OA20740A - Lassavirus vaccines. - Google Patents

Abstract

The present invention relates to polynucleotides 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 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, - 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.

Description

ORCAHIÏATIOH AFRICAINE CE LA PROPRIETE INTÉLLÉCrUÉLLE OAPI

ORGANISATION AFRICAINE DE LA PROPRIETE INTELLECTUELLE

Inter. Cl.

AGI K 39/12 (2018.01) A61P 31/14 (2018.01)

N° 20740

FASCICULE DE BREVET D'INVENTION

Numéro de dépôt : 1202200088

PCT/EP2020/075541

Date de dépôt : 11/09/2020 © Titulaire(s) :

KATHOLIEKE UNIVERSITEIT LEUVEN,

KU Leuven R&D Waaistraat 6 - box 5105, 3000 LEUVEN Vlaams-Brabant (BE)

Priorité(s) :

EP n° 19197202.5 du 13/09/2019

Inventeur(s) :

NEYTS, Johan (BE) DALLMEIER, Kai (BE) LEMMENS, Viktor (BE) SANCHEZ FELIPE, Lorena (BE)

Délivré le : 11/01/2023

Publié le : 24/02/2023

Mandataire : Cabinet BONNY & ASSOCIES LAW FIRM, B.P. 869, YAOUNDE (CM)

Titre : Lassavirus vaccines.

Abrégé :

The présent invention relates to polynucleotides comprising a sequence of a live, infectious, attenuated Flavivirus wherein a nucléotide sequence encoding at least a part of a arenavirus glycoprotein protein is located at the intergenic région 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 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,

- 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.

O.A.P.I. - B.P. 887, YAOUNDE (Cameroun) - Tel. (237) 222 20 57 00-Site web: http:/www.oapi.int- Email: oapi@oapi.int

LASSAVIRUS VACCINES

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 hâve been generated involving several platform technologies. The most advanced candidates are VSV based LASV (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 replicationcompetent VSVs expressing the glycoprotein of LASV. ML29 is a reassortant between Lassa and Mopeia viruses that carnes the L-segment of MOPV and the S- segment (nucleoprotein and glycoprotein) from LASV. INO-45Q0 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 yeilow fever virus 17D has been used as vector for Lassa virus glycoprotein (GPC) or its subunits GP1 and GP2 (Bredenbeek et al. (2006) Virology 345, 299-304 and Jiang 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/NSl. These constructs hâve 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.

The only vaccine that hâve just started a phase I clinical trial is INO-4500 (pLASVGPC). This vaccine requires multiple high doses delivered via derma! electroporation in order to achieve full protection and enhance the vaccine immune response. This multidose administration regimen will be very challenglng to implement in the rural areas of West Africa where LASV is endemic and the main outbreaks hâve 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 of this vaccine. VSVLASV-GPC still requires a cold chain to preserve it which involves high cost 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).

In addition, this vaccine candidate showed issues of genetic instability that did not allow to scale-up the technology as required for vaccine production.

Summary of the invention

We hâve 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 cieavage site R246A to keep GP1 and GP2 bound and additional mutations R207C, G360C and E329P) into yellow fever E /NSI întergenic région as follows: N-terminai (Nt) signal peptide was deleted, first 9 aminoacids of NSI (27 nucléotides) 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 resuiting PLLAV-YFV17D-LASV-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 stmilar construct has been generated in which the cieavage site has been restored (R246A mutation was restored to R246R).(Additional information in the attached data)

PLLAV-YFV 17 D-LASV-G PC is a dual vaccine inducing YFV and Lassa virus spécifie 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 modality, but also unexpectedly the recombinant YFV17D-LASV-GPC virus appears to be genetically more than that disclosed in prior art by Bredenbeek et al. and Jiang et ai. (cited above).

The invention is further summarized in the following statements:

1. A polynucleotide comprising a sequence of a live, infectious, attenuated Flavivirus wherein a nucléotide sequence encoding at least a part of a arenavirus glycoprotein protein is located at the întergenic région between the E and NSI 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 terminally of the signal peptide of the NSI protein of said Flavivirus comprises in the following order; - a further signal peptide of a Flavivirus NSI protein,

-an arenavirus Glycoprotein protein lacking the N terminal signal sequence and the GP2 transmembrane domain,

- a TM1 and TM2 domain of a fîaviviral E protein.

2. The polynucleotide according to claim 1, wherein the sequence of the iive, 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 is a chimeric backbone of two different flaviviruses.

4. The polynucleotide according to any one of daims 1 to 3, wherein the arena virus a Mammarena virus.

5. The polynudeotide according to any one of daims 1 to 4, wherein the arenavirus a Lassa virus.

6. The polynudeotide according to any one of daims 1 to 3, wherein the Lassa strain is the Josiah strain.

7. The polynucieotide according to any one of daims 1 to 6, wherein the glycoprotein comprises the R207C, G360C and E329P stabilizing mutations.

8. The polynucieotide according to any one of daims 1 to 7, wherein the glycoprotein comprises the R246A proteolytic cleavage site.

9. The polynudeotide according to any one of daims 1 to 8, wherein the nudeotide sequence of the G protein is codon optimised for improved expression in mammalian cells.

10. The polynudeotide according to any one of daims 1 to 9, wherein the signal peptide of the NSI protein comprises or consists of the sequence DQGCAINFG [SEQ ID NO: 10].

11. The polynudeotide according to any one of daims 1 to 10, wherein the TM1 and TM2 domain of a flaviviral E protein are from West Nile virus.

