WO 2021/048402
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`PCT/EP2020/075541
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`LASSAVIRUS VACCINES
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`The invention relates to chimeric Flavivirus based vaccines. The invention further
`
`relates to vaccines against viruses such as Lassa Virus.
`
`BACKGROUND OF THE INVENTION
`
`Currently,
`
`there is no licensed human vaccine approved against Lassa virus
`
`(LASV). A number of different vaccine candidates have been generated involving
`
`several platform technologies. The most advanced candidates are VSV based LASV
`
`10
`
`(VSV-LASV-GPC), a Mopeia virus (MOPV)/LASV reassortant virus (clone ML29) and
`
`a DNA vaccine called INO-4500 (pLASV-GPC). VSV based LASV vaccine candidate
`
`consists in a replication-competent VSVs expressing the glycoprotein of LASV.
`
`ML29 is a reassortant between Lassa and Mopeia viruses that carries the L-
`
`segment of MOPV and the S- segment (nucleoprotein and glycoprotein) from
`
`15
`
`LASV. INO-4500 is a DNA vaccine encoding the LASV-GPC gene from Josiah strain
`
`f and it is from Inovio company (pLASV-GPC).
`
`Besides the use of the different approaches mentioned above, also yellow fever
`
`virus 17D has been used as vector for Lassa virus glycoprotein (GPC) or its
`
`subunits GP1 and GP2 (Bredenbeek et a/, (2006) Virology 345, 299-304 and Jiang
`
`20
`
`et al. (2011) Vaccine 29, 1248-1257)). In these constructs the GP gene (lack of
`
`signal peptide, SSP) (or either GP1 or GP2 sequences) were inserted between YF-
`
`E/NS1. These constructs have at the C-terminus of the insert fusion sequences
`
`derived from YF-E, WNV-E or artificial designed sequences. These constructs need
`
`to be transfected in cells and the viruses derived from them are used as vaccines.
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`25
`
`The only vaccine that have just started a phaseI clinical trial is INO-4500 (pLASV-
`
`GPC). This vaccine
`
`requires multiple
`
`high doses delivered via
`
`dermal
`
`electroporation in order to achieve full protection and enhance the vaccine immune
`
`response. This multi-dose administration regimen will be very challenging to
`
`30
`
`implement in the rural areas of West Africa where LASV is endemic and the main
`
`outbreaks have occurred.
`
`Regarding the other candidates, ML29 is classified in risk group 2 by the EU and
`
`risk group 3 by US CDC what is an obstacle for further development ofthis vaccine.
`
`VSV-LASV-GPCstill requires a cold chain to preserve it which involves high cost
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`35
`
`and still there are no studies concerning its safety. The approach involving YF17D
`
`as vector to express Lassa glycoprotein precursor was not successful
`
`in NHP
`
`studies (0% survival, marmosets).
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`In addition, this vaccine candidate showed issues of genetic instability that did not
`
`allow to scale-up the technology as required for vaccine production.
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`2
`
`Summary of the invention
`
`We have used our PLLAV (plasmid-launched live-attenuated vaccine) technology
`
`and the live-attenuated yellow fever vaccine strain (YFV-17D) as vector to
`
`engineer a transgenic vaccine by inserting LASV-GPC (with a mutation in the
`
`cleavage site R246A to keep GP1 and GP2 bound and additional mutations R207C,
`
`10
`
`G360C and E329P)
`
`into yellow fever E /NS1 intergenic region as follows: N-
`
`terminal
`
`(Nt)
`
`signal peptide was deleted,
`
`first 9 aminoacids of NS1i
`
`(27
`
`nucleotides) were added Nt of LASV-GPC to allow proper release of LASV-GPC
`
`protein, the transmembrane domain was deleted and the ectodomain was fused
`
`to the WNV transmembrane domain 1 and 2. The resulting PLLAV-YFV17D-LASV-
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`15
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`GPC launches viable live-attenuated viruses expressing functional LASV-GPC and
`
`YFV-17D proteins. The PLLAV-YFV17D-LASV-GPC construct can be used directly
`
`as vaccine what involves that this vaccine is thermostable. The vaccine induces
`
`immune responses against both LASV and YFV after one-single shot. A second
`
`similar construct has been generated in which the cleavage site has been restored
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`20
`
`(R246A mutation was restored to R246R).(Additional information in the attached
`
`data)
`
`PLLAV-YFV17D-LASV-GPC is a dual vaccine inducing YFV and Lassa virus specific
`
`immunity. PLLAV-YFV17D-LASV-GPC can also be used as stable seed for the
`
`production of tissue culture-derived live-attenuated vaccine not only in the PLLAV
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`25
`
`modality, but also unexpectedly the recombinant YFV17D-LASV-GPCvirus appears
`
`to be genetically more than that disclosed in prior art by Bredenbeek et al. and
`
`Jiang et al. (cited above).
