LASSA VACCINE

Abstract

The invention relates to recombinant measles virus expressing Lassa virus polypeptides, and concerns in particular immunogenic LASV particles expressed by a measles virus and/or virus like particles (VLPs) that contain proteins of a Lassa virus. These particles are recombinant infectious particles able to replicate in a host after an administration. The invention provides means, in particular nucleic acid constructs, vectors, cells and rescue systems to produce these recombinant infectious particles. The invention also relates to the use of these recombinant infectious particles, in particular under the form of a composition, more particularly in a vaccine formulation, for the treatment or prevention of an infection by Lassa virus.

Claims

1. A nucleic acid construct which comprises: (1) a cDNA molecule encoding a full length antigenomic (+) RNA strand of a measles virus (MeV); (2) a first heterologous polynucleotide encoding at least one polypeptide, or an antigenic fragment thereof, of a Lassa virus (LASV), said at least one polypeptide being selected from the group consisting of the nucleoprotein (NP), a mutated nucleoprotein (mNP), the glycoprotein precursor (GPC) and the zinc-binding protein (Z); and wherein the first heterologous polynucleotide is operatively cloned within an additional transcription unit (ATU) inserted within the cDNA of the antigenomic (+) RNA, in particular an ATU localized between the P gene and the M gene of the MeV, in particular the ATU2 inserted between the P gene and the M gene of the MeV.

2. A nucleic acid construct according to claim 1 further comprising a second heterologous polynucleotide encoding at least one polypeptide, or an antigenic fragment thereof, of the LASV, said at least one polypeptide being selected from the group consisting of the GPC protein, the NP protein, the mNP protein and Z protein, the second heterologous polynucleotide being operatively cloned within another ATU at a location distinct from the location of the first cloned heterologous polynucleotide, preferentially upstream the N gene of the MeV, said another ATU being in particular the ATU1 inserted upstream the N gene of the MeV, the second heterologous polynucleotide encoding in particular at least one polypeptide or antigenic fragment thereof different from at least one polypeptide encoded by the first heterologous polynucleotide.

3. The nucleic acid construct according to claim 1 or 2, wherein the heterologous polynucleotide(s) encoding the GPC protein, the NP protein, the mNP and/or the Z protein, or an antigenic fragment thereof, is(are) from the LASV strain Josiah, or is(are) derived from the LASV strain Josiah, in particular the heterologous polynucleotide(s) is(are) issued or derived from the sequence of Genbank J04324.1 and/or U73034.2.

4. The nucleic acid construct according to any one of claims 1 to 3, wherein the first and/or second heterologous polynucleotide(s) encode(s) the mNP protein, or an antigenic fragment thereof, wherein the mNP protein has a mutated exonuclease domain, in particular wherein the amino acid sequence of the encoded mNP protein is mutated on amino acid residue 389 and/or 392, preferentially by substitution on amino acid residues 389 and 392, of SEQ ID No: 3, in particular the encoded mNP protein is the sequence of SEQ ID No: 5, in particular the heterologous polynucleotide encoding the mNP protein comprises SEQ ID No: 6.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the heterologous polynucleotide(s) encoding the GPC protein, the NP protein, the mNP protein and/or the Z protein, or an antigenic fragment thereof, has (have) codon-optimized open reading frame(s) (ORF), in particular, the heterologous polynucleotide(s) comprise(s) at least one of the following sequence: SEQ ID No: 2 which encodes the GPC protein; and/or SEQ ID No: 4 which encodes the NP protein; and/or SEQ ID No: 6 which encodes the mNP protein; and/or SEQ ID No: 8 which encodes the Z protein.

6. The nucleic acid construct according to any one of claims 1 to 5, wherein the heterologous polynucleotide(s) encode(s): the GPC protein of SEQ ID No: 1 or an antigenic fragment thereof; and/or the NP protein of SEQ ID No: 3 or an antigenic fragment thereof; and/or the mNP protein of SEQ ID No: 5 or an antigenic fragment thereof; and/or the Z protein of SEQ ID No: 7 or an antigenic fragment thereof.

7. The nucleic acid construct according to any one of claims 1 to 6 comprising from 5 to 3 end the following polynucleotides: (a) a polynucleotide encoding the N protein of the MeV; (b) a polynucleotide encoding the P protein of the MeV; (c) the first heterologous polynucleotide encoding at least one polypeptide selected from the group consisting of the GPC protein, the NP protein, the mNP protein and the Z protein of the LASV, or an antigenic fragment thereof, in particular encoding a single polypeptide which is the GPC protein or an antigenic fragment thereof, or encoding at least two polypeptides, which are the GPC protein or an antigenic fragment thereof and either the NP protein or the mNP protein, or an antigenic fragment thereof, in particular encoding the GPC protein and the mNP protein, wherein the first polynucleotide is in particular operatively cloned within an ATU, in particular ATU2; (d) a polynucleotide encoding the M protein of the MeV; (e) a polynucleotide encoding the F protein of the MeV; (f) a polynucleotide encoding the H protein of the MeV; (g) a polynucleotide encoding the L protein of the MeV; and wherein said polynucleotides are operatively linked within the nucleic acid construct and under the control of a viral replication and transcriptional regulatory elements such as MeV leader and trailer sequence(s).

8. The nucleic acid construct according to any one of claims 2 to 6 comprising from 5 to 3 end the following polynucleotides: (a) the second heterologous polynucleotide encoding at least one polypeptide selected from the group consisting of the GPC protein, the NP protein, the mNP protein and the Z protein of the LASV, or an antigenic fragment thereof, in particular encoding the Z protein, or an antigenic fragment thereof, wherein the second polynucleotide is operatively cloned within an ATU localized upstream the N gene of the MeV, in particular within the ATU1; (b) a polynucleotide encoding the N protein of the MeV; (c) a polynucleotide encoding the P protein of the MeV; (d) the first heterologous polynucleotide encoding at least one polypeptide selected from the group consisting of the GPC protein, the NP protein, the mNP protein and the Z protein of the LASV, or an antigenic fragment thereof, in particular encoding a single polypeptide which is the GPC protein or an antigenic fragment thereof, or encoding at least two polypeptides, which are the GPC protein or an antigenic fragment thereof and either the NP protein or the mNP protein, or an antigenic fragment thereof, in particular encoding the GPC protein and the mNP protein, wherein the first polynucleotide is in particular operatively cloned within an ATU, in particular ATU2; (e) a polynucleotide encoding the M protein of the MeV; (f) a polynucleotide encoding the F protein of the MeV; (g) a polynucleotide encoding the H protein of the MeV; (h) a polynucleotide encoding the L protein of the MeV, and wherein said polynucleotides are operatively linked within the nucleic acid construct and under the control of a viral replication and transcriptional regulatory elements such as MeV leader and trailer sequence(s).

9. The nucleic acid construct according to any one of claims 1 to 8, wherein the first heterologous polynucleotide comprises from 5 to 3 end: (a) a nucleic acid encoding the NP protein or the mNP protein of the LASV, or an antigenic fragment thereof, in particular whose nucleic acid is SEQ ID No: 4 for the NP protein or SEQ ID No: 6 for the mNP protein; and (b) a nucleic acid encoding the GPC protein of the LASV, or an antigenic fragment thereof, in particular whose nucleic acid is SEQ ID No: 2; and wherein the first heterologous polynucleotide is operatively cloned between the P gene and the M gene of the MeV, in particular within an ATU, in particular ATU2.

10. The nucleic acid construct according to any one of claims 1 to 9, wherein the first heterologous polynucleotide comprises from 5 to 3 end: (a) the nucleic acid of SEQ ID No: 6 encoding the mNP protein; and (b) the nucleic acid of SEQ ID No: 2 encoding the GPC protein; and wherein the first heterologous polynucleotide sequence is operatively cloned between the gene P and the gene M of the MeV, in particular within an ATU, in particular ATU2.

11. The nucleic acid construct according to any one of claims 2 to 10, wherein the second heterologous polynucleotide encodes the Z protein of the LASV, or an antigenic fragment thereof, and wherein the first heterologous polynucleotide encodes the GPC protein of the LASV, or an antigenic fragment thereof, in particular wherein the sequence of the second heterologous polynucleotide comprises the sequence of SEQ ID No: 8 and the sequence of the first heterologous polynucleotide comprises the sequence of SEQ ID No: 2.

