WO 2021/048402
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`PCT/EP2020/075541
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`24
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`CLAIMS
`
`A polynucleotide comprising a sequence of a live, infectious, attenuated
`
`Flavivirus wherein a nucleotide sequence encoding at least a part of a
`
`arenavirus glycoprotein protein is located at the intergenic region between
`
`the E and NS1 gene of said Flavivirus, such that a chimeric virus is
`
`expressed, characterised in that the encoded sequence C terminally of the
`
`10
`
`E protein of said Flavivirus and N terminally of the signal peptide of the NS1
`
`
`
`
`
`
`
`protein the_following§orderof said Flavivirus comprises in
`
`
`
`
`
`
`
`- a further signal peptide of a Flavivirus NS1 protein,
`
`-an arenavirus Glycoprotein protein lacking the N terminal signal sequence
`
`and the GP2 transmembrane domain,
`
`15
`
`- a TM1 and TM2 domain ofa flaviviral E protein.
`
`The polynucleotide according to claim 1, wherein the sequence of the live,
`
`infectious, attenuated Flavivirus is Yellow Fever virus, typically the YF17D
`
`strain.
`
`20
`
`The polynucleotide according to claim 1 or 2, wherein the arena virus a
`
`Mammarena virus.
`
`The polynucleotide according to any one of claims 1 to 3, wherein the
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`25
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`arenavirus a Lassa virus.
`
`The polynucleotide according to any one of claims 1 to 4, wherein the
`
`encoded protein sequence comprises the sequence of SEQ ID NO: 2 or SEQ
`
`ID NO:4.
`
`30
`
`The polynucleotide according to any one of claims 1 to 5, wherein the
`
`glycoprotein comprises the R207C, G360C and E329P stabilizing mutations.
`
`The polynucleotide according to any one of claims 1 to 6, wherein the
`
`35
`
`glycoprotein comprises the R246A proteolytic cleavage site.
`
`SUBSTITUTE SHEET (RULE 26)
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`
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`WO 2021/048402
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`PCT/EP2020/075541
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`25
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`The polynucleotide according to any one of claim 1 to 7, wherein the signal
`
`peptide of
`
`the NS1 protein comprises or consists of
`
`the sequence
`
`DQGCAINFG [SEQ ID NO: 10].
`
`The polynucleotide according to any one of claims 1 to 8, wherein the TM1
`
`domain of a flaviviral E protein has the sequence of SEQ ID: NO 14, or
`
`wherein the TM2 domain of a flaviviral E protein has the sequence of SEQ
`
`ID NO: 15, or wherein the sequence of the chimeric virus at the junction
`
`of the NS1 signal sequence and the GP1 domain comprises the sequence
`
`of SEQ ID NO:11.
`
`10.
`
`The polynucleotide according to any one of claims 1 to 9, wherein the
`
`sequence of the chimeric virus at the junction of the GP2 domain and the
`
`TM1 domain comprises the sequence of SEQ ID NO:12, or wherein the
`
`sequence of the chimeric virus at the junction of the TM2 domain and NS1
`
`protein comprises the sequence of SEQ ID NO:13.
`
`10
`
`15
`
`11.
`
`20
`
`The polynucleotide according to any one of claims 1 to 10, encoding a
`
`sequence comprising SEQ ID NO: 11, SEQ ID: NO 12 and SEQ ID NO13;
`
`and outside SEQ ID NO: 11, SEQ ID: NO 12 and SEQID NO: 13 , a sequence
`
`identity of at least 95 %, 96 %, 97 %, 98% or 99 % with SEQ ID NO:2 or
`
`SEQ ID NO:4.
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`25
`
`12.
`
`A chimeric live, infectious, attenuated Flavivirus wherein at least a part of
`
`an arenavirus Glycoprotein is located between the E and NS1 protein of
`
`said Flavivirus, such that C terminally of the E protein and N terminally of
`
`the signal peptide of the NS1 protein the virus comprises in the following
`
`order :
`
`30
`
`- a further signal peptide of a Flavivirus NS1 protein,
`
`-an arenavirus Glycoprotein protein lacking the N terminal signal sequence
`
`and the GP2 transmembrane domain,
`
`- a TM1 and TM2 domain of a flaviviral E protein.
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`35
`
`13.
`
`The chimeric Flavivirus according to claim 12, wherein the Flavivirus is YFV.
`
`
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`WO 2021/048402
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`PCT/EP2020/075541
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`14.
`
`The chimeric Flavivirus according to claim 12 or 13, wherein the arenavirus
`
`is Lassa virus.
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`26
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`15.
`
`A polynucleotide according to any of claims 1 to 11, or a chimeric virus in
`
`accordanceto any one of claims 12 to 14, for use as a medicament.
`
`