`
`The invention is further summarized in the following statements:
`
`30
`
`1.
`
`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 E protein of said Flavivirus and N
`
`35
`
`terminally of the signal peptide of the NS1 protein of said Flavivirus comprises in
`
`the following order :
`
`- a further signal peptide of a Flavivirus NS1 protein,
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`-an arenavirus Glycoprotein protein lacking the N terminal signal sequence and
`
`the GP2 transmembrane domain,
`
`- a TM1 and TM2 domain ofa flaviviral E protein.
`
`2.
`
`The polynucleotide according to claim 1, wherein the sequenceof the live,
`
`infectious, attenuated Flavivirus is Yellow Fever virus, typically the YF17D strain.
`
`3.
`
`The polynucleotide according to claim 1, wherein the live,
`
`infectious,
`
`
`
`attenuated Flavivirus backbone two_differentis a chimeric backbone of
`
`
`
`
`
`
`
`flaviviruses.
`
`4.
`
`The polynucleotide according to any one of claims 1 to 3, wherein the arena
`
`10
`
`virus a Mammarena virus.
`
`5.
`
`The polynucleotide according to any one of claims 1 to 4, wherein the
`
`arenavirus a Lassa virus.
`
`6.
`
`The polynucleotide according to any one of claims 1 to 3, wherein the Lassa
`
`strain is the Josiah strain.
`
`15
`
`7.
`
`The polynucleotide according to any one of claims 1 to 6, wherein the
`
`glycoprotein comprises the R207C, G360C and E329P stabilizing mutations.
`
`8.
`
`The polynucleotide according to any one of claims 1 to 7, wherein the
`
`glycoprotein comprises the R246A proteolytic cleavage site.
`
`9.
`
`The polynucleotide according to any one of claims 1 to 8, wherein the
`
`20
`
`nucleotide sequence of the G protein is codon optimised for improved expression
`
`in mammalian cells.
`
`10.
`
`The polynucleotide according to any one of claims 1 to 9, wherein the signal
`
`peptide of the NS1 protein comprises or consists of the sequence DQGCAINFG
`
`[SEQ ID NO: 10].
`
`25
`
`11.
`
`The polynucleotide according to any one of claims 1 to 10, wherein the TM1
`
`and TM2 domain of a flaviviral E protein are from West Nile virus.
`
`12.
`
`The polynucleotide according to any one of claims 1 to 11, wherein the TM1
`
`domain of a flaviviral E protein has the sequence of SEQ ID: NO 14.
`
`13.
`
`The polynucleotide according to any one of claims 1 to 12, wherein the TM2
`
`30
`
`domain of a flaviviral E protein has the sequence of SEQ ID NO 15.
`
`14.
`
`The polynucleotide according to any one of claims 1 to 13, 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.
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`35
`
`15.
`
`The polynucleotide according to any one of claims 1 to 14, 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.
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`16.
`
`The polynucleotide according to any one of claims 1 to 14, 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.
`
`In preferred embodiments the junctions connecting the flavirus NSi1_ signal
`
`sequence, the Lassavirus G protein, the TM2 protein and the second NS1 signal
`
`sequence provide a fingerprint for the encoded proteins. Thus embodiments of
`
`encoded sequences can be defined by sequences having the sequence of SEQ ID
`
`NO:2 or SEQ ID NO: 4, comprising the sequences with SEQ ID NO: 11, SEQ ID:
`
`NO 12 and SEQ ID NO13; and wherein outside SEQ ID NO: 11, SEQ ID: NO 12
`
`10
`
`and SEQ ID NO13 , a number of amino acids may differ from SEQ ID NO:2 or SEQ
`
`ID NO:4, e.g. differing up to 20, up to 10, or up to 5 compared to SEQ ID NO:2
`
`or SEQ ID NO: 4, or e.g. having a sequenceidentity of at least 95 %, 96 %, 97
`
`%, 98% or 99 % with SEQ ID NO:2 or SEQ ID NO:4.