12. The nucleic acid construct according to claim 11, wherein the first heterologous polynucleotide and the second heterologous polynucleotide are operatively cloned within ATUs located at different position in the cDNA molecule of the MeV, in particular the first heterologous polynucleotide is operatively cloned with the ATU2 and the second heterologous polynucleotide is operatively cloned within the ATU1.

13. The nucleic acid construct according to any one of claims 1 to 12, wherein the measles virus is an attenuated virus strain selected from the group consisting of the Schwarz strain, the Zagreb strain, the AIK-C strain, the Moraten strain, the Philips strain, the Beckenham 4A strain, the Beckenham 16 strain, the Edmonston seed A strain, the Edmonston seed B strain, the CAM-70 strain, the TD 97 strain, the Leningrad-16 strain, the Shanghai 191 strain and the Belgrade strain, in particular the Schwarz strain.

14. The nucleic acid construct according to any one of claims 1 to 13, wherein the sequence of the first heterologous polynucleotide comprises at least one of SEQ ID No: 2, SEQ ID No: 4, SEQ ID No: 6, and/or SEQ ID No: 8, and preferentially at least SEQ ID No: 2, in particular SEQ ID No: 2 and SEQ ID No: 6.

15. The nucleic acid construct according to any one of claims 1 to 14 whose recombinant cDNA sequence is selected from the group consisting of: SEQ ID No: 9 (construct MeV-GPC); SEQ ID No: 10 (construct MeV-NP-GPC); SEQ ID No: 11 (construct MeV-mNP-GPC); SEQ ID No: 12 (construct Z-MeV-GPC); SEQ ID No: 13 (construct Z-MeV-NP-GPC); and SEQ ID No: 14 (construct Z-MeV-mNP-GPC).

16. A transfer plasmid vector comprising the nucleic acid construct according to any one of claims 1 to 15, in particular comprising or consisting of a sequence selected from the group consisting of: SEQ ID No: 9 (Plasm MeV-GPC); SEQ ID No: 10 (Plasm MeV-NP-GPC); SEQ ID No: 11 (Plasm MeV-mNP-GPC); SEQ ID No: 12 (Plasm Z-MeV-GPC); SEQ ID No: 13 (Plasm Z-MeV-NP-GPC); and SEQ ID No: 14 (Plasm Z-MeV-mNP-GPC).

17. A recombinant measles virus, said virus comprising in its genome a nucleic acid construct according to any one of claims 1 to 15, or a transfer plasmid vector according to claim 16, or whose genome consists of the transfer plasmid vector of claim 16.

18. The recombinant measles virus according to claim 17 expressing at least one polypeptide selected from the group consisting of the GPC protein, the NP protein, the mNP protein and the Z protein of the LASV, or an antigenic fragment thereof, in particular expressing at least two polypeptide selected from the group consisting of the GPC protein, the NP protein, the mNP protein and the Z protein of the LASV, or an antigenic fragment thereof.

19. The recombinant measles virus according to claim 17 or 18 expressing the GPC protein and the mNP protein of the LASV, or antigenic fragments thereof.

20. The recombinant measles virus according to claim 17 or 18 expressing the GPC protein and the Z protein of the LASV, or antigenic fragments thereof.

21. The recombinant measles virus according to any one of claims 17 to 20 furthermore expressing at least one of the N protein, the P protein, the M protein, the F protein, the H protein and the L protein of the MeV.

22. The recombinant measles virus according to any one of claims 17 to 21, which elicits a cellular and/or humoral and cellular response, in particular after a single immunization, against the antigenic fragment(s) of the GPC protein, the NP protein, the mNP protein and/or the Z protein of LASV, in particular against the antigenic fragments of the N protein, the P protein, the M protein, the F protein, the H protein and/or the L protein of MeV, in particular a T cell response, in particular a CD4+ and CD8+ T cell response.

23. A host cell transfected with the nucleic acid construct according to any one of claims 1 to 15 or with the transfer plasmid vector according to claim 16, or infected with the recombinant measles virus according to any one of claims 17 to 22, in particular a mammalian cell, a VERO NK cells, CEF cells, human embryonic kidney cell line 293 or MRC5 cells.

24. Recombinant virus like particles (VLPs) comprising a Z protein and optionally a GPC protein and/or a NP protein and/or a mNP protein, or an antigenic fragment thereof, of the LASV, in particular expressing at least the Z protein and the GPC protein, or an antigenic fragment thereof, wherein the protein(s) or antigenic fragments thereof is(are) encoded by the first and/or second heterologous polynucleotide(s) of the nucleic acid construct according to claims 1 to 15, or of the transfer plasmid vector of claim 16, or the recombinant measles virus according to any one of claims 17 to 22, or produced within the host cell of claim 23.

25. An immunogenic composition, especially a virus vaccine composition, comprising the recombinant VLPs according to claim 24, or the recombinant measles virus according to any one of claims 17 to 22, or the recombinant VLPs according to claim 24 and the recombinant measles virus according to any one of claims 17 to 22, and a pharmaceutically acceptable vehicle.

26. The composition according to claim 25 for use in the elicitation of a protective, and preferentially prophylactic, immune response against the Lassa virus by the elicitation of antibodies directed against LASV protein(s) or antigenic fragment(s) thereof, and/or a cellular and/or humoral and cellular response against the Lassa virus, in a host in need thereof, in particular a human host, in particular a child.

27. The composition of claim 26 for use in the elicitation of a protective, and preferentially prophylactic, immune response against measles virus by the elicitation of antibodies directed against measles virus protein(s), and/or a cellular and/or humoral and cellular response against the measles virus, in a host in need thereof, in particular a human host, in particular a child.

28. A process for rescuing recombinant Lassa virus like particles (VLPs) and/or recombinant measles virus expressing at least one of polypeptide selected from the group consisting of the GPC protein, the NP protein, the mNP protein and the Z protein of LASV, or an antigenic fragment thereof, comprising: (a) transfecting cells, in particular helper cells, in particular HEK293 helper cells, stably expressing T7 RNA polymerase and measles virus N and P proteins with the nucleic acid construct according to any one of claims 1 to 15 or with the transfer plasmid vector according to claim 16; (b) maintaining the transfected cells in conditions suitable for the production of recombinant measles virus and/or LASV VLPs; (c) infecting cells enabling propagation of the recombinant measles virus and/or the LASV VLPs by co-cultivating them with the transfected cells of step (b); (d) harvesting the recombinant measles virus expressing at least one of the GPC protein, the NP protein, the mNP protein and/or the Z protein of LASV, in particular expressing at least the GPC protein and another LASV protein, in particular expressing the GPC protein and the mNP protein, and/or the LASV VLPs expressing at least the Z protein and optionally at least one of the GPC protein, the NP protein and/or the mNP protein of LASV, in particular expressing the Z protein and the GPC protein.

29. A method for preventing a Lassa virus related disease, said method comprising the immunization of a mammalian, especially a human, in particular a child, by the injection, in particular by subcutaneous injection, of recombinant Lassa virus VLPs according to claim 24, and/or a measles virus according to any one of claims 17 to 22.

30. A method for treating a Lassa virus related disease, said method comprising the immunization of a mammalian, especially a human, in particular a child, by the injection, in particular subcutaneous injection, of recombinant Lassa virus VLPs according to claim 24, and/or a measles virus according to any one of claims 17 to 22.

Description

DESCRIPTION OF THE FIGURES

[0179] Some of the figures, to which the present application refers, are in color. The application as filed contains the color print-out of the figures, which can therefore be accessed by inspection of the file of the application at the patent office.

[0180] FIG. 1. Schematic representation of nucleic acid constructs. A: MeV vector. B: nucleic acid constructs according to the invention. MeV genes are indicated in grey and LASV genes are indicated in green, blue and red. For the MV genes: N (nucleoprotein); P/V/C (phosphoprotein and V/C proteins); M (matrix); F (Fusion protein); H (hemagglutinin): L (polymerase). For the LASV genes: NP (nucleoprotein); NP.sub.ExoN (also referenced NP.sub.KO on some figures; mutated sequence encoding a mutated NP with its exonuclease activity knocked down); GPC (glycoprotein precursor); Z (zinc-binding protein). ATUs are indicated by the black arrows. ATU1 is localized on the left, upstream the N gene of MeV while ATU2 is localized centrally, between P and M MeV genes.