12. The polynudeotide according to any one of daims 1 to 11, wherein the TM1 domain of a flaviviral E protein has the sequence of SEQ ID: NO 14.

13. The polynudeotide according to any one of daims 1 to 12, wherein the TM2 domain of a flaviviral E protein has the sequence of SEQ ID NO 15.

14. The polynudeotide according to any one of daims 1 to 13, wherein the sequence of the chimeric virus at the junction of the NSI signal sequence and the GP1 domain comprises the sequence of SEQ ID NO:11.

15. The polynudeotide according to any one of daims 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.

16. The polynucieotide according to any one of daims 1 to 14, wherein the sequence of the chimeric virus at the junction of the TM2 domain and NSI protein comprises the sequence of SEQ ID NO:13.

In preferred embodîments the junctions connecting the flavirus NSI signal sequence, the Lassavirus G protein, the TM2 protein and the second NSI signal sequence provide a fingerprint for the encoded proteins. Thus embodîments 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 whereîn outside SEQ ID NO: 11, SEQ ID: NO 12 and SEQ ID NO13 , a number of amino acids may differ from SEQ ID NO:2 or SEQ ID N0:4, e.g. differing up to 20, up to 10, or up to 5 comparée! to SEQ ID NO:2 or SEQ ID NO: 4, or e.g. having a sequence identity of at least 95 %, 96 %, 97 %, 98% or 99 % with SEQ ID NO:2 or SEQ ID NO:4. 17. The polynucleotîde according to any one of the claims 1 to 16, which is a bacterial artificiel chromosome.

18. A polynucleotîde in accordance to any one of claims 1 to 17, for use as a médicament.

19. The polynucleotîde for use as a médicament in accordance with claim 18, wherein the médicament is a vaccine.

20. A polynucleotîde 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 NSI protein of said Flavivirus, such that C terminally of the E protein and N terminally of the signal peptide of the NSI protein the virus comprises in the following order :

- a further signal peptide of a Flavivirus NSI 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.

22. The chimeric Flavivirus according to claim 21, wherein the Fiavivîrus is YFV.

23. 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 médicament.

25. A chimeric virus in accordance to any one of claims 21 to 24, for use in the prévention of an Arenaviral infection.

26. A chimeric virus encoded by a nucléotide in accordance to any one of claims 21 to 23, for use in the prévention of an Arenaviral infection and in the prévention 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 virus according to any one of claims 1 to 16, and comprising cis-regulatory éléments for transcription of said virai 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 ceils,

- 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.

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 daims 27 to 29, wherein the vector is a BAC, which comprises an inducible bacteria! ori sequence for amplification of said BAC to more than 10 copies per bacterial celf.

DETAILED DESCRIPTION

Figure legends

Figure 1: Schematic représentation of 1) PLLAV-YFV17D-LASV-GPC and 2) PLLAVYFV17D-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 passaging (in BHK-21J and VeroE6) of the YFV17D-LASV-GPC virus. C+, control positive PLLAVYFV17D-LASV-GPC; -RT: RT-PCR reaction without reverse transcriptase; RNA: RT-PCR reaction with the virus RNA.

Figure 3: Schematic vaccination schedule. AG129 mice were vaccinated with PLLAVYFV17D-LASV-GPC (25 ug, i.p.) ΟΓ YFV17D-LASV-GPC (375 PFU).

Figure 4: Analysis of cellular immunity in vaccinated AG129 mice. A) Représentative IFN-γ ELÏSPOT wells after 48 hours of stimulation of splénocytes with the indicated antigen. B) Spots per six hundred thousand splénocytes in IFN-γ 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) Coexpression of LASV-GPC and YFV antrgens detected by immunofluorescence of BHK21J cells infected with supernatant of cells transfected with PLLAV-YFV17D-LASV-GPCcs .Cells were fixed 48h post-infection and staîned for LAV-GPC (red) and YFV (green).

Figure 6: A) Schematic vaccination schedule. AG129 mice were vaccinated subcutaneous (SC) with YFV17D-LASV-GPCcs (250 PFU). B) Analysis of cellular immunity in vaccinated AG129 mice. Représentative IFN-gamma ELISPOT wells after hours splénocyte stimulation with the indicated antigen. Spots per six hundred thousand splénocytes in IFN-gamma 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 peptide stimulated).

The présent invention is exemplified for Yeliow Fever virus, but is also applicable using other viral backbones of Flavivirus species such, but not limited to, Japanese 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, Wesseisbron and Omsk Hémorrhagie Fever virus.

The invention is further applicable to Flaviviridae, which comprises the genus Flavivirus but also the généra, 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).

The genus Pestivirus comprises e.g. Bovine virus diarrhea virus 1 and Classical swine fever virus (previously hog choiera virus).

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 région are from Yeliow Fever and the prME région 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 nucléotide sequence and encoded proteins wherein within the RNA or copy DNA (cDNA) of a flavivirus a glycoprotein of an arenavirus is inserted

Giycoproteins of Arenavirus are discussed in Burr et al. (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 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).

Based on serological, genetic and geographicai 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 (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 ît contalns the major viruses causing hémorrhagie fevers (HF) in South America, i.e. Junin (JUNV), Machupo (MACV), Guanarito (GTOV) and Sabia (SABV) viruses but also 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. The present invention envisages chimeric constructs based on G proteins of Reptarenavirusse or Hartmanivirusses are used.

The present invention is exemplified with G protein of Lassa virus strain Josiah. This sequence of this protein is accessible for example as UniProtKB P08669 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 présentation of the encoded 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 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 NSI protein.