`
`17.
`
`The polynucleotide according to any one of the claims 1 to 16, which is a
`
`15
`
`bacterial artificial chromosome.
`
`18.
`
`A polynucleotide in accordance to any one of claims 1 to 17, for use asa
`
`medicament.
`
`19.
`
`The polynucleotide for use as a medicament in accordance with claim 18,
`
`wherein the medicament is a vaccine.
`
`20
`
`20.
`
`A polynucleotide sequence in accordance to any one of claims 1 to 17, for
`
`use in the vaccination against an arenavirus infection.
`
`21.
`
`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
`
`25
`
`peptide of the NS1 protein the virus comprises in the following order :
`
`- 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 ofa flaviviral E protein.
`
`30
`
`22.
`
`23.
`
`The chimeric Flavivirus according to claim 21, wherein the Flavivirus is YFV.
`
`The chimeric Flavivirus according to claim 21 or 22, wherein the arenavirus
`
`is Lassa virus.
`
`24.
`
`A chimeric virus in accordance to any one of claims 21 to 23, for use as a
`
`medicament.
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`35
`
`25.
`
`A chimeric virus in accordance to any one of claims 21 to 24, for use in the
`
`prevention of an Arenaviral infection.
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`26.
`
`A chimeric virus encoded by a nucleotide in accordance to any one of claims
`
`21 to 23, for use in the prevention of an Arenaviral infection and in the prevention
`
`of the Flavivirus.
`
`27.
`
`A method of preparing a vaccine against an arenaviral infection, comprising
`
`the steps of:
`
`providing a BAC which comprises:
`
`an inducible bacterial ori sequence for amplification of said BAC to more than 10
`
`copies per bacterial cell, and
`
`a viral expression cassette comprising a cDNA of a arenaviral-flaviviral chimeric
`
`10
`
`virus according to any one of claims 1 to 16, and comprising cis-regulatory
`
`elementsfor transcription of said viral CDNA in mammalian cells and for processing
`
`of the transcribed RNA into infectious RNA virus,
`
`- transfecting mammalian cells with the BAC of step a) and passaging the infected
`
`cells,
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`15
`
`- validating replicated virus of the transfected cells of step b) for virulence and the
`
`capacity of generating antibodies and inducing protection against said arenaviral
`
`infection,
`
`- cloning the virus validated in step c into a vector, and
`
`- formulating the vector into a vaccine formulation.
`
`20
`
`28.
`
`The method according to claim 27, wherein the Flavivirus is yellow fever
`
`virus.
`
`29.
`
`The method according to claim 27 or 28, wherein the arenavirus is Lassa
`
`virus.
`
`30.
`
`The method according to any one of claims 27 to 29, wherein the vector is
`
`25
`
`a BAC, which comprises an inducible bacterial ori sequence for amplification of
`
`said BAC to more than 10 copies per bacterial cell.
`
`DETAILED DESCRIPTION
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`30
`
`Figure legends
`
`Figure 1: Schematic representation of 1) PLLAV-YFV17D-LASV-GPC and 2)
`
`PLLAV-YFV17D-LASV-GPCcs.
`
`Figure 2: A) Plaque phenotype of YFV17D-LASV-GPC compared to YFV17D. B)
`
`Virus stability: RT-PCR analysis of the virus samples harvested during serial
`
`35
`
`passaging (in BHK-21]J and VeroE6) of the YFV17D-LASV-GPC virus. C+, control
`
`positive
`
`PLLAV-YFV17D-LASV-GPC;
`
`-RT: RT-PCR reaction without
`
`reverse
`
`transcriptase; RNA: RT-PCR reaction with the virus RNA.
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`Figure 3: Schematic vaccination schedule. AG129 mice were vaccinated with
`
`PLLAV-YFV17D-LASV-GPC (25 ug, i.p.) or YFV17D-LASV-GPC (375 PFU).