[0181] FIG. 2. Growth kinetics of viruses on Vero E6 cells. MeV-GFP corresponds to a construct wherein a polynucleotide encoding a Green Fluorescent Protein has been inserted within ATU2. MeV-GPC.sub.LASV corresponds to a construct wherein a polynucleotide encoding the GPC protein of LASV has been inserted within ATU2. MeV-NP+GPC.sub.LASV corresponds to a construct wherein genes encoding the GPC protein and the NP protein of LASV has been inserted within ATU2. MeV-NP.sub.ExoN+GPC.sub.LASV corresponds to a construct wherein polynucleotide encoding the GPC protein and a mutated NP protein (exonuclease activity knocked down) of LASV has been inserted within ATU2. MeV-Z+GPC.sub.LASV corresponds to a construct wherein a polynucleotide encoding the GPC protein has been inserted within ATU2, and wherein a polynucleotide encoding the Z protein has been inserted within ATU1. Titers obtained in typical experiments, measured by TCID.sub.50 from 3 independent experiments. Means and standard errors are represented.

[0182] FIG. 3. Expression of LASV proteins (GPC, NP and Z) and MeV protein (F) in infected Vero E6 cells and in the supernatants of infected Vero E6 cells. The effect of each construct was assessed by Western blot as detailed in the material and method. NI: non-infected cells. ns: non specific.

[0183] FIG. 4. MeV-GFP entry and replication in immune antigen presenting cells derived from human peripheral blood mononuclear cells.

[0184] FIG. 5. Expression of type I IFN in human primary macrophages infected with different MeV-LASV vectors. Quantitative RNA analysis by qPCR analyses of type I IFN response (quantitative expression of IFNa1, IFNa2 and IFNb). Expression 24 h post-infection. All results are normalized to GAPDH gene and expressed as fold induction relative to GAPDH.

[0185] FIG. 6. Cell surface expression of cluster of differentiation markers CD80, CD86 and CD83 CD40 in macrophages infected with different MeV-LASV vectors. Flow cytometry for the cell surface expression of co-activation molecules 48 h post-infection.

[0186] FIG. 7. Expression of type I IFN in human primary dendritic cells infected with different MeV-LASV vectors. Quantitative RNA analysis by qPCR analyses of type I IFN responses (quantitative expression of IFNa1, IFNa2 and IFNb). Expression 24 h post-infection. All results are normalized to GAPDH gene and expressed as fold induction relative to GAPDH.

[0187] FIG. 8. Cell surface expression of cluster of differentiation markers CD80, CD86, CD83 and CD40 in human primary dendritic cells infected with different MeV-LASV vectors. Flow cytometry for the cell surface expression of co-activation molecules 48 h post-infection.

[0188] FIG. 9. Body temperature in cynomolgus monkeys (Macaca fascicularis) during a 30-day period post immunization. 3, 4 and 4 monkeys were subcutaneously immunized with 2.10.sup.6 Tissue culture Infective Dose 50 (TCID.sub.50) of respectively a recombinant MeV strain Schwarz vaccine, a recombinant MeV-NP.sub.ExoN-GPC vaccine and a recombinant Z-MeV-GPC vaccine.

[0189] FIG. 10. LASV antigens-specific CD4 and CD8 T cells responses in vaccinated cynomolgus monkeys (Macaca fascicularis). Flow cytometry after stimulation of whole blood by overlapping peptides specific to GPC, NP and Z.

[0190] FIG. 11. Clinical scores in cynomolgus monkeys (Macaca fascicularis) after challenge with a lethal dose of LASV strain Josiah. Clinical score is based on body temperature, body weight, capacity to feed and hydrate normally, behavior, clinical signs. A score of 15 is the endpoint for killing. Lethal dose of LASV strain consists in 1.500 FFU of LASV strain Josiah subcutaneously injected to the animals.

[0191] FIG. 12. Body temperature in cynomolgus monkeys (Macaca fascicularis) challenged with a lethal dose of LASV strain Josiah.

[0192] FIG. 13. Liver enzymes (AST and ALT) levels in plasma of immunized cynomolgus monkeys.

[0193] FIG. 14. Plasma level of LDH (A), CRP (B) and albumin (C) in cynomolgus monkeys (Macaca fascicularis) challenged with a lethal dose of LASV strain Josiah.

[0194] FIG. 15. Viremia (RNA(A) and Titer (B)) in cynomolgus monkeys challenged with a lethal dose of LASV strain Josiah. RNA quantification by qPCR. Titration according to known method in the art.

[0195] FIG. 16. Viral RNA quantification in the nasal (A) and oral secretions (B) and in the urine (C) of cynomolgus monkeys challenged with a lethal dose of LASV strain Josiah. RNA quantification by qPCR.

[0196] FIG. 17. LASV RNA levels detected in organs of challenged cynomolgus monkeys previously immunized with different MeV-LASV.

[0197] FIG. 18. LASV infectious titers detected in organs of challenged cynomolgus monkeys previously immunized with different MeV-LASV.

[0198] FIG. 19. IgM and IgG responses against LASV in cynomolgus monkeys challenged with a lethal dose of LASV strain Josiah. A. IgM LASV specific. B: IgG LASV specific. Immunoglobulin levels measured by ELISA. Optical density calculated according to the absorbance at 450 nM.

[0199] FIG. 20. LASV GP- and NP-specific CD8+ and CD4+ T cell responses after immunization. The percentage of CD8+ and CD4+ T cells that produced IFNg, TNFa and/or IL-2 after stimulation with overlapping peptides covering the whole LASV GP and NP have been determined using flow cytometry.

[0200] FIG. 21. LASV antigens-specific CD4 and CD8 T cell responses of immunized cynomolgus monkeys challenged with a lethal dose of LASV strain Josiah. Flow cytometry after stimulation of whole blood by overlapping peptides specific to GPC and NP.

[0201] FIG. 22. Proliferation and activation of CD4 and CD8 T cells in of cynomolgus monkeys challenged with a lethal dose of LASV strain Josiah. CD8 proliferation assessed by Ki67 staining (A). CD4 (B) and CD8 (C) Activation assessed by quantification of granzyme B expression.

[0202] FIG. 23. LASV GP- and NP-specific CD8+ T cell responses after LASV challenge. The percentage of CD8+ T cells that produced IFNg, TNFa and/or IL-2 after LASV challenge with overlapping peptides covering the whole LASV GP (23A) and NP (23B) have been determined with flow cytometry. The proportion of different sub-populations of responding T cells is presented using pie chart.

[0203] FIG. 24. LASV GP- and NP-specific CD4+ T cell responses after LASV challenge. The percentage of CD4+ T cells that produced IFNg, TNFa and/or IL-2 after LASV challenge with overlapping peptides covering the whole LASV GP (23A) and NP (23B) have been determined with flow cytometry. The proportion of different sub-populations of responding T cells is presented using pie chart.

[0204] FIG. 25. KEGG pathway analysis performed on the transcriptomic data obtained from cynomolgus monkeys PBMC collected at different time points post-immunization with MeV-NP.sub.ExoN-GPC.sub.LASV.

[0205] FIG. 26. KEGG pathway analysis performed on the transcriptomic data obtained from cynomolgus monkeys PBMC collected at different time points post-immunization with MeV-Z+GPC.sub.LASV.

[0206] FIG. 27. Quantification of cytokines in the plasma of immunized monkeys after LASV challenge. Different cytokines have been quantified in the plasma of MeV-, MeV-NP.sub.ExoN-GPC.sub.LASV, and MeV-Z+GPC.sub.LASV immunized cynomolgus monkeys after LASV challenge. Significant differences (p<0.05) between different conditions are indicated: n-c (MeV-NP.sub.ExoN-GPC.sub.LASV and MeV), n-z (MeV-NP.sub.ExoN-GPC.sub.LASV and MeV-Z+GPC.sub.LASV) and n-cz (MeV-NP.sub.ExoN-GPC.sub.LASV and MeV; MeV-NP.sub.ExoN-GPC.sub.LASV and MeV-Z+GPC.sub.LASV).

[0207] FIG. 28. IgM and IgG responses against MeV in cynomolgus monkeys challenged with a lethal dose of LASV strain Josiah. A: IgM MeV specific. B: IgG MeV specific. IgG and IgM MeV-specific were not measures at days 7 and 14 for the monkeys immunized with the MeV construct.

