13. RNA according to any one of the preceding claims, wherein the RNA comprises at least one poly(A) sequence, preferably comprising 30 to 200 adenosine nucleotides, preferably about 64 adenosine nucleotides (A64), about 100 adenosine nucleotides (A100) or about 150 adenosine nucleotides.
14. RNA according to any one of the preceding claims, wherein the RNA comprises at least one histone stem-loop, wherein the histone stem-loop preferably comprises a nucleic acid sequence according to SEQ ID NOs: 13842 or 13843 or a fragment or variant thereof.
15. RNA according to any one of the preceding claims comprising the following elements: a) 5′-cap structure, preferably as defined in claim 12; b) 5′-UTR and/or 3′-UTR as defined in claims 1 to 3; c) at least one coding sequence, preferably as defined by any one of claims 7 to 11 or encoding a protein as defined by any one of claims 4 to 6 or; d) optionally, a poly(A) sequence, preferably as defined by claim 13; e) optionally, a poly(C) sequence, preferably as defined by claim 13; f) optionally, a histone stem-loop, preferably as defined by any one of claim 14; g) optionally, a 3′-terminal sequence according to SEQ ID NOs: 13848-13867, 13873-13879.
16. RNA according claims to any one of the preceding claims comprising the following elements, preferably in 5′- to 3′-direction: a) 5′-cap structure, preferably as defined in claim 12; b) 5′-UTR and/or 3′-UTR according to a-1, a-2, b-4, i-2, or i-3; c) at least one coding sequence as defined by any one of claims 7 to 11 wherein said coding sequence is located between said 5′-UTR and said 3′-UTR, preferably downstream of said 5′-UTR and upstream of said 3′-UTR; d) optionally, a poly(A) sequence, preferably as defined by claim 13; e) optionally, poly(C) sequence, preferably as defined by claim 13; f) optionally, histone stem-loop, preferably as defined by any one of claim 14; g) optionally, a 3′-terminal sequence according to SEQ ID NOs: 13848-13867, 13873-13879.
17. RNA according to any one of the preceding claims wherein said RNA comprises or consists of an RNA sequence which is identical or at least 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: 2287-3566, 14056-15207 (encoding GPC) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 6107-7546, 15208-16743 (encoding GPCmut1) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 23109-24644 (encoding GPCmut2) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 24805-26340 (encoding GPCmut3) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 26501-28036 (encoding GPCmut4) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 28197-29732 (encoding GPCmut5) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 29893-31428 (encoding GPCmut6) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 31589-33124 (encoding GPCmut7) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 33285-34820 (encoding GPCmut8) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 34981-36516 (encoding GPCmut9) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 36677-38212 (encoding GPCmut10) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 38373-39908 (encoding GPCmut11) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 40069-41604 (encoding GPCmut12) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 41765-43300 (encoding GPCmut13) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 9806-11165, 16744-17967 (encoding NP) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 18104-19327, 19464- 20687, 20825-22048 (encoding SP-NP) or a fragment or variant of any of these sequences; said RNA comprises or consists of an RNA sequence which is identical or at least 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: 12804-13803, 22049-22948 (encoding Z) or a fragment or variant of any of these sequences.
18. A composition comprising at least one RNA as defined in any one of claims 1 to 17, wherein the composition optionally comprises at least one pharmaceutically acceptable carrier.
19. Composition according to claim 18, wherein the composition comprises more than one or a plurality, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of different RNAs each defined in any one of claims 1 to 18.
20. Composition according to claim 19, wherein each of the different RNAs encodes a different antigenic peptide or protein derived from the same LASV, or wherein each of the RNAs encodes a different antigenic peptide or protein derived from different proteins of the same LASV, or wherein each of the RNAs encodes a different antigenic peptide or protein derived from different proteins of different LASV.
21. Composition according to claim 19 or 20, wherein the composition comprises at least one RNA encoding at least one antigenic peptide or protein derived from GPC or prefusion-stabilized GPC or a variant or fragment thereof and at least one additional RNA encoding at least one antigenic peptide or protein derived from NP (or SP-NP) or a variant or fragment thereof, and/or at least one additional RNA encoding at least one antigenic peptide or protein derived from Z or a variant or fragment thereof.
22. Composition according to claims 19 to 21, wherein the composition comprises at least one RNA encoding at least one antigenic peptide or protein derived from GPC or prefusion-stabilized GPC or a variant or fragment thereof and at least one additional RNA encoding at least one antigenic peptide or protein derived from NP (or SP-NP) or a variant or fragment thereof.
23. Composition according to claims 19 to 22, wherein said antigenic peptides or proteins are derived from the same LASV or from different LASV or combinations thereof.
24. Composition according to claim 23, wherein the different LASV belong to different LASV clades or different LASV lineages, preferably to the LASV clades I, II, III, IV, V, and VI or to the LASV lineages I, II, III, IV, V, and VI.