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 sequence identity between G proteins of different arenavirus présents no problems to the skilled person to identify in related sequences the sequence éléments corresponding to those présent in Lassa virus G protein.

Flaviviruses hâve a positive single-strand RNA genome of approximately 11,000 nucléotides in length. The genome contains a 5' untranslated région (UTR), a long openreadlng frame (ORF), and a 3' UTR. The ORF encodes three structural (capsid [C], precursor membrane [prM], and envelope [E]) and seven nonstructural (NSI, ΝΞ2Α, ΝΞ2Β, NS3, NS4A, NS4B, and NS5) proteins. Along with genomic RNA, the structural proteins form viral particles. The nonstructural proteins participate in viral polyprotein Processing, réplication, virion assembly, and évasion of host immune response. The signal peptide at the C terminus of the C protein (C-sîgnal peptide; also called C-anchor domain) régulâtes Flavivirus packaging through coordination of sequential cleavages at the N terminus (by viral NS2B/NS3 protease in the cytoplasm) and C terminus (by host signalase in the endoplasmic réticulum [ER] lumen) of the signal peptide sequence.

The positive-sense single-stranded genome is 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 (NSI, NS2A, ΝΞ2Β, NS3, NS4A, NS4B, ΝΞ5) proteins, The structural proteins are responsible for forming the (spherical) structure ofthe 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 responsible for viral réplication, modulation and évasion of immune responses in 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, indicated as TM1 and TM2.

NSI 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 al. (1989) Virology 169, 365-376). The NSI comprises at its C terminal a 8-9 amino acids signal sequence which contains a récognition site for a protease (Muller & Young (2013) Antiviral Res. 98, 192-208) The constructs ofthe présent invention are chimeric viruses wherein a Lassa G protein is inserted at the boundary between the E and NSI protein. However additional sequence éléments are provided N terminally and C termînally of the G protein insert. The invention relates to polynucleotide comprising a sequence of a live, infections, attenuated Flavivirus wherein a nucléotide sequence encoding at least a part of a arenavirus G protein is inserted at the intergenic région between the E and NSI 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 NSI protein of said Flavivirus comprises in the foliowing order :

- a sequence element allowing the proteolytic Processing of the G protein from the E protein by a signal peptidase.

- a G protein lacking its signai peptide and a GP2 transmembrane protein, and - a two TM domains of the E protein of a flavivrus

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 NSI protein at its C terminal, sequence éléments 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 et al. cited above. A discussion on suitable récognition sites for signalling proteases is found in Nielsen et al. (1997) Protein Eng. 10, 1-6.

Typically, at the C terminus of the G protein, the signal peptide at the N terminus of the NSI protein will be used (or a fragment which allows proteolytic processing).

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 of a live, infections, attenuated Flavivirus. Herein a nucléotide sequence encoding at least a part of an arenavirus G protein is inserted at the intergenic région between the E and NSI 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 NSI protein comprises in the following order:

a further signal peptide (or deavable fragment thereof) of a Flavivirus NSI gene, C terminal to the E protein and N terminal to the NSI protein.

a arenavirus G protein iacking a functional signal peptide and a transmembrane sequence of the GP2 domain. This G protein is C terminally positioned from a NSI 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 sequence follows the NSI 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 NSI sequence. In the embodiments disclosed in the examples the protein and DNA sequence of both NSI are identical.

In typical embodiments both NSI signal sequences hâve the sequence DQGCAINFG [SEQ ID NQ:10].

The constructs of the présent invention did not show recombination due to the presence of this répétitive sequence. Sequence modifications can be introduced or NSI 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 which processes these NSI Nterminal signal sequences.

In typical embodiments, as disclosed in the examples, the G protein is of Lassa virus, preferably ofthe Josiah strain of Lassa virus.

To facîlitate the production of virus in the mammalian hosts, the nucléotide sequence ofthe 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 éléments. For example, amino acids substitutions wherein hydrophobie side chains are preserved in the transmembrane domain, or truncated versions ofthe cytoplasmic domain with sufficient length to allow proper localisation ofthe transmembrane domains at the N terminus and C terminus ofthe cytoplasmatic domain.

It has been found that the presence of a functional signal peptide of the G protein results in a négative sélective pressure whereby a part ofthe G protein comprising its signal peptide is deleted or mutated. Thus the constructs of the présent invention typically contain a defective G protein signal by partial or complété removal of this sequence or by the introduction of mutations which render the signal protein nonfunctional.

The TM domains which are located C terminal ly of the G protein and N terminally ofthe ΝΞ1 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 of an E protein are from a different Flavivirus than the virus forming the backbone. The examples of présent invention descri be the TM1 and TM2 domain of the E protein of the West Ni le virus. These domain hâve the sequence GGMSWITQGLLGALLLWMGINARD [SEQ ID NO: 14] and RSIAMTFLAVGGVLLFLSVNVHA [SEQ ID NO: 15].

In the examples section below and in the schematic représentation ail sequence éléments form a continuous sequence without any intervening sequence éléments. It is submitted that in between these sequence éléments, additional amino acids may be présent as long as the localisation ofthe protein at either the ER lumen or cytosol is not disturbed and proteolytic Processing is maintained.

The above described nudeotide sequence can be that of the virus itself or can refer to a sequence in a vector. A suîtable 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 hâve médicinal application, whereby the virus or a vector encoding the virus can be used to vaccinate against the S arenavirus which contains the G protein that was cloned in the Fiavivirus. In addition, the proteins from the Fiavivirus equally provide protection such that the compounds of the present invention can be used to vaccinate against a Fiavivirus and an arenavirus using a single virus or DNA vaccine.