`
`Figure 4: Analysis of cellular
`
`immunity in vaccinated AG129 mice. A)
`
`Representative IFN-y ELISPOT wells after 48 hours of stimulation of splenocytes
`
`with the indicated antigen. B) Spots per six hundred thousand splenocytes in IFN-
`
`y ELISPOT after 48 hours of stimulation with the indicated antigen. For each
`
`mouse, samples were analyzed in duplicates and values are normalized by
`
`subtracting the number of spots in control wells (ovalbumin stimulated).
`
`Figure 5: A) Plaque phenotype of YFV17D-LASV-GPCcs compared to YFV17D. B)
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`10
`
`Co-expression of LASV-GPC and YFV antigens detected by immunofluorescence of
`
`BHK21J cells infected with supernatant of cells transfected with PLLAV-YFV17D-
`
`LASV-GPCcs .Cells were fixed 48h post-infection and stained for LAV-GPC (red)
`
`and YFV (green).
`
`Figure 6: A) Schematic vaccination schedule. AG129 mice were vaccinated
`
`15
`
`subcutaneous (SC) with YFV17D-LASV-GPCcs (250 PFU). B) Analysis of cellular
`
`immunity in vaccinated AG129 mice. Representative IFN-gamma ELISPOT wells
`
`after 48 hours splenocyte stimulation with the indicated antigen. Spots per six
`
`hundred thousand splenocytes
`
`in
`
`IFN-gamma ELISPOT after 48 hours of
`
`stimulation with the indicated antigen. For each mouse, samples were analyzed in
`
`20
`
`duplicates and values are normalized by subtracting the number of spots in control
`
`wells (ovalbumin peptide stimulated).
`
`The present invention is exemplified for Yellow Fever virus, but is also applicable
`
`using other viral backbonesof Flavivirus species such, but not limited to, Japanese
`
`25
`
`Encephalitis, Dengue, Murray Valley Encephalitis (MVE), St. Louis Encephalitis
`
`(SLE), West Nile (WN), Tick-borne Encephalitis (TBE), Russian Spring-Summer
`
`Encephalitis (RSSE), Kunjin virus, Powassan virus, Kyasanur Forest Disease virus,
`
`Zika virus, Usutu virus, Wesselsbron and Omsk Hemorrhagic Fever virus.
`
`The invention is further applicable to Flaviviridae, which comprises the genus
`
`30
`
`Flavivirus but also the genera, Pegivirus, Hepacivirus and Pestivirus.
`
`The genus Hepacivirus comprises e.g. Hepacivirus C (hepatitis C virus) and
`
`Hepacivirus B (GB virus B)
`
`The genus Pegivirus comprises eg Pegivirus A (GB virus A), Pegivirus C (GB virus
`
`C), and Pegivirus B (GB virus D).
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`35
`
`The genus Pestivirus comprises e.g. Bovine virus diarrhea virus 1 and Classical
`
`swine fever virus (previously hog cholera virus).
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`The Flavivirus which is used as backbone can itself by a chimeric virus composed
`
`of parts of different Flavivirus.
`
`For example the C and NS1-5 region are from Yellow Fever and the prME region
`
`is of Japanese encephalitis or of Zika virus.
`
`The present invention is exemplified for the G protein of Lassa virus but is also
`
`applicable to G proteins of other arenaviruses.
`
`The present
`
`invention relates to nucleotide sequence and encoded proteins
`
`wherein within the RNA or copy DNA (cDNA) of a flavivirus a glycoprotein of an
`
`10
`
`arenavirus is inserted
`
`Glycoproteins of Arenavirus are discussed in Burr et a/, (2012) Viruses 4, 2162-
`
`2181 and in Nurnberg & Yorke (2012) Viruses 4, 83-101. Arenaviruses are
`
`comprised of two RNA genome segments and four proteins, the polymerase L, the
`
`envelope glycoprotein GP (also referred to in the present invention as G protein or
`
`15
`
`GPC), the matrix protein Z, and the nucleoprotein NP.
`
`In the arenavirus life-cycle the biosynthesis and maturation of the GP precursor
`
`(GPC) is performed by cellular signal peptidases and the cellular enzyme Subtilisin
`
`Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP
`
`complex formed by GP1/GP2 and a stable signal peptide (SSP).