[0208] FIG. 29. Determination of the Exonuclease activity of native and mutated NP protein. Fold induction of Luciferase activity virus-induced and immunostimulatory RNAs-induced interferon-beta activation. CT: control. NP.sub.LASV: native NP protein. NP.sub.ExoNLASV: mutated NP protein of SEQ ID No: 5. SeV: Sendai virus at moi=1.

[0209] FIG. 30. Analysis of MeV-NP.sub.ExoN-GPC.sub.LASV tropism. CHO cell lines were infected with either a Mopeia virus pseudotyped with LASV GPC or with MeV-NP.sub.ExoN-GPC.sub.LASV. Expression of GPC was analyzed by staining with an anti-GP1 antibody. The nuclei are in blue, while the anti-GP1 stained is in green.

[0210] FIG. 31. Schematic representation of transfer vector plasmid according to a first embodiment of the invention. The transfer vector has the sequence of SEQ ID No: 9. The measles gene encoding the N protein is localized between nucleotides 189 and 1767. The measles gene encoding the P protein is localized between nucleotides 1889 and 3412. The codon-optimized heterologous polynucleotide of SEQ ID No: 2 encoding the GPC is localized between nucleotides 3532 and 5007. ATU2 is localized between nucleotides 3487 and 5071 minus the heterologous polynucleotide insert. The measles gene encoding the M protein is localized between nucleotides 5104 and 6111.

[0211] FIG. 32. Schematic representation of transfer vector plasmid according to a second embodiment of the invention. The transfer vector has the sequence of SEQ ID No: 10. The measles gene encoding the N protein is localized between nucleotides 190 and 1767. The measles gene encoding the P protein is localized between nucleotides 1889 and 3412. The codon-optimized heterologous polynucleotide of SEQ ID No: 4 encoding the NP protein is localized between nucleotides 3532 and 5241. The codon-optimized heterologous polynucleotide of SEQ ID No: 2 encoding the GPC is localized between nucleotides 5386 and 6861. A linker sequence comprising regulatory sequence of the measles virus is localized between nucleotides 5242 and 5385. ATU2 is localized between nucleotides 3487 and 6925 minus the heterologous polynucleotide insert and the linker sequence. The measles gene encoding the M protein is localized between nucleotides 6958 and 7965.

[0212] FIG. 33. Schematic representation of transfer vector plasmid according to a third embodiment of the invention. The transfer vector has the sequence of SEQ ID No: 11. The measles gene encoding the N protein is localized between nucleotides between 190 and 1767. The measles gene encoding the P protein is localized between nucleotides 1889 and 3412. The codon-optimized heterologous polynucleotide of SEQ ID No: 6 encoding the mutated NP protein is localized between nucleotides 3532 and 5241. The codon-optimized heterologous polynucleotide of SEQ ID No: 2 encoding the GPC is localized between nucleotides 5386 and 6861. A linker sequence comprising regulatory sequence of the measles virus is localized between nucleotides 5242 and 5385. ATU2 is localized between nucleotides 3487 and 6925 minus the heterologous polynucleotide insert and the linker sequence. The measles gene encoding the M protein is localized between nucleotides 6958 and 7965.

[0213] FIG. 34. Schematic representation of transfer vector plasmid according to a fourth embodiment of the invention. The transfer vector has the sequence of SEQ ID No: 12. The codon-optimized heterologous polynucleotide of SEQ ID No: 8 encoding the Z protein is localized between nucleotides 193 and 504. The measles gene encoding the N protein is localized between nucleotides 646 and 2223. The measles gene encoding the P protein is localized between nucleotides 2345 and 3868. The codon-optimized heterologous polynucleotide of SEQ ID No: 2 encoding the GPC is localized between nucleotides 3988 and 5463. The measles gene encoding the M protein is localized between nucleotides 5560 and 6567.

[0214] FIG. 35. Schematic representation of transfer vector plasmid according to a fifth embodiment of the invention. The transfer vector has the sequence of SEQ ID No: 13. The codon-optimized heterologous polynucleotide of SEQ ID No: 8 encoding the Z protein is localized between nucleotides 193 and 504. The measles gene encoding the N protein is localized between nucleotides 646 and 2223. The measles gene encoding the P protein is localized between nucleotides 2345 and 3868. The codon-optimized heterologous polynucleotide of SEQ ID No: 4 encoding the NP protein is localized between nucleotides 3988 and 5697. The codon-optimized heterologous polynucleotide of SEQ ID No: 2 encoding the GPC is localized between nucleotides 5842 and 7317 The measles gene encoding the M protein is localized between nucleotides 7414 and 8421.

[0215] FIG. 36. Schematic representation of transfer vector plasmid according to a sixth embodiment of the invention. The transfer vector has the sequence of SEQ ID No: 14. The codon-optimized heterologous polynucleotide of SEQ ID No: 8 encoding the Z protein is localized between nucleotides 193 and 504. The measles gene encoding the N protein is localized between nucleotides 646 and 2223. The measles gene encoding the P protein is localized between nucleotides 2345 and 3868. The codon-optimized heterologous polynucleotide of SEQ ID No: 6 encoding the mutated NP protein is localized between nucleotides 3988 and 5697. The codon-optimized heterologous polynucleotide of SEQ ID No: 2 encoding the GPC is localized between nucleotides 5842 and 7317 The measles gene encoding the M protein is localized between nucleotides 7414 and 8421.

EXAMPLES

[0216] Materials and Methods

[0217] Cells and Viruses

[0218] 293T7/N/P cells expressing stably the T7 polymerase and the measles N and P proteins were used to rescue recombinant measles viruses and were maintained as described before {Combredet, 2003 #76}. Vero NK cells were grown in Glutamax Dulbecco Modified Eagle's Medium (DMEM, Life Technologies) supplemented with 5% FCS, and 0.5% Penicillin-Streptomycin. Blood samples were obtained from the Etablissement Frangais du Sang (EFS, Lyon, France). Mononuclear cells were purified by Ficoll density gradient centrifugation (GE Healthcare). Monocytes were first separated from peripheral blood mononuclear cells by centrifugation on a cushion of 50% Percoll (GE Healthcare, Velizy, France) in PBS and then purified using the Monocyte isolation kit II according to the manufacturer's instructions (Miltenyi Biotec, Paris, France). Macrophages were obtained by incubating monocytes for 6 days in RPMI, 10% SVF, 10% autologous serum supplemented with 50 ng/mL of M-CSF. M-CSF was added every 2 days and 40% of the culture medium was replaced.

[0219] Plasmid Constructs

[0220] Codon-optimized ORF of LASV GPC, NP and Z (LASV strain Josiah) were cloned in the pTM1-MVSchwarz vector in additional transcriptional units (ATU) placed upstream of Nucleoprotein (N) (ATU1 for Z) or between the phosphoprotein (P) and the matrix (M) genes of the Schwarz MV genome (ATU2, GPC alone or NP+GPC) like previously described (Combredet, C., et al., A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. J Virol, 2003. 77(21): p. 11546-54). All plasmid constructs were verified by sequencing.

[0221] Western Blot and Antibodies

[0222] Vero NK cells infected with recombinant MeV-GFP, MeV-GPC.sub.LASV, MeV-NP+GPC.sub.LASV, MeV-NP.sub.ExoN+GPC.sub.LASV or MeV-Z+GPC.sub.LASV were lysed in Co-IP buffer and cleared by centrifugation. Lysates and culture supernatants were then separated on 4-12% precast gels (Biorad) under denaturing conditions and transferred to PVDF membrane. Membranes were stained with primary antibodies to GP1 (in house mouse monoclonal production), NP (mouse anti-LASV serum), Z (in house rabbit polyclonal production) or F (rabbit polyclonal Fcyt, a kind gift of R. Cattaneo). Cell lysates were also stained with an anti-actin antibody coupled to the horseradish peroxidase (HRP). After staining with secondary antibodies coupled to HRP, membranes were revealed using West Dura substrate (Pierce) and photographed using a LAS4000 imager (GE Healthcare).