The use of Bacterial Artificial Chromosomes, and especialiy the use of inducible BACS 10 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 présent invention.

A BAC as described in this publication BAC comprises:

- an inducible bacterial ori sequence for amplification of said BAC to more than 10 copies 15 per bacterial cell, and

- a viral expression cassette comprisîng a cDNA of an the RNA virus genome and comprisîng cis-regulatory éléments for transcription of said viral cDNA in mammalian cells and for Processing of the transcribed RNA into infectious RNA virus.

As is the case in the present invention the RNA virus genome is a chimeric viral cDNA 20 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 RNA virus genome, an typicalty a RNA polymerase driven promoter preceding the 5' end of said cDNA for initiating the transcription of said cDNA, and

- an eiement for RNA self-cleaving following the 3' end of said cDNA for cleaving the RNA transcript of said viral cDNA at a set position.

The BAC may further comprise a yeast autonom ou si y replicating sequence for shuttling to and maintaîning said bacterial artificial chromosome in yeast. An example of a yeast ori sequence is the 2μ plasmid origin or the ARS1 (autonomously replicating sequence 30 1) or functionally homologous dérivatives thereof.

The RNA polymerase driven promoter of this first aspect of the invention can be an RNA polymerase II promoter, such as Cytomégalovirus Immédiate Early (CMV-IE) promoter, or the Simian virus 40 promoter or functionally homologous dérivatives thereof.

The RNA polymerase driven promoter can equaliy be an RNA polymerase I or III 35 promoter.

The BAC may also comprise an eiement for RNA self-cleaving such as the cDNA of the genomic ribozyme of hepatitis delta virus or functionally homologous RNA éléments.

The formulation of DNA into a vaccine préparation 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, NJ. 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 préparation 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 immunothera py.

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 dérivatives, malt, gélatine, talc, calcium sulphate and carbonates, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonie saline and salts such as minerai acid salts including hydrochlorides, bromides and sulphates, organic acids such as acétates, propionates and malonates and pyrogen-free water.

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 empioyed for providing a patient with the DNA vaccine. For example, oral, rectal, parentéral, sublingual, buccal, intravenous, intraartlcular, intra-muscular, intra-dermal, subeutaneous, inhalational, (ntraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be empioyed. Intra-muscular and subeutaneous injection may be appropriate, for example, for administration of immunotherapeutic compositions, proteinaceous vaccines and nucleic acid vaccines. It is also contemplated that microparticle bombardment or electroporatlon may be particularly useful for delivery of nucleic acid vaccines.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aérosols, 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 additionaily in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobie polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose dérivatives such as hydroxypropylmethyl cellulose. In addition, the controtled release may be effected by using other poiymer matrices, liposomes and/or microspheres.

DNA vaccines suitable for oral or parentéral administration may be presented as discrète units such as capsules, sachets or ta blets each conta ining a pre-determined amount of plasmid DNA, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water émulsion or a water-in-oil liquid émulsion. Such compositions may be prepared by any of the methods of pharmacy but ail methods include the step of brlngîng into association one or more agents as described above with the carrier which constitutes one or more necessary ingrédients. In general, the compositions are prepared by uniformly and intimately admixing the DNA plasmids with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired présentation.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is effective. The dose administered to a patient, should be sufficient to effect a bénéficiai response in a patient over an appropriate period of time. The quantity of agent (s) to be administered may dépend on the subject to be treated inclusive of the âge, sex, weight and general health condition thereof, factors that will dépend on the judgement of the practitioner.

Furthermore DNA vaccine may be delivered by bacterial transduction as using liveattenuated strain of Salmonella transformed with said DNA plasmids as exemplified by Darji et al. (2000) FEMS Immunoi Med Microbiol 27, 341-349 and Cicin-Sain et al. (2003) J Vire! 77, 8249-8255 given as reference.

Typically the DNA vaccines are used for prophylactic or therapeutic immunisation of humans, but can for certain viruses also be applied on vertebrate animais (typically mammals, birds and fish) inciuding domestic animais such as livestock and companion animais. The vaccination is envisaged of animais which are a live réservoir of viruses (zoonosis) such as monkeys, dogs, mice, rats, birds and bats.

In certain embodîments vaccines may include an adjuvant, i.e. one or more substances that enhances the immunogenicity and/or efficacy of a vaccine composition However, life vaccines may eventually be harmed by adjuvants that may stimulate innate immune response independent of viral réplication. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of anima! origin); block copoiymers; détergents such asTween-80; Quil! A, minerai oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acné; Mycobacterium bovis (Bacille Calmette and Guérin or BCG); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOMt) and ISCOMATRIX (B) adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other dérivatives; Avridine; Lipid A dérivatives; dextran sulfate; DEAEDextran or with aluminium phosphate; carboxypolymethylene such as Carbopol'EMA; acrylic copolymer émulsions such as Neocryl A640; vaccinia or animal poxvirus proteins; sub-viral particle adjuvants such as choiera toxin, or mixtures thereof.