25. Composition according to claims 18 to 24, wherein at least one RNA is complexed or associated with or at least partially complexed or partially associated with one or more cationic or polycationic compound, preferably cationic or polycationic polymer, cationic or polycationic polysaccharide, cationic or polycationic lipid, cationic or polycationic protein, cationic or polycationic peptide, or any combinations thereof.
26. Composition according to claim 25, wherein the at least one RNA is complexed or associated with one or more lipids, thereby forming liposomes, lipid nanoparticles, lipoplexes, and/or nanoliposomes.
27. Composition according to claim 26, wherein the at least one RNA is complexed with one or more lipids thereby forming lipid nanoparticles (LNP).
28. Composition according to claim 26 or 27, wherein the LNP comprises a cationic lipid with the formula III: ##STR00097## or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L.sup.1 or L.sup.2 is each independently —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O).sub.x—, —S—S—, —C(═O)S—, —SC(═O)—, —NR.sup.aC(═O)—, —C(=O)NR.sup.a—, —NR.sup.aC(═O)NR.sup.a—, —OC(=O)NR.sup.a— or —NR.sup.aC(═O)O—, preferably L.sup.1 or L.sup.2 is —O(C═O)—or —(C═O)O—; G.sup.1 and G.sup.2 are each independently unsubstituted C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene; G.sup.3 is C.sub.1-C.sub.24 alkylene, alkenylene, C.sub.3-C.sub.8 cycloalkylene, or C.sub.3-C.sub.8 cycloalkenylene; R.sup.a is H or C.sub.1-C.sub.12 alkyl; R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24 alkenyl; R.sup.3 is H, OR.sup.5, CN, —C(═O)OR.sup.4, —OC(═O)R.sup.4 or —NR.sup.5C(═O)R.sup.4; R.sup.4 is C.sub.1-C.sub.12 alkyl; R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and x is 0, 1 or 2;
29. Composition according to claim 28, wherein the cationic lipid is a compound of formula III, and wherein: L.sup.1 and L.sup.2 are each independently —O(C═O)— or (C═O)—O—; G.sup.3 is C.sub.1-C.sub.24 alkylene or C.sub.1-C.sub.24 alkenylene; and R.sup.3 is H or OR.sup.5.
30. Composition according to any one of claims 28 to 29, wherein the cationic lipid is a compound of formula III, and wherein: L.sup.1 and L.sup.2 are each independently —O(C═O)— or (C═O)—O—; and R.sup.1 and R.sup.2 each independently have one of the following structures: ##STR00098##
31. Composition according to any one of claims 28 to 30, wherein the cationic lipid is a compound of formula III, and wherein R.sup.3 is OH.
32. Composition according to any one of claims 28 to 31, wherein the cationic lipid is selected from structures III-1 to III-36: TABLE-US-00035 No. Structure III-1
33. Composition according to any one of claims 28 to 32, wherein the cationic lipid is ##STR00135##
34. Composition according to any one of claims 27 to 33, wherein the LNP comprises a PEG lipid with the formula (IV): ##STR00136## wherein R.sup.8 and R.sup.9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
35. Composition according to claim 34, wherein in the PEG lipid R.sup.8 and R.sup.9 are saturated alkyl chains.
36. Composition according to claim 34 or 35, wherein the PEG lipid is ##STR00137## wherein n has a mean value ranging from 30 to 60, preferably wherein n has a mean value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, most preferably wherein n has a mean value of 49.
37. Composition according to any one of claims 27 to 36, wherein the LNP comprises one or more neutral lipids and/or a steroid or steroid analogues.
38. Composition according to claim 37, wherein the neutral lipid is selected from the group comprising distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
39. Composition according to claim 37 or 38, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and wherein the molar ratio of the cationic lipid to DSPC is optionally in the range from about 2:1 to 8:1.
40. Composition according to claim 37, wherein the steroid is cholesterol, and wherein the molar ratio of the cationic lipid to cholesterol is optionally in the range from about 2:1 to 1:1
41. Composition according to any one of claims 27 to 40, wherein the LNP essentially consists of (i) at least one cationic lipid, preferably as defined in any one of claims 28 to 33; (ii) a neutral lipid, preferably as defined in any one of claims 37 to 39; (iii) a steroid or steroid analogue, preferably as defined in claim 40; and (iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA, preferably as defined in any one of claims 34 to 36, wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.
42. A vaccine comprising at least one RNA as defined in any one of claims 1 to 17, or the composition as defined in any one of claims 18 to 41.
43. Vaccine according to claim 42, wherein the at least one RNA as defined in any one of claims 1 to 17, or the composition as defined in any one of claims 18 to 41 elicits an adaptive immune response.
44. A Kit or kit of parts comprising at least one RNA as defined in any one of claims 1 to 17, the composition as defined in any one of claims 18 to 41, and/or the vaccine as defined in any one of claim 42 or 43, optionally comprising a liquid vehicle for solubilising, and optionally technical instructions providing information on administration and dosage of the components.