[0223] Virus Rescue and Titration

[0224] Recombinant measles viruses expressing LASV antigens were rescued as previously described (Combredet, 2003; Radecke, F., et al., Rescue of measles viruses from cloned DNA. Embo J, 1995. 14(23): p. 5773-84; WO2008/078198). Briefly, 293T7/N/P cells were transfected with plasmids encoding the measles L polymerase and the antigenomic segment of the desired MeV vector. Clonal syncytia were picked and used to infect Vero NK cells in 6-well plates. When syncytia has reached about 50% of the well superficy, cells were detached and overlaid on Vero NK cells in 10 cm dishes to produce passage 1 (P1) stocks. To prepare higher passage virus stocks, Vero NK cells were infected at a multiplicity of infection (MOI) of 0.01 and then incubated at 32 C. for 2 to 3 days. To harvest virus, cells were scraped into Opti-MEM I reduced-serum medium and freeze-thawed twice. Titers were determined by 50% tissue culture infective dose (TCID50) titration on Vero NK cells.

[0225] Quantitative RNA Analysis

[0226] For RT-qPCR experiments, total RNA was isolated from mock or infected cells using the Rneasy Mini Kit (Qiagen, Courtaboeuf, France), according to the manufacturer's instructions, and a supplementary DNase step added using the Turbo DNA free kit Ambion (Thermo Fisher Scientific). Synthesis of cDNA was performed using SuperScript III and amplification was performed using the Gene Expression Master Mix kit (Applied Biosystems, Thermo Fisher Scientific). For type I IFN, the primer/probe mix was developed in house. Runs of qPCR assays were performed in a LightCycler 480 (Roche Diagnostics, Meylan, France). The expression of all genes was standardized to that of the GAPDH gene, and expressed as fold induction relative to GAPDH. For viral RNA quantification, an RNA probe of the 771-934 pb region of the NP ORF was cloned into the pGEM vector (Promega) to generate T7 polymerase driven transcripts. The RNA probe was DNAse treated, purified, and quantified (Dropsense96, Trinean, Gent, Belgium). Quantitative PCR for viral RNA was performed with the EuroBioGreen qPCR Mix Lo-ROX (Eurobio, Les Ulis, France), using LASV specific primers.

[0227] Flow Cytometry for MP Activation, T Cell Activation and Proliferation

[0228] Mock and MOI 1-infected MP were detached 48 h after infection, saturated with human IgG and surface stained with antibodies to CD40, CD83, CD80, and CD86 (BD Biosciences, Le-Pont-de-Claix, France) before final fixation in PBS/1% PFA. LASV antigen-specific T cells were analysed from fresh whole blood. Cells were incubated with a pool of GPC, NP or Z overlapping peptides in the presence of CD28 and CD49d antibodies (2 g/ml) and Brefeldin A (10 g/ml) for 6 h at 37 C. SEA (1 g/ml) or PBS were respectively used as positive or negative control of activation. Peptides are 15-mer amino acids long (1 g/ml each) with an overlap of 11 residues and spanned the complete GPC, NP or Z ORF of LASV strain Josiah. PBS-EDTA 20 mM was added to samples before cell-surface staining for CD3, CD4 and CD8 (BD Biosciences). Red blood cells were then lysed using PharmLyse (BD Biosciences). Cells were then fixed and permeabilized for intracellular staining with antibodies to IFN (Biolegend). For proliferation and activation, wells were stained using antibodies to Ki67 or Granzyme B. Cells were analysed by flow cytometry using an LSR Fortessa cytometer (BD Biosciences) or a 10-color Gallios cytometer (Beckman Coulter). Data were analysed using Kaluza software (Beckman Coulter).

[0229] Cynomolgus Monkey Challenge with LASV

[0230] Groups of 4 male cynomolgus monkeys (Macaca Fascicularis, 32-39 month-old, 3-4 kg) were immunized in A2 facilities (SILABE, France) by subcutaneous injection of 2.106 TCID50 of MeV-NP.sub.ExoN+GPC.sub.LASV or MeV-Z+GPC.sub.LASV, respectively. Another control group of 3 monkeys was immunized with the MeV vaccine strain Schwarz. Blood draws, oral and nasal swabs and urine sampling were performed every 2-3 days during the first two weeks then once a week up to day 37 in order to assess vaccine replication and shedding, IgM and IgG responses and T cell responses against LASV GPC, NP or Z. After 37 days, monkeys were transported to BSL-4 facilities (Laboratoire P4-Inserm Jean Merieux) where they were challenged subcutaneously using 1,500 FFU of LASV strain Josiah. Animals were followed for clinical signs of the disease and were euthanized according to scoring made based on body temperature, body weight, feeding, hydrating, behaviour and clinical signs. Experimentation endpoint was placed at day 28 post challenge and all animals that had survived to this point were euthanized according to validated experimental procedures. Blood draws, oral and nasal swabs and urine sampling were performed every 2-3 days during the first two weeks then once a week up to day 28 in order to assess LASV virus replication and shedding, IgM and IgG responses and T cell responses against LASV GPC, NP or Z. This study has been approved by the Comite Regional d'Ethique en Matiere d'Experimentation Animale de Strasbourg (APAFIS #6543-20160826144775) and by the Comite Regional d'Ethique pour l'Experimentation Animale Rhone Alpes (CECCAPP 20161110143954).

[0231] Determination of the Exonuclease Activity of Native and Mutated NP Protein (FIG. 29):

[0232] 293T cells were cotransfected using calcium phosphate with 100 ng of a vector that expresses the firefly luciferase (Fluc) reporter gene from a known functional promoter sequence of the IFN-beta gene (pIFNbeta-LUC), variable amounts of either native (wild type) or mutant LASV NP vectors, and 50 ng of a 1-gal-expressing plasmid for transfection normalization. At 24 h post-transfection, cells were infected with Sendai virus (at moi=1) in order to induce IFN- expression. At 24 hpi, cell lysates were prepared for luciferase and 1-gal assays. Fluc activities were normalized by the 1-gal values. To determine whether NP has an exonuclease activity or not, its effect on the suppression of the immunostimulatory RNAs-induced IFN production is analysed, HEK293 cells were transfected with pIFNbeta-LUC, variable amounts of either native (WT) or mutant LASV NP vectors, and a beta-gal-expressing plasmid for transfection normalization. 18 h later, cells were transfected with either 1 g of Poly(I:C) or 250 ng of Pichinde virion RNAs by lipofectamine 2000. Luciferase activity was determined at 18 h after the immunostimulatory RNA transfection and normalized by the beta-gal activity. A mutated NP protein with its exonuclease activity knocked down does not suppress the immunostimulatory RNAs-induced IFN production.

Example 1Generation of Recombinant MeV Viruses Expressing LASV Antigens

[0233] In order to determine what is the best combination of LASV antigens to introduce in the MeV vector to get the best immunogenicity, we have generated several MeV/LASV vaccine candidates using the Schwarz MeV vaccine platform (FIG. 1A) expressing GPC alone or in combination with NP (mutated or not in the exonuclease domain to abrogate the immunosuppressive function contained in the LASV NP) or GPC and Z in order to produce antigenic LASV virus-like particles (VLPs) in vivo. We also made constructs expressing Z, GPC and NP mutated or not in the exonuclease domain. The GPC and NP genes were cloned between the MeV P and M genes in the additional transcriptional unit 2 (ATU2).

[0234] The Z gene was cloned upstream of the N gene in the ATU1 (FIG. 1B). All viruses were rescued and grew to similar titers on Vero E6 cells than a control MeV-GFP expressing GFP from ATU2 (FIG. 2), except for the MeV/LASV-Z+GPC that was attenuated by about a log compared to other constructs. Expression of the different LASV antigens was controlled by western blot using specific antibodies against LASV GPC, NP or Z, or against the measles fusion protein F (FIG. 3). Expression of GPC was detected in MeV-GPC.sub.LASV, MeV-NP+GPC.sub.LASV, MeV-NP.sub.ExoN+GPC.sub.LASV and MeV-Z+GPC.sub.LASV infected Vero E6 cells; expression of NP in MeV-NP+GPC.sub.LASV, MeV-NP.sub.ExoN+GPC.sub.LASV and MeV-Z+GPC.sub.LASV infected Vero E6 cells; expression of Z only in MeV-Z+GPC.sub.LASV infected cells. MeV F expression was detected in all MeV infected cells. As expected, GPC was also detected in the supernatants of MeV-Z+GPC.sub.LASV infected cells along with Z, supporting the release of GPC along with Z under the form of VLPs. All vectors were passaged 10 times without loss of LASV antigens expression.