EXAMPLES

EXAMPLE 1 YFV17D/ LASSA CONSTRUCTS

The Lassa glycoprotein precursor (LASV-GPC) from Josiah strain was inserted between YF-E/NS1 to generate two constructs as follows (Figure 1):

1) PLLAV-YFV17D-LASV-GPC: Lassa glycoprotein with the N-terminal signal peptide sequence (SSP) and the GP2 transmembrane domain (TM) deleted. The LASV glycoprotein cleavage site was mutated (R246A) to keep the precursor GPC (GP1 and GP2 linked). These point mutations R207C and G360C (bind GP1 and GP2 covalently) and E329P (described in Hastie et al (2017) Science 356, 923-928) were introduce to improve stability. This Lassa-GPC with the mutations was fused to the transmembrane domains of WNV (TM1 and TM2) to keep the polyprotein topology required to replicate YFV17D and allow the proper expression of LASV-GPC. In addition, the sequence that codified for the first 9 amino acids of YF-NS1 was introduced before LASV-GPC sequence to allow the correct processing of the antigen.

2) PLLAV-YFV17D-LASV-GPCcs: Similar construct to the one described above but, in this construct, the cleavage site was restored (R246A mutation was restored to R246R). The rest of the mutations were similar, mutations R207C and G360C (bind GP1 and GP2 covalently) and E329P (described in Hastie et al. (2017) Science 356, 923928) were introduced to improve stability.

Example 2 CONSTRUCT #1 PLLAV-YFV17D-LASV-GPC

PLLAV-YFV17D-LASV-GPC was transfected into BHK21J cells and typical CPE was observed as well as the virus supernatant harvested from them formed markedly smaller plaques compared to the plaque phenotype of YFV17D (Figure 2A). Therefore, the resulting transgenic virus (YFV17D-LASV-GPC) is further attenuated, and virus yields were at least 10-fold less compared to YFV17D.

The stability of PLLAV-YFV17D-LASV-GPC was determined by performing RT-PCR to detect the transgene insert in virus samples that were harvested during serial passage of the YFV17D-LASV-GPC (Figure 2B). Sequencing of the RT-PCR products showed that

LASV-GPC insert with no mutations can be detected at least until passage 5 in SHK21J cells.

Example 3 Immunogenicity of PLLAV-YFV17D-LASV-GPC in AG129 mice

The immunogenicity of PLLAV-YFV17D-LASV-GPC and the derived lîve-attenuated virus (LAV) was assessed in vivo in AG129 mice. Animais (n=9/group) were vaccinated with either 25 pg of PLLAV-YFV17D-LASV-GPC or 375 PFU of YFV17D-LASV-GPC (Figure 3). The YFV- and LASV-specific antibody responses were quantified by indirect immunofluorescence assay (IIFA) and the cell mediated immune response was quantified by ELISPOT (Figure 4).

Vaccinated mice were monitored daily for morbidity/mortality and blood was sampled for seralogical analysis at baseline and with two-week intervals. The vaccine was safe as no adverse effects were observed in any of the vaccinated mice. Some animais (4 of the 9 mice) were boosted two weeks after first inoculation with the PLLAV or LAV YFV17D-LASV-GPC using same dose and route than in the first vaccination (Figure 3). The immunogenicity analysis for YFV17D-LASV-GPC (PLLAV or LAV) revealed that at 14 days post vaccination there were spécifie antibodies against LASV in 3 and 1 mice vaccinated with PLLAV or LAV respectively. Of note, for LASV it is currentiy thoughtthat the CD8+ T-ceil response is the main déterminant responsible for providing protection against LASV infection. Therefore, the T celis responses were analyzed in both groups at 4 months post vaccination, This analysis showed that there was T cells responses against LASV and YFV in ail the mice vaccinated with YFV17D-LASV-GPC (LAV) and in 7 out of 9 after vaccination with the PLLAV version (Figure 4). These T cell responses can hence be considered to confer immunity and protection against LASV infection.

Example 4 CONSTRUCT #2 PLLAV-YFV17D-LASV-GPCcs (cleavage site)

A second construct, similar to the above described, was generated. In this PLLAVYFV17D-LASV-GPCcs in which the natural cleavage site between GP1 and GP2 was restored. This construct was transfected in BHK21J cells and typicai CPE was observed as well as the virus supernatant harvested from them formed markedly smaller plaques compared to the plaque phenotype of YFV17D (Figure 5A). Therefore, similar to the previous YFV17D/LASV construct, the resulting transgenic virus (YFV17D-LASV-GPC) is further attenuated, and virus yields were at least 10-fold less compared to YFV17D. Co-expression of LASV-GPC along with the YFV polyprotein could be confirmed (Figure 5B) indicating proper expression and folding of LASV-GPC.

To assess the immunogenicity of this construct, AG129 mice were vaccinated subcutaneously (s.c.) with YFV17D-LASV-GPCcs (LAV) and the T- cell responses were determîned at 28 days post-vaccination (Figure 6). The analysis show that there was strong spécifie T celis responses against both, LASV and YFV. These results suggests that the vaccine can work as a bivalent vaccine against both viruses, LASV and YFV. The vaccine was safe as no adverse effects were observed in any of the vaccinated mice.