45. Kit or kit of parts according to claim 44 comprising at least the following components a) at least one RNA as defined in any one of claims 1 to 17 encoding at least one antigenic peptide or protein derived from a LASV, preferably derived from GPC or prefusion-stabilized GPC, wherein said RNA is preferably complexed with one or more lipids thereby forming lipid nanoparticles (LNP); and b) at least one, two or more further RNA species as defined in any one of claims 1 to 17 each encoding an antigenic peptide or protein derived from a LASV, preferably derived from NP or Z, wherein each of said further RNA species are preferably complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein components a) and b) are provided as separate entities or as a single entity.
46. Kit or kit of parts according to claim 45 or 46 further comprising Ringer lactate solution.
47. RNA as defined in any one of claims 1 to 17, the composition as defined in any one of claims 18 to 41, the vaccine as defined in any one of claim 42 or 43, or the kit or kit of parts as defined in claims 44 to 46 for use as a medicament.
48. RNA as defined in any one of claims 1 to 17, the composition as defined in any one of claims 18 to 41, the vaccine as defined in any one of claim 42 or 43, or the kit or kit of parts as defined in claims 44 to 46 for use in the treatment or prophylaxis of an infection with a virus, preferably with LASV, or a disorder related to such an infection.
49. A method of treating or preventing an infection with a LASV, or a disorder related to such an infection, wherein the method comprises applying or administering to a subject in need thereof the at least one RNA as defined in any one of claims 1 to 17, the composition as defined in any one of claims 18 to 41, the vaccine as defined in any one of claim 42 or 43, or the kit or kit of parts as defined in claim 44 to 46.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[1092] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.
Example 1
Preparation of DNA and RNA Constructs and Compositions for In Vitro and In Vivo Experiments
[1093] The present Example provides methods of obtaining the RNA of the invention as well as methods of generating a composition or a vaccine of the invention.
[1094] 1.1. Preparation of DNA and RNA constructs:
[1095] DNA sequences encoding different LASV antigenic proteins were prepared and used for subsequent RNA in vitro transcription. Said DNA sequences were prepared by modifying the wild type cds sequences by introducing an optimized cds (opt1, opt2, opt4, opt6, opt11). Sequences were introduced into a plasmid vector to comprise (i) advantageous 3′-UTR sequences derived from PSMB3, ALB7, alpha-globin (“muag”), or CASP1 and (ii) advantageous 5′-UTR sequences selected from HSD17B4, RPL32, NDUFA4, additionally comprising (iii) a stretch of adenosines, and optionally a histone-stem-loop structure and/or a stretch of 30 cytosines (Table 7).
[1096] Obtained plasmid DNA was transformed and propagated in bacteria using common protocols and plasmid DNA was extracted, purified, and used for subsequent RNA in vitro transcription (see section 1.2.).
[1097] Alternatively, DNA plasmids prepared according to paragraph 1 are used as DNA template for PCR-based amplification. The generated PCR products are purified and used for subsequent RNA in vitro transcription (see section 1.3.).
[1098] 1.2. RNA In Vitro Transcription from Plasmid DNA Templates:
[1099] DNA plasmids prepared according to paragraph 1.1 were enzymatically linearized using a restriction enzyme (e.g. EcoRI, sapI or HindIII) and used for DNA dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (e.g., m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG or 3′-O-Me-m7G(5′)ppp(5′)G)) under suitable buffer conditions. The obtained RNA constructs were purified using RP-HPLC (PureMessenger®, CureVac AG, Tübingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. RNA for clinical development is produced under current good manufacturing practice e.g. according to WO2016/180430, implementing various quality control steps on DNA and RNA level. The generated RNA sequences/constructs are provided in Table 7 with the encoded antigenic protein and the respective UTR elements indicated therein. In addition to the information provided in Table 7, further information relating to specific RNA SEQ-ID NOs may be derived from the information provided under <223> identifier provided in the ST.25 sequence listing.
[1100] Alternatively, enzymatically linearized DNA is used for DNA dependent RNA in vitro transcription using an RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N(1)-methylpseudouridine (m1ψ), pseudouridine (ψ) or 5-methoxyuridine) and cap analog (m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG) under suitable buffer conditions. The obtained m1ψ-modified, ψ-modified or 5-methoxyuridine modified RNA is purified e.g. as explained above and used for further experiments.
[1101] Some RNA constructs are in vitro transcribed in the absence of a cap analog. The cap-structure (cap0 or cap1) is added enzymatically using capping enzymes as commonly known in the art. In short, in vitro transcribed RNA is capped using a capping kit to obtain cap0-RNA. Cap0-RNA may be additionally modified using Cap specific 2′-O-methyltransferase to obtain cap1-RNA. CapO-RNA or Cap1-RNA is purified e.g. as explained above and used for further experiments.