`
`20
`
`
`
`
`
`
`
`Based on_serological, genetic and geographical data, Mammarenavirus
`
`
`
`arenaviruses are divided into two major subgroups: the Old World (OW) and the
`
`New World (NW) complex. The Old World lineage consists of the prototypic LCMV
`
`and other viruses endemic to the African continent, including Lassa (LASV), Mopeia
`
`25
`
`(MOPV), Ippy, and Mobala (MOBV) viruses.
`
`The larger New World complex is further divided into three clades, A, B and C.
`
`Clade B is the most important in term of human disease, since it contains the
`
`major viruses causing hemorrhagic fevers (HF)
`
`in South America,
`
`i.e. Junin
`
`(JUNV), Machupo (MACV), Guanarito (GTOV) and Sabia (SABV) viruses but also
`
`30
`
`other non-pathogenic viruses, like Tacaribe (TCRV) and Amapari virus (AMPV).
`
`The present invention envisages chimeric constructs based on G proteins of any
`
`of the above groups, subgroups or species are used. Preferred embodiment are
`
`constructs based on G proteins of the LASV inserted within a flavivirus RNA or
`
`cDNA.
`
`35
`
`The present
`
`invention envisages chimeric constructs based on G proteins of
`
`Reptarenavirusse or Hartmanivirusses are used.
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`The present invention is exemplified with G protein of Lassa virus strain Josiah.
`
`This sequence of this protein is accessible for example as UniProtKB PO8669
`
`database entry or as NCBI NP_694870.1 database entry.
`
`In alternative embodiment the arenavirus envisaged is a virus wherein the protein
`
`sequence of the G protein has a sequence identity of at least 70, at least 80, at
`
`least 90, at least 95, or least 99 %identity with the G protein of Lassa virus strain
`
`Josiah, as disclosed in the above cited database entries.
`
`The constructs of the present invention allow a proper presentation of the encoded
`
`10
`
`insert into the ER lumen and proteolytic processing. As exemplified by Lassa G
`
`protein, the encoded protein by the insert lacks the N terminal signal sequence
`
`and a GP2 transmembrane domain. To preserve the required topology two
`
`transmembrane domains of e.g. WNV are fused c terminally to the glycoprotein
`
`sequence . Based on this principle any immunogenic protein can be presented via
`
`15
`
`the vector of the present invention that the protein lacks an N terminal membrane
`
`targeted domain and contains at the C terminus a targeting membrane followed
`
`by a cytoplasmic sequence to allow the connection with the transmembrane
`
`membrane preceding the NS1 protein.
`
`20
`
`The invention is now further described for embodiments wherein a Flavivirus is
`
`used as backbone and a G protein of Lassa virus as insert.
`
`The high sequenceidentity between G proteins of different arenavirus presents no
`
`problems to the skilled person to identify in related sequences the sequence
`
`25
`
`elements corresponding to those present in Lassa virus G protein.
`
`Flaviviruses have a positive single-strand RNA genome of approximately 11,000
`
`nucleotides in length. The genome contains a 5’ untranslated region (UTR), a long
`
`open-reading frame (ORF), and a 3’ UTR. The ORF encodesthree structural (capsid
`
`30
`
`[C], precursor membrane [prM], and envelope [E]) and seven nonstructural (NS1,
`
`NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. Along with genomic RNA, the
`
`structural proteins form viral particles. The nonstructural proteins participate in
`
`viral polyprotein processing, replication, virion assembly, and evasion of host
`
`immune response. The signal peptide at the C terminus of the C protein (C-signal
`
`35
`
`peptide; also called C-anchor domain) regulates Flavivirus packaging through
`
`coordination of sequential cleavages at the N terminus (by viral NS2B/NS3
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`protease in the cytoplasm) and C terminus (by host signalase in the endoplasmic
`
`reticulum [ER] lumen) of the signal peptide sequence.