SEQUENCES DEPICTED IN THE APPLICATION construct#!:PLLAV-YFV17D-LASV-GPC (signal peptide deleted, transmembrane domain g p2deleted,clsavage sitemutated(r256a)and mutations r207c ,e329p and g360c)

- end YF-E (amino acids 1-40)

- first 27 nuclectide (9 amino acids) of SNS1 (amino acids 41-49) bold underlined

- lasv-gpl domain[without signal peptide and with mutation tgt (r207c)] (amino acids 50-250)

- cleavage site mutated gcaagattgcta(r256a) [SEQ ID NO:16] (amino acids 247-250)

- lasv- p2 [without tm and mutations cca(e329p) ,tgt(g360c) ] (amino acids 251-418)

- WNV TM1 (amino acids 418-442)underlined

- WNV TM2 (amino acids 443-465) underlined - beginning yf-nsl (amino acids 466-527)

SEQ ID NO:1 DNA

SEQ ID NO:2 protein

AAGGTCATCATGGGGGCGGTACTTATATGGGTTGGCATCAACACAAGAAACATGACAATG KVIMGAVLIWVGINTRNMTM20 TCCATGAGCATGATCTTGGTAGGAGTGATCATGATGTTTTTGTCTCTAGGAGTTGGcGCc

SMSMILVGVIMMFLSLGVGA40 40 GAC CAGGGC T GCGCGATAAATTTCGGTaccagtc111ataaagggg111atgagc11cag

DQGCAINFGT SLYKGVYELQ60 41 49 51 actctggaactaaacatggagacactcaatatgaccatgcctctctcctgcacaaagaac

TLELNMETLNMTMPLSCTKN80 aacagtcatcattatataatggtgggcaatgagacaggactagaactgaccttgaccaac

NSHHYIMVGNETGLELTLTN 100 acgagcattattaatoaoaaattttgcaatctgtctgatgcccacaaaaagaacctctat

TS I INHKFCNLSDAHKKNLY 120 gaccacgctcttatgagcataatctcaactttccacttgtccatccccaacttcaatcag

DHALMSIISTFHLSIPNFNQ 140 tatgaggcaatgagctgcgattttaatgggggaaagattagtgtgcagtacaacctgagt

YEAMSCDFNGGKISVQYNLS 160 cacagctatgctggggatgcagccaaccattgtggtactgttgcaaatggtgtgttacag

HSYAGDAANHCGTVANGVLQ 180 acttttatgaggatggcttggggtgggagctacattgctcttgactcaggcTcftggcaac

TFMRMAWGGSYIALDSG C_G N 200 R207C tgggactgtattatgactagttatcaatatctgataatacaaaatacaacctgggaagat WDCIMTSYQYLI IQNTTWED 220 cactgccaattctcgagaccatctcccatcggttatctcgggctcctctcacaaaggact

HCQFSRPSPIGYLGLLSQRT 240 agaga ta t1tatattaqtgcAAGATTGCTAGGCA CA TTCACA TGGACACTGTCAGATTCT R D I Y ! S A R L L G TFTWTLSDS 260

R256A 247 250

GAAGGTAAAGACACACCAGGGGGATATTGTCTGACCAGGTGGATGCTAATTGAGGCTGAA

EGKDTPGGYCLTRWMLIEAE 280

CTAAAA TGCTTCGGGAACA CAGCTGTGGCAAAA TGTAA TGAGAAGCATGATGAGGAA TTT

LKCFGNTAVAKCNEKHDEEF 300

TGTGACATGCTGAGGCTGTTTGACTTCAACAAACAAGCCATTCAAAGGTTGAAAGCTccA

CDMLRLFDFNKQA I Q R L K A P 320

E329P

GCACAAA TGAGCATTCAGTTGATCAACAAAGCAGTAAA TGCTTTGATAAATGACCAACTT A QMSIQLINKA V N A L I N D Q L 340

ATAA TGAAGAACCATCTACGGGACATCATGÇGtATTCCATACTGTAATTACAGCAAGTA T

IMKNHLRDIMCIPYCNYSKY 360

G360C

TGGTACCTCAACCACACAA CTACTGGGAGAACA TCACTGCCCAAA TGTTGGCTTGTA TCA

WYLNHTTTGRTSLPKCWLVS 380

AATGGTTCATACTTGAACGAGACCCACTTTTCTGATGATATTGAACAACAAGCTGACAAT N G S YLNETHFSDDIEQQADN 400

ATGATCACTGAGATGTTACAGAAGGAGTATATGGAGAGGCAGGGGAAGACACCAGGAGGG MI T E M L Q K E YMERQGKTP G G 4 2 0 418 419

ATGTCCTGGATCACACAGGGACTTCTGGGAGCTCTTCTGTTGTGGATGGGAATCTiATGCC

MSWITQGLLGALLLWMGINA 440

CGTGACAGGTCAATTGCTATGACGTTTCTTGCGGTTGGAGGAGTTTTGCTCTTCCTTTCG R D R S IAMTFLAVGGVLLFLS 460

GTCAACGTCCATGCTGATCAAGGATGCGCCATCAACTTTGGCAAGAGAGAGCTCAAGTGC

V N V H A D Q G CA I N F G K R E L K C 480

485 466 474 475

GGAGATGGTATCTTCATATTTAGAGACTCTGATGACTGGCTGAACAAGTACTCATACTAT GDGIFIFRDSDDWLNKYSYY 500

CCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCTCTTTTGAAGAAGGGAAGTGT

PEDPVKLASIVKASFEEGKC 520

GGCCTAAATTCAGTTGACTCC

G L N S V D S 527

527 construct#2iPLLAV-YFV17D-LASV-GP CCS signal peptide deleted, transmembrane domain gp2 deleted,cleavage site restored(R246R)aND MutatloNS R207C.E329P aND G360C)

-End YFE (amino acids 1-40)

-first 27 nucléotides nsi(9 aminoacids) (amino acids 41-49

-lasv-gpl[without signal peptide and mutation tgt(r207c)] (amino acids 50 to 250)

- cleavage site restored agaagattgcta (r256r) [SEQ ID NO:17]

-lasv-gp2[without tm and mutations cca(e329p),tgt(g360c)] (amino acids 251 to 418)

-WNV-TM1 (amino acids 419-442

-WNV-TM2 (amino 462-465)