[1102] 1.3. RNA In Vitro Transcription from PCR Amplified DNA Templates:
[1103] Purified PCR amplified DNA templates prepared according to paragraph 1.1 are transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG or 3′-O-Me-m7G(5′)ppp(5′)G)) under suitable buffer conditions. Alternatively, PCR amplified DNA is transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N(1)-methylpseudouridine (m1ψ), pseudouridine (ψ) or 5-methoxyuridine) and cap analog (m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG or 3′-O-Me-m7G(5′)ppp(5′)G)) under suitable buffer conditions. Some RNA constructs are in vitro transcribed in the absence of a cap analog and the cap-structure (cap0 or cap1) is added enzymatically using capping enzymes as commonly known in the art. The obtained RNA is purified e.g. as explained above and used for further experiments.
TABLE-US-00025 TABLE 7 RNA constructs used in the present examples 5′-UTR/ SEQ ID SEQ ID Virus Virus LASV CDS 3′-UTR; NO: NO: RNA ID Strain Clade Antigen opt. UTR Design RNA Protein R6023/R6630 Josiah IV GPC opt1 RPL32/ALB7; i-2 3087 1 R6024 Josiah IV NP opt1 RPL32/ALB7; i-2 10656 7547 R6025 Josiah IV Z opt1 RPL32/ALB7; i-2 13429 11166 R6026/R6631 LP I GPC opt1 RPL32/ALB7; i-2 3088 2 R6027/R6632 803213 II GPC opt1 RPL32/ALB7; i-2 3089 3 R6028/R6633 GA391 III GPC opt1 RPL32/ALB7; i-2 3090 4 R6717 Josiah IV GPC opt1 —/muag; i-3 3343 1 R7278 Josiah IV GPC opt1 HSD17B4/PSMB3; a-1 2319 1 R7279 Josiah IV GPC opt1 HSD17B4/CASP1; b-4 2831 1 R7280 Josiah IV GPC opt1 Ndufa4/PSMB3; a-2 2575 1 R7286 Josiah IV GPC opt2 —/muag; i-3 3375 1 R7287 Josiah IV GPC opt4 —/muag; i-3 3439 1 R7288 Josiah IV GPC opt6 —/muag; i-3 3503 1 R7289 Josiah IV GPC opt11 —/muag; i-3 3535 1 R7390 Josiah IV GPCmut1 opt1 —/muag; i-3 7291 3567 R8477 Josiah IV GPC opt1 —/muag; i-3 14055 1 R8471 Josiah IV GPC opt1 —/muag; i-3 14920 1 R8472 Josiah IV GPCmut1 opt1 —/muag; i-3 16360 3567 R8513 Josiah IV GPCmut2 opt1 —/muag; i-3 24261 22949 R8516 Josiah IV GPCmut3 opt1 —/muag; i-3 25957 24645 R8473 Josiah IV GPC opt1 —/muag; i-3 14920 1 R8474 Josiah IV GPCmut1 opt1 —/muag; i-3 16360 3567 R8514 Josiah IV GPCmut2 opt1 —/muag; i-3 24261 22949 R8517 Josiah IV GPCmut3 opt1 —/muag; i-3 25957 24645 R8475 Josiah IV GPC opt1 HSD17B4/PSMB3; a-1 14152 1 R8476 Josiah IV GPCmut1 opt1 HSD17B4/PSMB3; a-1 15336 3567 R8515 Josiah IV GPCmut2 opt1 HSD17B4/PSMB3; a-1 23237 22949 R8518 Josiah IV GPCmut3 opt1 HSD17B4/PSMB3; a-1 24933 24645 R8479 Josiah IV GPCmut13 opt1 —/muag; i-3 41893 41605
[1104] 1.4. Preparation of an LNP Formulated RNA Composition:
[1105] Lipid nanoparticles (LNP), cationic lipids, and polymer conjugated lipids (PEG-lipid) were prepared and tested essentially according to the general procedures described in WO2015/199952, WO2017/004143 and WO2017/075531, the full disclosures of which are incorporated herein by reference. LNP formulated RNA was prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. Briefly, cationic lipid compound of formula 111-3, DSPC, cholesterol, and PEG-lipid of formula IVa were solubilized in ethanol at a molar ratio (%) of approximately 50:10:38.5:1.5 or 47.4:10:40.9:1.7. LNPs comprising cationic lipid compound of formula III-3 and PEG-lipid compound of formula IVa were prepared at a ratio of RNA to total Lipid of 0.03-0.04 w/w. The RNA was diluted to 0.05 mg/mL to 0.2 mg/mL in 10 mM to 50 mM citrate buffer, pH4. Syringe pumps were used to mix the ethanolic lipid solution with the RNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15 ml/min. The ethanol was then removed and the external buffer replaced with a PBS buffer comprising Sucrose by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 um pore sterile filter and the LNP-formulated RNA composition was adjusted to about 1 mg/ml total RNA. Lipid nanoparticle particle diameter size was 60-90 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK). For other cationic lipid compounds mentioned in the present specification, the formulation process is essentially similar. The obtained LNP-formulated RNA composition (1 mg/ml total RNA) was diluted to the desired target concentration using Saline before in vivo application.