`
`The positive-sense single-stranded genomeis translated into a single polyprotein
`
`that is co- and post translationally cleaved by viral and host proteins into three
`
`structural
`
`[Capsid (C), premembrane (prM), envelope (E)], and seven non-
`
`structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins. The structural
`
`proteins are responsible for forming the (spherical) structure of the virion,
`
`initiating virion adhesion, internalization and viral RNA release into cells, thereby
`
`initiating the virus life cycle. The non-structural proteins on the other hand are
`
`10
`
`responsible for viral replication, modulation and evasion of immune responsesin
`
`infected cells, and the transmission of viruses to mosquitoes. The intra- and inter-
`
`molecular interactions between the structural and non-structural proteins play key
`
`roles in the virus infection and pathogenesis.
`
`The E protein comprises at its C terminal end two transmembrane sequences,
`
`15
`
`indicated as TM1 and TM2.
`
`NS1 is translocated into the lumen of the ER via a signal sequence corresponding
`
`to the final 24 amino acids of E and is released from E at its amino terminus via
`
`cleavage by the ER resident host signal peptidase (Nowak et a/. (1989) Virology
`
`169, 365-376). The NS1 comprises at its C terminal a 8-9 amino acids signal
`
`20
`
`sequence which contains a recognition site for a protease (Muller & Young (2013)
`
`Antiviral Res. 98, 192-208)
`
`The constructs of the present invention are chimeric viruses wherein a Lassa G
`
`protein is inserted at the boundary between the E and NS1 protein. However
`
`25
`
`additional sequence elements are provided N terminally and C terminally of the G
`
`protein insert.
`
`The invention relates to polynucleotide comprising a sequence ofa live, infectious,
`
`attenuated Flavivirus wherein a nucleotide sequence encoding at least a part of a
`
`30
`
`arenavirus G protein is inserted 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 E protein of said Flavivirus and N
`
`terminal the NS1 protein of said Flavivirus comprises in the following order :
`
`- a sequence element allowing the proteolytic processing of the G protein from the
`
`35
`
`E protein by a signal peptidase.
`
`- a G protein lacking its signal peptide and a GP2 transmembrane protein, and
`
`- a two TM domains of the E protein of a flavivrus
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`
`To allow proteolytic processing of the arenavirus G protein from the Flavivirus E
`
`protein at its aminoterminal end and allow proteolytic processing of the arenavirus
`
`G protein from the Flavivirus NS1 protein at its C terminal, sequence elements are
`
`provided which are substrates for a signal peptidase. These can vary in length and
`
`in sequence, and can be as short as one amino acid as shown in Jang etal. cited
`
`above. A discussion on suitable recognition sites for signalling proteases is found
`
`in Nielsen et a/. (1997) Protein Eng. 10, 1-6.
`
`Typically, at the C terminus of the G protein, the signal peptide at the N terminus
`
`of the NS1 protein will be used (or a fragment which allows proteolytic processing).
`
`10
`
`Typically, at the N terminus of the G protein, the same signal peptide (or fragment)
`
`of the NSi protein of the Flavivirus backbone is introduced.
`
`The invention equally relates to polynucleotides comprising a sequence ofa live,
`
`infectious, attenuated Flavivirus. Herein a nucleotide sequence encoding at least
`
`a part of an arenavirus G protein is inserted at the intergenic region between the
`
`15
`
`E and NS1 gene of said Flavivirus. Additional sequences are provided such that
`
`when the chimeric virus is expressed such that the encoded sequence from the C
`
`terminally of the E protein to the N terminus of the signal peptide of the NS1
`
`protein comprises in the following order:
`
`a further signal peptide (or cleavable fragment thereof) of a Flavivirus NS1 gene,
`
`20
`
`C terminal to the E protein and N terminal to the NS1 protein.
`
`a arenavirus G protein lacking a functional signal peptide and a transmembrane
`
`sequenceof the GP2 domain. This G protein is C terminally positioned from a NS1
`
`signal peptide. C terminally of the G protein is the sequence of a Flavivirus TM1
`
`and TM2 transmembrane domain of a Flavivirus. C terminally of these TM
`
`25
`
`sequence follows the NS1 protein, including its native signal peptide sequence.
`
`Thus, the G protein and the TM domains are flanked at N terminus and C terminus
`
`by an NS1 sequence. In the embodiments disclosed in the examples the protein
`
`and DNA sequence of both NSi areidentical.
`
`In typical embodiments both NS1
`
`signal
`
`sequences have the sequence
`
`30
`
`DQGCAINFG [SEQ ID NO:10].