Example 2Immunogenicity of MeV Viruses Expressing LASV Antigens in Human Primary Antigen Presenting Cells

[0235] To characterize the immunogenic properties of the different MeV vectors in human immune cells, we infected monocyte derived macrophages and dendritic cells. MeV enters and replicate in these cells, as shown by the expression of GFP on FIG. 4. However, viruses did not replicate efficiently in these cells as infectious titers were barely detectable at day 1 post infection and did not increase over time, likely due to the induction of an antiviral innate response.

[0236] We analysed the immune responses of macrophages and dendritic cells to the different recombinant viruses by combining flow cytometry analyses of activation markers and qPCR analyses of the type 1 IFN response. We analysed the type I IFN responses induced by the different vectors by qPCR at 24 hrs post infection (FIG. 5). In macrophages, MeV-GPC.sub.LASV and MeV-Z+GPC.sub.LASV induced the same levels of IFN alpha-1, alpha-2 and beta than the control MeV-GFP. However, addition of the LASV NP reduced by almost 3 logs the induction of type I IFN (MeV-NP+GPC.sub.LASV) but mutation of the ExoN domain of LASV NP (MeV-NP.sub.ExoN+GPC.sub.LASV) restored the induction of type I IFN to levels comparable to MeV-GFP and -GPC.sub.LASV. This results demonstrate that the LASV NP can control the induction of type I IFN through its ExoN activity, likely by digesting dsRNA molecules expressed during MeV replication (Son, 2015). We then looked at the induction of co-activation molecules by the different vectors at 48 hrs post infection in macrophages (FIG. 6). Importantly, cell surface expression of co-activation molecules is critical to activate the T cell responses. As shown on FIG. 6, all vectors induced strong cell surface expression of CD80, CD86 and CD83. Notably, expression was reduced in MeV-NP+GPC.sub.LASV infected macrophages compared the expression in macrophages infected by the other vectors but was restored when the ExoN domain of LASV NP was mutated.

[0237] Similar experiments were performed on dendritic cells (FIGS. 7 and 8). As observed in macrophages, MeV-GPC.sub.LASV and MeV-Z+GPC.sub.LASV induced the same levels of IFN alpha-1, alpha-2 and beta than the control MeV-GFP (FIG. 7). Addition of LASV NP also reduced the induction of type I IFN, but mutation of the ExoN domain of LASV NP restored the induction of type I IFN to levels comparable to MeV-GFP, MeV-Z+GPC.sub.LASV and MeV-GPC.sub.LASV. The expression of the co-activation molecules CD80, CD86, CD83 and CD40 were also similarly induced in dendritic cells infected with MeV-GFP, MeV-Z+GPC.sub.LASV and MeV-GPC.sub.LASV (FIG. 8).

[0238] The vaccine strains of MeV-LASV induce a type I IFN response and cell surface expression of co-activation molecules; the presence of wild type NP strongly reduces the ability of the vaccine to induce these effects, but mutation within the ExoN domain restore the ability of the vaccine to induce these effects.

Example 3Safety, Immunogenicity and Efficacy of Two Vaccines in Cynomolgus Monkeys

[0239] Based on the results obtained in human macrophages, we decided to test two vaccine candidates in cynomolgus monkeys, the gold standard model to study LASV pathogenesis. Three control animals were immunized subcutaneously with 2.106 TCID50 of a recombinant MeV strain Schwarz vaccine and two groups of 4 animals were immunized subcutaneously with 2.106 TCID50 of MeV-NP.sub.ExoN+GPC.sub.LASV and MeV-Z+GPC.sub.LASV, respectively. The health of the animals was then followed for 37 days post immunization (body temperature, body weight, respiratory rate) and no adverse effects were noted. Notably, the body temperature of the animals, continuously monitored thanks to intraperitoneal devices, was not altered by the immunization (FIG. 9).

[0240] We also assessed the viremia in immunized animals every 2-3 days during the two weeks following immunization then once a week and could not detect any trace of viral RNA, neither in plasma nor among PBMC. Similarly, we could not detect any viral RNA in the nasal and oral secretions or in the urine of vaccinated animals. Thus, it appears that the vaccine candidates are safe in monkeys and are not shed at any moment post immunization.

[0241] In order to assess the immunogenicity of the vectors, we performed ELISA to detect LASV-specific IgM and IgG. We could not detect specific IgM and IgG in MeV-Z+GPC.sub.LASV immunized animals and we only detected low levels of LASV-specific IgG in 3 out of 4 MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals at day 37 post immunization. In addition, one MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animal had neutralizing antibodies as demonstrated by plaque reduction neutralization assay (1:100 titer). We also assessed the LASV-specific T cell responses by flow cytometry after stimulation of whole blood by overlapping peptides specific to GPC, NP or Z. In MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals, we detected both CD4 and CD8 T cell responses against GP starting at day 7 and decreasing by day 14 post immunization (FIGS. 10A and 10C, orange bars). In these animals, we also detected both CD4 and CD8 T cell responses against NP between days 10 and day 21 post immunization (FIGS. 10B and 10D, orange bars). In MeV-Z+GPC.sub.LASV immunized animals, the CD4 T cell response against GPC was delayed compared to MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals, starting by day 10 post immunization but lasting until day 21 post inoculation (FIG. 10A, green bars). GPC-specific CD8 T cell responses started at day 7 at low levels but peaked at day 21 post immunization (FIG. 10C, green bars). Both CD4 and CD8 Z-specific T cell responses were also detected starting at day 7 and until day 30, with a peak at day 21 post immunization (FIGS. 10B and 10D, green bars). Importantly, no T cell responses against LASV antigens were detected in MeV-vaccinated control animals. Thus, both vaccine candidates induce LASV antigen-specific T cell responses, with MeV-NP.sub.ExoN+GPC.sub.LASV inducing earlier responses than MeV-Z+GPC.sub.LASV.

Example 4Efficacy of the Vaccines

[0242] In order to test the efficacy of the vaccine candidates, immunized animals were challenged 37 days post immunization with a lethal dose (1,500 ffu, subcutaneous) of LASV strain Josiah. Animals were then monitored for up to 30 days and were attributed clinical scores based on their body temperature, body weight, capacity to feed and hydrate normally, behaviour, clinical signs, with a score of 15 being the endpoint for killing. The three control animals had scores increasing from day 3 and had to be euthanized respectively at day 12, 14 and 15 post challenge (FIG. 11A).

[0243] On the contrary, all vaccinated animals survived the LASV infection but the clinical outcomes were different depending on the vaccine. Indeed, MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals had a small increase in clinical score by day 5 (max score of 3, FIG. 11B), mainly due to an elevated temperature (see FIG. 12, center graph). On the contrary, MeV-Z+GPC.sub.LASV immunized animals experienced severe symptoms such as high fever between day 3 and day 12 (see FIG. 12, lower graph) and while 2 animals totally recovered by day 12, 2 other animals had difficulties feeding and hydrating, showed prostration and one animal had balance issues and lost more than 7.5% of his body weight by day 30, reaching a score of 14 (FIG. 11C).

[0244] We also followed several biological parameters in plasma over the course of the infection, such as liver enzymes levels (ALT and AST), lactate deshydrogenase (LDH), C-reactive protein (CRP) and albumin, among other parameters. In control animals, the levels of liver enzymes were increasing continuously starting at day 6 and until the death of the animals (FIG. 13, left panels). On the contrary, liver enzyme levels remained normal at any time in MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals (FIG. 13, center panels) and only slightly increased in MeV-Z+GPC.sub.LASV immunized animals between day 6 and 15 (FIG. 13, right panels).

[0245] The plasma levels of LDH are a marker of tissue damages. In control animals, LDH levels started to increase at day 6 post challenge and thus till the end of the animals (FIG. 14A, left panel). No increase was noted in MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals over the course of the infection (FIG. 14A, center panel). However, LDH levels were elevated in MeV-Z+GPC.sub.LASV immunized animals between day 6 and day 15, especially for two animals having LDH values similar to the control animals at day 9 (FIG. 14A, right panel), suggesting some tissular damage in these animals. The levels of CRP, a marker of inflammation, also rapidly increased in control animals until death (FIG. 14B, left panel). CRP values remained low in the MeV-NP.sub.ExoN+GPC.sub.LASV group except for one animal that showed a transient increase in CRP levels by day 6 (FIG. 14B, center panel). On the contrary, all the animals from the MeV-Z+GPC.sub.LASV group had increased CRP levels between day 3 and day 15 (FIG. 14B, right panel). In this group, one animal showed a second wave of CRP synthesis between day 15 and day 30, suggesting that LASV virus was still replicating in this animal (FIG. 14B, right panel, light green). We also followed the plasma levels of albumin, a marker of kidney and liver dysfunction, in immunized monkeys. On control animals, albumin levels constantly decreased starting at day 3 (FIG. 14C, left panel) while these levels remained steady in MeV-NP.sub.ExoN+GPC.sub.LASV immunized animals (FIG. 14C, center panel). In the MeV-Z+GPC.sub.LASV group, all animals experienced a decrease in albumin plasma levels between day 3 and day 12 but these levels eventually went back to normal by day 15 (FIG. 14C, right panel).