Example 2
Vaccination of Mice with LNP-Formulated mRNA Encoding LASV GPC
[1106] The present Example shows that LNP-formulated mRNA encoding LASV GPC induces strong humoral and cellular immune responses in mice after i.m. immunization.
[1107] RNA encoding LASV GPC was generated according to Example 1 (R6717; see Table 7) and formulated in LNPs according to Example 1.4. Female Balb/c mice were vaccinated on day 0 and day 21 intramuscularly (i.m) with doses provided in Table 8. Mice injected with saline (0.9% NaCl buffer) served as negative controls.
TABLE-US-00026 TABLE 8 Animal groups and vaccination schedule of Example 2 SEQ ID NO: SEQ ID NO: Group Mice Treatment Protein mRNA Dose Route 1 6 R6717; LNP formulated 1 3343 10 ug i.m. 2 6 R6717; LNP formulated 1 3343 5 ug i.m. 3 6 R6717; LNP formulated 1 3343 2 ug i.m. 4 6 0.9% NaCl buffer — — — i.m.
[1108] Determination of Binding Antibody Titers:
[1109] Serum samples were collected (on day 21 and day 35) and binding antibodies (IgG1 and IgG2a) were determined by ELISA using LASV GP2 protein (LASV GA391, clade I; The Native Antigen Company) for coating. Coated plates were incubated using given serum dilutions. Binding of specific antibodies to the GA391 protein was detected using biotinylated isotype specific anti-mouse antibodies in combination with streptavidin-HRP (horse radish peroxidase) with Amplex UltraRed substrate. ELISA results are shown in
[1110] Intracellular Cytokine Staining (ICS):
[1111] Splenocytes from vaccinated and control mice were isolated according to a standard protocol. 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 were seeded into 96-well plates (2×10.sup.6 cells/well). Cells were stimulated with LASV-specific CD4 and CD8 peptide mix and 2.5 ug/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of the mixture of GolgiPlug™/GolgiStop™ (Protein transport inhibitors containing Brefeldin A and Monensin, respectively; BD Biosciences). After stimulation cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcy-block diluted 1:100. Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells were collected using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analysed using FlowJo software (Tree Star, Inc.). The result of the experiment is shown in
[1112] Results:
[1113] As can be seen from
Example 3
Vaccination of Mice with Optimized LNP-Formulated mRNA Encoding LASV GPC
[1114] As demonstrated in Example 2, LNP-formulated mRNA encoding LASV GPC induces both specific humoral and specific cellular immune responses in mice. To further optimize the LASV RNA construct, e.g. to increase the expression of the RNA or the stability of the antigen, constructs comprising advantageous 3′ UTR and 5′ UTR elements (SEQ ID NO: 2319, 2831, 2575), different cds optimizations (SEQ ID NOs: 3375, 3439, 3503, 3535), or an optimized antigen (RNA construct encoding stabilized GPC; (SEQ ID NO: 7291)) are generated according to Example 1 and tested in vivo. Female Balb/c mice are vaccinated with said optimized RNA constructs on day 0 and day 21 intramuscularly (i.m) with doses provided in Table 8. Serum samples from day 21 and 35 are analyzed for binding antibody titers (as explained above) and analysis of T-cell responses is performed on splenocytes (day 35; as explained above).
TABLE-US-00027 TABLE 9 Animal groups and vaccination schedule of Example 3 SEQ ID NO: Group Mice Treatment mRNA Dose Route 1 6 R7278; LNP formulated 2319 5 ug i.m. 2 6 R7279; LNP formulated 2831 5 ug i.m. 3 6 R7280; LNP formulated 2575 5 ug i.m. 4 6 R7286; LNP formulated 3375 5 ug i.m. 5 6 R7287; LNP formulated 3439 5 ug i.m. 6 6 R7288; LNP formulated 3503 5 ug i.m. 7 6 R7289; LNP formulated 3535 5 ug i.m. 8 6 R7290; LNP formulated 7291 5 ug i.m. 9 6 0.9% NaCl buffer — — i.m.
Example 4
Vaccination of Mice with Optimized LNP-Formulated mRNA Encoding LASV GPC
[1115] The present Example shows that LNP-formulated mRNA designs encoding LASV GPC induced strong humoral and cellular immune responses in mice after i.m. immunization.
[1116] RNA encoding LASV GPC was generated according to Example 1 (see Table 7) and formulated in LNPs according to Example 1.4. Female Balb/c mice were vaccinated on day 0 and 21 intramuscularly (i.m) with doses of 5 ug (further details provided in Table 10).