`
`The constructs of the present invention did not show recombination due to the
`
`presence of this repetitive sequence. Sequence modifications can be introduced or
`
`NS1 sequences from different Flavivirus can be used to avoid presence of identical
`
`sequences, as long as the encoded peptide remains a target from the protease
`
`35
`
`which processes these NS1i Nterminal signal sequences.
`
`In typical embodiments, as disclosed in the examples, the G protein is of Lassa
`
`virus, preferably of the Josiah strain of Lassa virus.
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`To facilitate the production of virus in the mammalian hosts,
`
`the nucleotide
`
`sequence of the G protein is codon optimized.
`
`It is submitted that minor sequence modifications in the G protein and in the C
`
`terminal
`
`tail can be introduced without
`
`loss of function of these sequence
`
`elements. For example, amino acids substitutions wherein hydrophobic side chains
`
`are preserved in the transmembrane domain, or truncated versions of the
`
`cytoplasmic domain with sufficient
`
`length to allow proper localisation of the
`
`transmembrane domains at the N terminus and C terminus of the cytoplasmatic
`
`domain.
`
`10
`
`It has been found that the presence of a functional signal peptide of the G protein
`
`results in a negative selective pressure whereby a part of the G protein comprising
`
`its signal peptide is deleted or mutated. Thus the constructs of the present
`
`invention typically contain a defective G protein signal by partial or complete
`
`15
`
`removal of this sequence or by the introduction of mutations which render the
`
`signal protein non-functional.
`
`The TM domains which are located C terminally of the G protein and N terminally
`
`of the NS1 is generally of a Flavivirus, typically from the E protein, and more
`
`typical a TM domains of an E protein. In preferred embodiments these TM domains
`
`20
`
`of an E protein are from a different Flavivirus than the virus forming the backbone.
`
`The examples of present invention describe the TM1 and TM2 domain of the E
`
`protein
`
`of
`
`the West Nile
`
`virus. These
`
`domain
`
`have
`
`the
`
`sequence
`
`GGMSWITQGLLGALLLWMGINARD
`
`[SEQ
`
`ID
`
`NO:
`
`14]
`
`and
`
`RSIAMTFLAVGGVLLFLSVNVHA [SEQ ID NO: 15].
`
`25
`
`In the examples section below and in the schematic representation all sequence
`
`elements form a continuous sequence without any intervening sequence elements.
`
`It is submitted that in between these sequence elements, additional amino acids
`
`may be present as long as the localisation of the protein at either the ER lumen or
`
`cytosol is not disturbed and proteolytic processing is maintained.
`
`30
`
`35
`
`The above described nucleotide sequence can be that of the virus itself or can
`
`refer to a sequencein a vector. A suitable vector for cloning Flavivirus and chimeric
`
`version are, amongst other technologies, Bacterial Artificial Chromosomes, as
`
`described in more detail below.
`
`The methods and compounds of the present invention have medicinal application,
`
`wherebythe virus or a vector encoding the virus can be used to vaccinate against
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`12
`
`the arenavirus which contains the G protein that was cloned in the Flavivirus. In
`
`addition, the proteins from the Flavivirus equally provide protection such that the
`
`compounds of the present invention can be used to vaccinate against a Flavivirus
`
`and an arenavirus using a single virus or DNA vaccine.
`
`The use of Bacterial Artificial Chromosomes, and especially the use of inducible
`
`BACS as disclosed by the present inventors in WO2014174078,
`
`is particularly
`
`suitable for high yield, high quality amplification of cDNA of RNA viruses such as
`
`chimeric constructs of the present invention.
`
`10
`
`A BAC as described in this publication BAC comprises:
`
`- an inducible bacterial ori sequence for amplification of said BAC to more than 10
`
`copies per bacterial cell, and
`
`- a viral expression cassette comprising a CDNA of an the RNA virus genome and
`
`comprising cis-regulatory elements for
`
`transcription of said viral
`
`cDNA in
`
`15
`
`mammalian cells and for processing of the transcribed RNA into infectious RNA
`
`virus.
`
`As is the case in the present invention the RNA virus genomeis a chimeric viral
`
`cDNA construct of an RNA virus genome and an arenavirus G protein .