[0246] The viremia in challenged animals was also monitored after challenge by both qRT-PCR and titration. As shown on FIG. 15A, the levels of RNA levels in the blood of the infected controls increased relentlessly from day 3 to the day of killing, peaking at 109 RNA copies per mL at day 15 for one animal (left panel). Infectious titers were also detected in these animals (FIG. 15B, left panel). In MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals, viral RNA was only detected at day 6 post challenge and at low levels compared to control animals (FIG. 15A, center panel) and no associated viremia was detected (FIG. 15B, center panel). The levels of viral RNA were higher in MeV-Z+GPC.sub.LASV immunized animals, peaking at day 6 around 106 RNA copies per mL and decreasing until day 15 (FIG. 15A, right panel). Notably, one animal had a rebound in the number of RNA copies in the blood by day 15 (FIG. 15A, right panel, light green), which can be correlated with the rebound in CRP observed in the same animal (FIG. 14B, right panel). In addition, infectious titers were detected in all animals at day 6 and up to day 15 for the animal showing prolonged RNA levels in the blood (FIG. 15B, right panel).

[0247] In addition to viremia, we assessed the presence of viral RNA in nasal and oral swabs of challenged animals. As shown on FIG. 16, levels of viral RNA peaked at day 9 in the nasal and oral secretions of control animals (FIGS. 16A and B, left panels) and decreased but were still detectable at the time of death. Similarly, levels of viral RNA peaked at day 9 in these secretions for MeV-Z+GPC.sub.LASV immunized animals, with a rebound for one or two animals by day 15 (FIGS. 16A and B, right panels). On the contrary, only small amount of LASV RNA was detected in the nasal swab of one animal at day 3 and another animal at day 6 (FIGS. 16A and B, center panels) and was not associated with the presence of infectious virus (data not shown). In addition, we followed shedding of viral RNA in the urine of challenged animals. LASV RNA levels were only detected in control and MeV-Z+GPC.sub.LASV immunized animals, respectively starting at day 9 or day 15 post challenge (FIG. 16C).

[0248] The amount of LASV RNA (FIG. 17), as well as the LASV infectious titers (FIG. 18), have been analysed in different organs collected at the time of necropsy of animals immunized with MeV, MeV-NP.sub.ExoN-GPC.sub.LASV and MeV-Z-GPC.sub.LASV. All MeV control animals presented a detectable amount of LASV RNA and a high virus titer in each organ tested, except in the bladder wherein only a single animal presented a detectable amount of LASV RNA. Highest infectious titers were found in the spleen, the liver and the lung. In all animals immunized with MeV-Z+GPC.sub.LASV, detectable amount of LASV RNA has been found in inguinal lymph node, mesenteric lymph and spleen, and detectable amount of LASV RNA has been detected in all organs, but never in a single animal, within this group. Two animals presented infectious titers of Lassa virus in the inguinal lymph node, while one animal presented infectious titer of Lassa virus in the spleen, but the other organs were free of infectious virus. In the group of animals immunized with MeV-NP.sub.ExoN+GPC.sub.LASV, detectable amount of LASV RNA was found in the lymphoid organs and in the lung of one to three animals, but the presence of RNA was not associated with the presence of infectious Lassa virus.

Example 5Immune Response to LASV

[0249] In order to determine the immune response to infection, we first measured the levels of LASV-specific immunoglobulins produced after the challenge with LASV. The IgM response started at day 9 in all animals, and peaked at day 12 (FIG. 19A). Interestingly, IgM levels were higher at day 12 in animals from the control group and from the MeV-Z+GPC.sub.LASV group, suggesting that the level IgM response does not positively correlate with the protection but rather with the viral load. In terms of IgG responses, we noted a strong induction of LASV-IgG in all MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals by day 9, while the IgG response in MeV-Z+GPC.sub.LASV immunized animals only reached similar levels at day 15 (FIG. 19B). The levels of LASV-specific IgG in control animals remained very low at any time point. In addition, the seroneutralisation titers have been determined in the plasma of immunized monkeys with MeV-NP.sub.ExoN-GPC.sub.LASV, MeV-Z+GPC.sub.LASV and MeV at different time points post-immunization (results are illustrated in table 1). At the time of challenge (i.e. 37 days post-immunization, J0 in table 1), at least one animal per group had a neutralization titer of 1/100e. Fifteen days post-challenge, and until the day of necropsy, all animal vaccinated with MeV-NP.sub.ExoN-GPC.sub.LASV and MeV-Z+GPC.sub.LASV had neutralizing antibodies between 1/100e and 1/500e, on the contrary to the animals vaccinated with MeV. Monkeys vaccinated with MeV-Z+GPC.sub.LASV had the highest titer at day 30. Neutralizing antibodies have been detected in all animals except in control animals.

TABLE-US-00001 MeV-NP.sub.ExoN-GPC.sub.LASV MeV-Z + GPC.sub.LASV CDE031 CDE041 CDF053 CDI009 CDK026 CDK086 CDK106 CDG058 J0 No No 1/100 No 1/100 No No No J6 No No 1/100 No No No No 1/100 J15 1/100 1/100 1/500 1/100 1/500 1/500 1/500 1/500 J28 1/500 1/500 1/100 1/100 J30 1/100 1/100 1/100 1/100 MeV CDH011 CDH028 CDG058 J0 No No No J6 No No No J15 No No No J28 J30

[0250] The induction of CD8+ and CD4+ T cells specific for LASV antigens was also monitored by quantifying the percentage of T cells producing IFNg, TNFa and/or IL-2 in response to overlapping peptides covering the whole LASV GP, NP and Z proteins (FIG. 20) after immunization. T cells failed to respond to Z peptide (data not shown). The number of cytokine-producing T cells in response to GP and NP peptides was only modestly increased in comparison with baseline levels (Day 0) and MeV-control animals, and TNFa was the main cytokine involved in this response. Nevertheless, a non-significant increase in the percentage of GP-specific cytokine-producing CD8+ and CD4+ T cells 21 days after immunization with MeV-NP.sub.Exon-GPC.sub.LASV, but not after immunization with MeV-Z+GPC.sub.LASV, has been observed. Furthermore, NP-specific cytokine-producing CD4+ and CD8+ T cells appeared after 14 days in immunized animals and were still present 22 days after.

[0251] Similarly, we followed the T cell response to LASV GPC, NP or Z after challenge in T cell activation assay using overlapping peptides. The CD8 and CD4 response against GPC and NP were early and robust in MeV-NP.sub.ExoN+GPC.sub.LASV vaccinated animals, peaking at day 9 then decreasing slowly (FIG. 21, orange bars). The CD8 and CD4 responses against GPC was delayed and less intense in MeV-Z+GPC.sub.LASV immunized animals, peaking at day 12 (FIG. 21, green bars). These animals did not present a LASV-Z specific cellular response. Control animals only experienced a very weak and transient CD8 and CD4 responses against GPC and NP between day 6 and 12 (FIG. 21, red bars).

[0252] The intensity of the CD8 responses was correlated with the proliferation of these cells as assessed by a Ki67 staining (FIG. 22A), with strong proliferation of CD8 T cells in MeV-NP.sub.ExoN+GPC.sub.LASV immunized animals by day 9 compared to control animals and MeV-Z+GPC.sub.LASV immunized animals that had a milder proliferation peaking only at day 15 (compare orange bars with red and green bars, respectively). This proliferation was also associated with cytotoxic phenotypes of the CD8 and CD4 T cell response, as shown by the early and robust expression of granzyme B in MeV-NP.sub.ExoN+GPC.sub.LASV immunized animals and the delayed response in control animals and MeV-Z+GPC.sub.LASV immunized animals (FIGS. 22B and C, compare orange bars and green bars).