-beginning yf-nsl (amino acids 466-527)

SEQ ID NO:3

SEQ ID NO:4

AAGGTCATCATGGGGGCGGTACTTATATGGGTTGGCATCAACACAAGAAACATGACAATG KVIMGAVLIWVGINTRNMTM20

TCCATGAGCATGATCTTGGTAGGAGTGATCATGATGTTTTTGTCTCTAGGAGTTGGcGCc

SMSMILVGVIMMFLSLGVGA40

GACCAGGGCTGCGCGATAAATTTCGGTaccagtctttataaaggggtttatgagcttcag DQGCAINFGT SLYKGVYELQ 60

4149 50 actctggaactaaacatggagacactcaatatgaccatgcctctctcctgcacaaagaac ΤΕΕΈΝΜΕΤΕΝΜΤΜΡΕΞΟΤΚΝ80 aacagtcatcattatataatggtgggcaatgagacaggaatagaactgaccttgaccaac NSHHYIMVGNETGLELTLTN100 acgagcattattaatcacaaattttgcaatctgtctgatgcccacaaaaagaacctctat T S I INHKFCNLSDAHKKNLY120 gaccacgctcttatgagcataatctcaactttccacttgtccatccccaacttcaatcag D H A L M S I ISTFHLSIPNFNQ140 tatgaggcaatgagctgcgattttaatgggggaaagattagtgtgcagtacaacctgagt

YEAMSCDFNGGKI S V Q Y N L S160 cacagctatgctggggatgcagccaaccattgtggtactgttgcaaatggtgtgttacag180

HSYAGDAANHCGTVANGVLQ acttttatgaggatggcttggggtgggagctacattgcÎLCttgactcaqgcTgtggcaac TFMRMAWGGSYIALDSG C_G N 200 tgggactgtattatgactagttatcaatatctgataatccaaaatacaacctgggaagat

WDCIMTSYQYLIIQNTTWED220 cactgccaattctcgagaccatctcccatcggttatctcgggctcctctcacaaaggact

HCQFSRPSPIGYLGLLSQRT240 agagat at 11 a t a t taqtagAAGATTGCTAGGCACATTCACATGGACACTGTCAGA TTCT

R D I Y I S R R L L· G TFTWTLSDS 260

R256 247250

GAAGGTAAAGACACACCAGGGGGATATTGTCTGACCAGGTGGATGCTAATTGAGGCTGAA EGKDTPGGYCLTR W M L I E A E230

CTAAAATGCTTCGGGAACACAGCTGTGGCAAAATGTAATGAGAAGCATGATGAGGAATTT IKCEGMTAVAKCNEKHOEEF300

TGTGACATGCTGAGGCTGTTTGACTTCAACAAACAAGCCATTCAAAGGTTGAAAGCTccA CDMLRLFDFNKQAIQRLKAp320

GCACAAATGAGCATTCAGTTGATCAACAAAGCAGTAAATGCTTTGATAAATGACCAACTT AQMSIQLINKA V N A L I N D Q L 340

ΑΤΆΑTGAAGAACCATCTACGGGACATCATGtGtATTCCATACTGTAATTACAGCAAGTA T IMKNHLRDIMcI P Y C N Y S K Y 360

TGGTACCTCAACCACACAACTACTGGGAGAACATCACTGCCCAAATGTTGGCTTGTATCA NYLNHTTTGR TSLPKCWL VS 380

AATGGTTCATACTTGAACGAGACCCACTTTTCTGATGATATTGAACAACAAGCTGACAAT

NGSYLNETHFSDDIEQQADN 400

ATGATCACTGAGATGTTACAGAAGGAGTATATGGAGAGGCAGGGGAAGACACCAGGAGGG MI T E M L· Q K E YMERQGKTP G G 420

ATGTCCTGGATCACACAGGGACTTCTGGGAGCTCTTCTGTTGTGGATGGGAATCAATGCC MSWITQGLLGALLLWMGINA 440

CGTGACAGGTCTîATTGCTATGACGTTTCTTGCGGTTGGAGGAGTTTTGCTCTTCCTTTCG R D R S IAMTFLAVGGVLLFLS 460

GTCAACGTCCATGCTGATCAAGGATGCGCCATCAACTTTGGCAAGAGAGAGCTCAAGTGC V N V H A D QGCAINFGKRELKC4 90

GGAGATGGTATCTTCATATTTAGAGACTCTGATGACTGGCTGAACAAGTACTCATACTAT GDGIFIFRDSDDWLNKYSYY 500

CCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCTCTTTTGAAGAAGGGAAGTGT PEDPVKLASIVKASFEEGKC520

GGCCTAAATTCAGTTGACTCC G L N S V D S527

Nucléotide sequence and amino acid sequence of the deleted signal peptide(ΞΞΡ) SEQ ID NO:5

SEQ ID NO: 6 atgggacaaatagtgacattcttccaggaagtgcctcatgtaatagaagaggtgatgaac

MGQIVTFFQEVPHVIEEVMN2Q attgttctcattgcactgtctgtactagcagtgctgaaaggtctgtacaattttgcaacg

IVLIALSVLAVLKGLYNFAT40 tStSSccttgttSStttaatcactttcctcctgttgtgtggtaggtcttgcaca

ΟΟΕνσΕνΤΓΕΕΒΟΟΕΞΟΤ 53

Nucléotide and amino acid sequence of the deleted LASV-GP2 transmembrane domain and cytoplasmic tail: SEQ ID NO:7 SEQ ID NO:8