`
`In these BACS, the viral expression cassette comprises a cDNA of a positive-strand
`
`20
`
`RNA virus genome, an typically
`
`-
`
`a RNA polymerase driven promoter preceding the 5’ end of said cDNA for
`
`initiating the transcription of said cDNA, and
`
`-
`
`an element for RNA self-cleaving following the 3’ end of said cDNA for
`
`cleaving the RNA transcript of said viral cDNA at a set position.
`
`25
`
`The BAC may further comprise a yeast autonomously replicating sequence for
`
`shuttling to and maintaining said bacterial artificial chromosome in yeast. An
`
`example of a yeast ori sequence is
`
`the 2u plasmid origin or
`
`the ARS1
`
`(autonomously replicating sequence 1) or functionally homologous derivatives
`
`thereof.
`
`30
`
`The RNA polymerase driven promoter of this first aspect of the invention can be
`
`an RNA polymerase II promoter, such as Cytomegalovirus Immediate Early (CMV-
`
`IE) promoter, or the Simian virus 40 promoter or functionally homologous
`
`derivatives thereof.
`
`The RNA polymerase driven promoter can equally be an RNA polymeraseI or III
`
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`promoter.
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`The BAC may also comprise an element for RNA self-cleaving such as the cDNA of
`
`the genomic ribozyme of hepatitis delta virus or functionally homologous RNA
`
`elements.
`
`The formulation of DNA into a vaccine preparation is Known in the art and is
`
`described in detail in for example chapter 6 to 10 of "DNA Vaccines" Methods in
`
`Molecular Medicine Vol 127, (2006) Springer Saltzman, Shen and Brandsma (Eds.)
`
`Humana Press. Totoma, N.J. and in chapter 61 Alternative vaccine delivery
`
`methods, P 1200-1231, of Vaccines (6th Edition) (2013) (Plotkin et al. Eds.).
`
`Details on acceptable carrier, diluents, excipient and adjuvant suitable in the
`
`10
`
`preparation of DNA vaccines can also be found in WO2005042014, as indicated
`
`below.
`
`"Acceptable carrier, diluent or excipient” refers to an additional substance that is
`
`acceptable for use in human and/or veterinary medicine, with particular regard to
`
`immunotherapy.
`
`15
`
`By way of example, an acceptable carrier, diluent or excipient may be a solid or
`
`liquid filler, diluent or encapsulating substance that may be safely used in systemic
`
`or topic administration. Depending upon the particular route of administration, a
`
`variety of carriers, well Known in the art may be used. These carriers may be
`
`selected from a group including sugars, starches, cellulose and its derivatives,
`
`20
`
`malt, gelatine, talc, calcium sulphate and carbonates, vegetable oils, synthetic
`
`oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline
`
`and salts such as mineral acid salts including hydrochlorides, bromides and
`
`sulphates, organic acids such as acetates, propionates and malonates and
`
`pyrogen-free water.
`
`25
`
`A useful reference describing pharmaceutically acceptable carriers, diluents and
`
`excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N. J. USA,
`
`2(091) which is incorporated herein by reference.
`
`Any safe route of administration may be employed for providing a patient with the
`
`DNA vaccine.
`
`For
`
`example, oral,
`
`rectal, parenteral,
`
`sublingual,
`
`buccal,
`
`30
`
`intravenous,
`
`intra-articular,
`
`intra-muscular,
`
`intra-dermal,
`
`subcutaneous,
`
`inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and
`
`the like may be employed. Intra-muscular and subcutaneous injection may be
`
`appropriate, for example, for administration of immunotherapeutic compositions,
`
`proteinaceous vaccines and nucleic acid vaccines. It is also contemplated that
`
`35
`
`microparticle bombardment or electroporation may be particularly useful
`
`for
`
`delivery of nucleic acid vaccines.
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`
`Dosage forms include tablets, dispersions, suspensions,
`
`injections, solutions,
`
`syrups, troches, capsules, Suppositories, aerosols, transdermal patches and the
`
`like. These dosage forms may also include injecting or implanting controlled
`
`releasing devices designed specifically for this purpose or other forms of implants
`
`modified to act additionally in this fashion. Controlled release of the therapeutic
`
`agent may be effected by coating the same, for example, with hydrophobic
`
`polymers including