TABLE-US-00028 TABLE 10 Animal groups and vaccination schedule of Example 4 5′-UTR/ 3′-UTR; SEQ ID NO: Group Mice RNA ID Treatment UTR Design mRNA A 5 — 0.9% NaCl buffer —/— — (negative control) B 5 R6717 GPC —/muag 3343 C 5 R7278 GPC HSD17B4/PSMB3, a-1 2319 D 5 R7279 GPC HSD17B4/CASP1, b-4 2831 E 5 R7280 GPC Ndufa4/PSMB3, a-2 2575
[1117] Determination of Binding Antibody Titers:
[1118] Serum samples were collected (on day 21 and day 35) and binding antibodies (IgG1 and IgG2a) were determined by ELISA using LASV GP2 protein (LASV GA391, clade I; The Native Antigen Company) for coating. Coated plates were incubated using given serum dilutions. Binding of specific antibodies to the GA391 protein was detected using biotinylated isotype specific anti-mouse antibodies in combination with streptavidin-HRP (horse radish peroxidase) with Amplex UltraRed substrate. ELISA results are shown in
[1119] Intracellular Cytokine Staining (ICS):
[1120] Splenocytes from vaccinated and control mice (see Table 10) were isolated according to a standard protocol. Briefly, isolated spleens were 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 were seeded into 96-well plates (2×10.sup.6 cells/well). Cells were stimulated with a LASV-specific CD4 and CD8 peptide mix and 2.5 ug/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of the mixture of GolgiPlug™/GolgiStop™ (Protein transport inhibitors containing Brefeldin A and Monensin, respectively; BD Biosciences). After stimulation cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcγ-block diluted 1:100. Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells are collected using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analysed using FlowJo software (Tree Star, Inc.). The result of the experiment is shown in
[1121] Results:
[1122] As can be seen from
[1123] Moreover, as shown in
Example 5
Vaccination of Mice with LNP-Formulated mRNA Encoding an Optimized LASV GPC Construct
[1124] The present Example shows that LNP-formulated RNA encoding a LASV prefusion stabilized GPC induce strong humoral and cellular immune responses in mice after i.m. immunization.
[1125] Determination of Binding Antibodies:
[1126] HeLa cells were transfected with 2 ug RNA prefusion-stablized GPC (GPCmut1, R7390) using Lipofectamine. 20 h post transfection cells were harvested, counted and seeded at 1×10.sup.5 cells/well of a 96 well U-bottom plate. Cells were incubated with serum (diluted 1:50) collected two weeks post second immunization of Balb/c mice (as previously described in Example 4) with 5 ug R7390 (RNA encoding GPCmut1, Group B of Table 11) or NaCl buffer (group A), followed by secondary anti-mouse FITC-conjugated antibody. Cells were acquired on a BD FACS Canto II and analyzed using FlowJo software. The result of the experiment is shown in
[1127] Intracellular Cytokine Staining (ICS):
[1128] Splenocytes from vaccinated and control mice (see Table 11, Group A-C) were isolated according to a standard protocol. 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 were seeded into 96-well plates (2×10.sup.6 cells/well). Cells were stimulated with a LASV-specific CD4 and CD8 peptide mix and 2.5 ug/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of the mixture of GolgiPlug™/GolgiStop™ (Protein transport inhibitors containing Brefeldin A and Monensin, respectively; BD Biosciences). After stimulation cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcγ-block diluted 1:100. Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells are collected using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analysed using FlowJo software (Tree Star, Inc.) The result of the experiment is shown in
TABLE-US-00029 TABLE 11 Animal groups and vaccination schedule of Example 5 Construct SEQ ID NO: SEQ ID NO: Group Mice Treatment Design mRNA mRNA A 5 0.9% NaCl — — buffer (negative control) B 5 R7390 GPCmut1 7291 3567 C 5 R6717 GPC 3343 1
[1129] Results:
[1130] As can be seen from
[1131] Additionally, as shown in
Example 6
In Vitro Expression Analysis of Optimized mRNA Designs Encoding LASV GPC and Prefusion-Stabilized GPC constructs
[1132] The present Example shows that optimized mRNA designs according to the invention (UTR-combination a-1, cap1, and/or poly(A)-sequence located (exactly) at the 3′ end) encoding different optimized LASV GPC constructs express GPC and prefusion-stablized GPC proteins. As a detection antibody, the mouse 37.7H monoclonal antibody was used, which is directed against the quaternary GPC-B epitope. The use of this antibody gives indication for a correct conformation of the heterotrimeric GPC protomer to potentially induce neutralizing antibodies that achieve neutralization by stabilizing LASV GPC in its prefusion conformation.