TTGGGTCTAGTTGACCTCTTTGTGTTCAGCACAAGTTTCTATCTTATTAGCATCTTCCTT

LGLVDLFVFSTSFYLISIFL20

CACCTAGTCAAAATACCAACTCATAGGCATATTGTAGGCAAGTCGTGTCCCAAACCTCAC

HLVKIPTHRHIVGKSCPKPH40

AGATTGAATCATATGGGCATTTGTTCCTGTGGACTCTACAAACAGCCTGGTGTGCCTGTG

RLNHMGICSCGLYKQPGVPV60

AAATGGAAGAGA

K W K R64

Lassa Josiah strain G protein sequence SEQ ID NO: 9 (amino acids 1-58 : signal sequence (amino acids 59-259: GP1 domain) (amino acids 260-437: GP2 domain) (amino acids 438-481: transmembrane domain and cytoplasmic tail)

MGQIVTFFQE VPHVIEEVMN IVLIALSVLA VLKGLYNFAT CGLVGLVTFL 50

LLCGRSCTTS LYKGVYELQT LELNMETLNM TMPLSCTKNN SHHYIMVGNE 100 58

TGLELTLTNT SIINHKFCNL SDAHKKNLYD HALMSIISTF HLSIPNFNQY 150

EAMSCDFNGG KISVQYNLSH SYAGDAANHC GTVANGVLQT FMRMAWGGSY 200

IALDSGRGNW DCIMTSYQYL IIQNTTWEDH CQFSRPSPIG YLGLLSQRTR 250

DIYISRRLLG TFTWTLSDSE GKDTPGGYCL TRWMLIEAEL KCFGNTAVAK 300

259

CNEKHDEEFC DMLRLFDFNK QAIQRLKAEA QMSIQLINKA VNALINDQLI 350

MKNHLRDIMG IPYCNYSKYW YLNHTTTGRT SLPKCWLVSN GSYLNETHFS 400

DDIEQQADNM ITEMLQKEYM ERQGKTPLGL VDLFVFSTSF YLISIFLHLV 450 437

KIPTHRHIVG KSCPKPHRLN HMGICSCGLY KQPGVPVKWK R 481

NSI signal sequence [SEQ ID NO:10] DQGCAINFG

Junction YFV NSI- Lassa GP1 domain [SEQ ID NO; 11] AINFG TSLYK

Junction Lassa GP2 domain - WNV TM1 domain [SEQ ID NO: 12] QGKTP GGMSW

Junction WNV TM2 domain - YFV NSI [SEQ ID NO; 13]

VNVHA DQGCA

WNV TM1 sequence [SEQ ID NO: 14] GGMSWITQGLLGALLLWMGINARD

WNV TM2 sequence [SEQ ID NO: 15) RSIΆΜΤΕΕΑναανΒΒΕΒΞVNVHA

Claims (10)

1. A polynucleotide comprising a sequence of a live, infectious, attenuated Flavivirus wherein a nucléotide sequence encoding at least a part of an arenavirus glycoprotein protein is located at the intergenic région between the E and NSI 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 terminally of the signal peptide of the NSI protein of said Flavivirus comprises in the following order :

- a further signal peptide of a Flavivirus NSI protein,

- an arenavirus glycoprotein lacking the N terminal signal sequence and the GP2 transmembrane domain,

- a TM1 and TM2 domain of a Flaviviral E protein.

2. The polynucleotide according to claim 1, wherein the sequence of the live, Infectious, attenuated Flavivirus is Yellow Fever virus, typically the YF17D strain.

3. The polynucleotide according to claim 1 or 2, wherein the arenavirus is a Mammarena virus or a Lassa virus.

4. The polynucleotide according to any one of claims 1 to 3, wherein the encoded protein sequence comprises the sequence of SEQ ID NO: 2 or SEQ ID N0:4.

5. The polynucleotide according to any one of claims 1 to 4, wherein the glycoprotein comprises:

- R207C, G360C and E329P stabilizing mutations, and/or

- a R246A proteolytic cleavage site.

6. The polynucleotide according to any one of claim 1 to 5, wherein:

- the signal peptide of the NSI protein comprises or consists of the sequence DQGCAINFG [SEQ ID NO: 10], and/or

- 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 NSI signal sequence and the G PI domain comprises the sequence of SEQ ID NO: 11, and/or

- 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 NSI protein comprises the sequence of SEQ ID NO: 13.

7. The polynucleotide according to any one of claims 1 to 6, encoding a sequence comprisîng SEQ ID NO: 11, SEQ ID: NO 12 and SEQ ID NO13; and outside SEQ ID NO: 11, SEQ ID: NO 12 and SEQ ID NO: 13, a sequence îdentity of at least 95 %, 96 %, 97 %, 98% or 99 % with SEQ ID NO:2 or SEQ ID NO:4.

8. A chimeric live, infectious, attenuated Fiavivirus wherein at least a part of an arenavirus glycoprotein is located between the E and NSI protein of said Fiavivirus, such that C terminally of the E protein and N terminalty of the signal peptide of the NSI protein the virus comprises in the following order:

- a further signal peptide of a Fiavivirus NSI protein,

- an arenavirus glycoprotein protein lacking the N terminal signal sequence and the GP2 transmembrane domain,

- a TM1 and TM2 domain of a Flavivîral E protein.

9. The chimeric Fiavivirus according to claim 12, wherein the Fiavivirus is YFV and/or the arenavirus is Lassa virus.

10. A polynucleotide according to any of claims 1 to 7, or a chimeric virus according to claim 8 or 9, for use as a médicament.