[1133] FACS Analysis:
[1134] HeLa cells were transfected with 2 ug of the different mRNA designs (see Table 12) using Lipofectamine. 20 h post transfection cells were harvested, and seeded in a 96 well U-bottom plate. Cells were incubated with α-GPC [37.7H] antibody which binds to a quaternary GPC-B epitope bridging the LASV GP1 and GP2 subunits,)) encoded by different mRNA constructs (see Table 12) followed by secondary anti-mouse FITC-conjugated antibody. Cells were acquired on a BD FACS Canto II and analyzed using FlowJo software. The result of the experiment is shown in
TABLE-US-00030 TABLE 12 Tested RNA constructs of Example 6 5′-UTR/ 5′-cap 3′-UTR; structure/ Modified SEQ ID Group RNA ID Construct Design UTR Design 3′ terminus nucleotides NO: mRNA A R8477 GPC full-length —/muag cap1/ — 14055 A64-N5-hSL-N5 B R8471 GPC full-length —/muag cap1/ — 14920 hSL-A64-N5 C R8472 GPCmut1 —/muag cap1/ — 16360 hSL-A64-N5 D R8513 GPCmut2 —/muag cap1/ — 24261 hSL-A64-N5 E R8473 GPC full-length —/muag cap1/ Ψ 14920 hSL-A64-N5 F R8474 GPCmut1 —/muag cap1/ Ψ 16360 hSL-A64-N5 G R8514 GPCmut2 —/muag cap1/ Ψ 24261 hSL-A64-N5 H R8475 GPC full-length HSD17B4/ cap1/ — 14152 PSMB3; a-1 hSL-A100 I R8476 GPCmut1 HSD17B4/ cap1/ — 15336 PSMB3; a-1 hSL-A100 J R8515 GPCmut2 HSD17B4/ cap1/ — 23237 PSMB3; a-1 hSL-A100 K Negative control (WFI = — — — — water for injection)
[1135] Western Blot Analysis:
[1136] HeLa cells were transfected with 2 ug of the respective mRNA designs encoding LASV GPC (see Table 13), with a negative control (water for injection) and as a positive control (irradiated LASV Josiah strain, clade IV). 20 h post transfection cells were harvested, lysed and subjected to SDS-PAGE followed by Western blot. For the detection a-GPC [37.7H] antibody was used and a goat anti-mouse IgG IRDye® 800CW antibody (1:10000; Li-Cor) as secondary antibody. The a-GPC [37.7H] antibody binds to a quaternary GPC-B epitope bridging the LASV GP1 and GP2 subunits. The result of the experiment is shown in
TABLE-US-00031 TABLE 13 mRNA designs for Western blot analysis 5′-UTR/ 5′-cap 3′-UTR; structure/ Modified mRNA Group RNA ID Construct Design UTR Design 3′ terminus nucleotides SEQ ID NO A R8471 GPC full-length —/muag cap1/ — 14920 hSL-A64-N5 B R8477 GPC full-length —/muag cap1/ — 14055 A64-N5-hSL-N5 C R8472 GPCmut1 —/muag cap1/ 16360 hSL-A64-N5 D R8513 GPCmut2 —/muag cap1/ 24261 hSL-A64-N5 E R8473 GPC full-length —/muag cap1/ Ψ 14920 hSL-A64-N5 F R8474 GPCmut1 —/muag cap1/ Ψ 16360 hSL-A64-N5 G R8514 GPCmut2 —/muag cap1/ Ψ 24261 hSL-A64-N5 H R8475 GPC full-length HSD17B4/ cap1/ — 14152 PSMB3; a-1 hSL-A100 I R8476 GPCmut1 HSD17B4/ cap1/ 15336 PSMB3; a-1 hSL-A100 J R8515 GPCmut2 HSD17B4/ cap1/ — 23237 PSMB3; a-1 hSL-A100 K Negative control (WFI = water for injection) L Irradiated LASV (positive control)
[1137] Results:
[1138]
[1139] As shown in
[1140] Through the detection with the 37.7H antibody directed to the quaternary GPC-B epitope, indication for a correct conformation of the GPC trimer to potentially induce neutralizing antibodies is given.
Example 7
Analysis of Polyvalent LASV Vaccine Compositions in Guinea Pigs (Challenge)
[1141] The present example shows that LASV mRNA vaccines induce protective immune responses against Lassa virus infection in Hartley guinea pigs. Hartley (outbred) guinea pigs are widely used for studying arenaviral hemorrhagic fevers and for testing potential therapeutics and vaccine candidates. Binding antibodies are measured using ELISA and moreover virus neutralizing antibodies to the vaccine are analyzed. In addition this example shows of the monovalent LASV-GPC lineage IV vaccine as well as the feasibility of a 1-dose regimen.
[1142] Different mRNA designs encoding different LASV GPC constructs are prepared according to Example 1. The mRNAs are formulated with LNPs (see Example 1.4.). The different mRNA vaccine candidates (see Table 14) are applied on day 0 and 28 or only at day 0 and administered intramuscular (i.m.) with different doses of RNA. Blood samples are collected at day 0, 28, 56 and post challenge for determination of humoral immune responses.
[1143] 4 weeks after the last vaccination the groups are challenged with 10.sup.5 PFU Josiah LASV strain (i.p. route). The groups are observed for 4 weeks after the challenge. The animals are monitored daily for survival, body weight, morbidity index, temperature and viremia. Additional the viral load in lung, spleen and kidney is measured at necropsy.
TABLE-US-00032 TABLE 14 Animal groups and vaccination schedule of Example 7 Administration Guinea Group Interval pigs Construct Design A 1 x 10 0.9% NaCl buffer (negative control) B 1 x 10 GPC or prefusion-stabilized GPC C 2 x 10 GPC or prefusion-stabilized GPC
Example 8
Analysis of Polyvalent LASV Vaccine Compositions in Guinea Pigs (Challenge)
[1144] The present example shows the efficacy of the mRNA LASV vaccine to provide protection against heterologous LASV strains or clades, especially that the polyvalent/tetravalent mRNA vaccine provides protection against two LASV viruses from phylogenetically most distant LASV lineages (I and IV) in guinea pigs.
[1145] mRNA designs encoding LASV GPC or prefusion-stabilized GPC are prepared according to Example 1. The mRNAs are formulated with LNPs (see Example 1.4.). The different mRNA vaccine candidates (see Table 15) are applied on day 0 and 28 and administered intramuscular (i.m.) with different doses of RNA. Blood samples are collected at day 0, 28, 56 and post challenge for determination of humoral immune responses.
[1146] 4 weeks after the last vaccination the groups are challenged with 10.sup.5 PFU Josiah LASV strain (i.p. route). The groups are observed for 4 weeks after the challenge. The animals are monitored daily for survival, body weight, morbidity index, temperature and viremia. Additional the viral load in lung, spleen and kidney is measured at necropsy.
TABLE-US-00033 TABLE 15 Animal groups and vaccination schedule of Example 8 Group Guinea pigs Virus Clade Vaccine composition A 10 — 0.9% NaCl buffer (negative control) B 10 IV GPC or prefusion-stabilized GPC C 10 I + II + Tetravalent GPC or tetravalent III + IV prefusion-stabilized GPC
Example 9
Analysis of Polyvalent LASV mRNA Vaccine
[1147] The present example shows a tetravalent mRNA vaccine covering Lassa clades I-IV as well as the inclusion of the nucleoprotein (NP) as an additional target of T cell responses to broaden the coverage of the vaccine.
[1148] RNA encoding different Lassa mRNA vaccine encoding GPC or a prefusion-stabilized GPC (see Table 16) was generated according to Example 1 and formulated in LNPs according to Example 1.4. Female CBA/J mice (9 mice per group) were vaccinated on day 0 and day 21 intramuscularly (i.m). Serum is collected at day 21 and 28 to test humoral immune responses. Splenocytes are collected at day 28 to test cellular immune responses via an ICS with LASV-GPC overlapping peptide libraries from lineages IV and I, II, III to analyze cross-reactivity.
TABLE-US-00034 TABLE 16 Animal groups and vaccination schedule of Example 9 Group Mice Virus Clade Vaccine composition A 9 — 0.9% NaCl buffer (negative control) B 9 IV GPC or prefusion-stabilized GPC and NP or SP-NP C IV NP or SP-NP D 9 I + II + Tetravalent GPC or tetravalent II + IV prefusion-stabilized GPC E 9 I GPC or prefusion-stabilized GPC F 9 II GPC or prefusion-stabilized GPC F 9 III GPC or prefusion-stabilized GPC G 9 IV GPC or prefusion-stabilized GPC
Example 9
Safety, Reactogenicity and Immunogenicity of LASV mRNA Vaccine in Healthy Adults
[1149] To demonstrate safety, reactogenicity and immunogenicity of LASV mRNA vaccine, a phase I clinical trial is initiated.
[1150] For clinical development, RNA is used that has been produced under GMP conditions (e.g. using a procedure as described in WO2016/180430).
[1151] In this LASV mRNA vaccine phase I trial different dosages of the candidate LASV mRNA vaccine will be administered in a one or two-dose schedule to healthy adult subjects. The subjects will be enrolled sequentially into the different trial groups to receive one or two doses of LASV mRNA vaccine. The subjects in the two-dose groups will be administered a second dose 28 days later. An additional group of control subjects will receive a single dose of saline on Day 1. Safety information for solicited (days 1-7 post-vaccination) and unsolicited (days 1-28 post-vaccination) adverse events (AEs) will be collected using diary cards. Serious AEs, AEs leading to premature withdrawal from the trial or receipt of the second dose, AEof Special Interest and medically-attended AEs will be collected throughout the trial (Day 1 to Day 365 post last vaccine dose). Specified safety data will be reviewed by an internal safety review team and a DSMB on a pre-defined schedule.