[0253] The induction of LASV GP- and NP-specific T cells in animals challenged with LASV has been monitored. No production of cytokines was observed after stimulation of PBMC with LASV Z peptides after the challenge (data not shown). The data regarding the CD8+ and CD4+ T cells producing cytokines in challenged animals are illustrated on FIGS. 23 and 24 respectively. In animals immunized with MeV, no significant amount of responding cells was found. In response to the LASV GPC peptides in animals immunized with MeV-NP.sub.ExoN+GPC.sub.LASV, the percentage of CD8+ and CD4+ T cells producing cytokines in response to GPC peptides rose to 2% and 0.6% respectively at day 12 post challenge, and then returned to basal level by day 22. The main part of T cells produced only IFNg, but the proportion of polyfunctional CD8+ and CD4+ T cells (Pf-T) which produce at least 2 cytokines increased from day 12 to day 30. Within the CD4+ T cells group, the percentage of IFNg-producing cells decreased until day 30 post-challenge, while the opposite was observed for the Pf-T, that rose to 59%. In the animals immunized with MeV-Z+GPC.sub.LASV, a moderate number of cytokine-producing CD4+ and CD8+ T cells was observed from day 15 and 12 respectively. Most T cells produced only IFNg, and the ratio of Pf-T remained around 20% for CD8+ T cells, but rose up to 40% for CD4+ T cells. At day 30 post-challenge, and for all immunized monkeys, a noticeable part of T cells produced only TNFa.

[0254] In response to the LASV NP peptide, trace amounts of cytokine-producing T cells from animals immunized with MeV were only detected 15 days post-challenge. Responding T cells from animals immunized with MeV-Z+GPC.sub.LASV were detected as soon as day 6 post-challenge, with a peak response at day 12. The phenotype of T cells was various after 6 days, while at day 9, mainly TNFa-secreting T cells were present. At day 12, IFNg-producing T cells dominated whereas Pf-T proportion increased until day 30.

[0255] The total cellular RNA content of PBMC of immunized monkeys with MeV-NP.sub.ExoN+GPC.sub.LASV and MeV-Z+GPC.sub.LASV has also been extracted at different times post-immunization to perform RNAseq and for analysing the differential expression of genes in PBMC at different time points. An enrichment analysis on differentially expressed genes has been performed using ClusterProfiler (KEGG analysis) in order to identify the pathway associated with those genes. The pathways differentially modulated through time after immunization with MeV-NP.sub.ExoN+GPC.sub.LASV and MeV-Z+GPC.sub.LASV are illustrated on FIGS. 25 and 26 respectively. In animals immunized with MeV-NP.sub.ExoN+GPC.sub.LASV, pathways involved in immune responses are activated in the first week following immunization (D2 vs D0, D4 vs D0 and D7 vs D0). The activation of the hematopoietic cell lineage and the phospholipase D pathways suggests a strong proliferation of immune cells during the first four days in association with phagocytic functions (FC gamma R-mediated phagocytosis) and chemokine signalling. The increased response of Th1, Th2 and Th17 until the seventh day suggests T cells proliferation. During the second week following immunization, the pathways involved in the modulation of the immune response are activated, notably the ubiquitin-mediated proteolysis, NF-kB signalling and the 11-17 signalling pathway. Immunization with MeV-Z+GPC.sub.LASV seems to induce a weaker immune response, with a weak and transient activation of Th1, Th2 and Th17 at day 4 post-immunization and a later activation of the NF-kB and IL-17 signalling pathways (at day 14) as compared to the results observed in animals immunized with MeV-NP.sub.ExoN+GPC.sub.LASV. Altogether, the activation of different pathways supports an active and effective cellular response to immunization.

[0256] The release of soluble mediators in plasma of animals after immunization and after LASV challenge has been observed. Among the 29 analytes quantified using Luminex assay, no difference in the levels of soluble mediator was found between animals after immunisation (data not shown). In challenged animals (FIG. 27), a transient release of IFNg was detected in the plasma of all animals, with levels peaking on day 6 and 9 post-infection in immunized animals and MeV-controls respectively. A lower concentration was nevertheless observed in the plasma of monkeys immunized with MeV-NP.sub.ExoN+GPC.sub.LASV. Concentrations of perforin rose until day 9 or 12 in all animals, and then decreased to a low level until day 22. Once again, the levels observed in monkeys immunized with MeV-NP.sub.ExoN+GPC.sub.LASV were lower. Elevated soluble CD137 (sCD137) levels were observed in monkeys immunized with MeV and MeV-Z+GPC.sub.LASV 9 days after infection, while only moderate concentrations of sCD137 were observed in animals immunized with MeV-NP.sub.ExoN+GPC.sub.LASV. IL-6 appeared in the plasma of all animals by day 6, and was still present at day 9 in animals vaccinated with MeV and MeV-Z+GPC.sub.LASV, while it was not detected anymore in the plasma of animals immunized with MeV-NP.sub.ExoN+GPC.sub.LASV. IL-6 levels were still rising in the plasma of MeV-animals, while the increase was moderate in the plasma of animals immunized with MeV-Z+GPC.sub.LASV. Elevated amount of IL-8 was observed in the plasma of animals immunized with MeV and MeV-Z+GPC.sub.LASV from day 6, whereas only low concentrations were detected between day 9 and day 12 in MeV-NP.sub.ExoN+GPC.sub.LASV monkeys. IL-18 was not detected in the plasma of MeV-NP.sub.ExoN+GPC.sub.LASV monkeys, while high levels and low levels was detected in animals immunized with MeV and MeV-Z+GPC.sub.LASV respectively. MCP1 remains at basal levels in MeV-NP.sub.ExoN+GPC.sub.LASV monkeys. In contrast, high concentrations were found in MeV and MeV-Z+GPC.sub.LASV animals, starting from day 6 and 9 respectively. Levels of IL-10 and IL-1 receptor antagonist (IL-1RA) increased until day 9 in MeV-Z+GPC.sub.LASV monkeys, and then decreases to reach a low level at day 22. IL-10 and IL-1RA levels were still rising in MeV animals, while except for small amount detected after 6 days, IL-10 and IL-1 RA were not released in the plasma of animals immunized with MeV-NP.sub.ExoN+GPC.sub.LASV.

Example 6: Immune Response to MeV

[0257] The levels of MeV-specific immunoglobulins produced against MeV-specific immunoglobulins was also assessed post-challenge by ELISA (FIG. 28A: IgM and 28B: IgG). The IgM and IgG MeV-specific are produced in all animals (MeV group, MeV-NP.sub.ExoN+GPC.sub.LASV group; MeV-Z+GPC.sub.LASV group). Similar MeV-specific IgM and IgG responses are induced by the MeV-NP.sub.ExoN-GPC.sub.LASV, the MeV-Z+GPC.sub.LASV and the MeV vaccines (FIG. 28). These animals are vaccinated MeV (FIG. 28), and animals which have been immunized with MeV-NP.sub.ExoN+GPC.sub.LASV or MeV-Z+GPC.sub.LASV are vaccinated against LASV (See FIG. 15) and MeV (FIG. 28).

Example 7: Tropism of the Vaccine Strains of MeV-LASV

[0258] The tropism of MeV-LASV has been analysed. Lassa virus uses -dystroglycan (-DG) as a receptor. The vaccine strains of MeV use CD46, SLAM and nectin-4 as receptors. A Mopeia virus has been used as a control to analyse if the introduction of Lassa antigens into the MeV vector has an impact on the tropism of the vaccine strains of MeV. Mopeia virus is an arenavirus closely related to the Lassa virus, and uses the same receptor. A Mopeia virus pseudotyped with the Lassa virus GPC replicates in CHO-K1 cells which express -DG and in CHO-hCD46 cells which express -DG and the human CD46, as illustrated on FIG. 30, wherein a staining with an anti-GP1 is positive in both cell lines. On the contrary, MeV-NP.sub.ExoN+GPC.sub.LASV are not able to replicate in CHO-K1 cells, while it replicates in CHO-hCD46 cell line (FIG. 30, bottom pictures). Therefore, the introduction of Lassa antigens into the MeV vector does not extend the tropism the MeV.

CONCLUSION

[0259] To conclude, both MeV-NP.sub.ExoN+GPC.sub.LASV and MeV-Z+GPC.sub.LASV vaccines were safe, immunogenic and efficacious in non-human primates. Both protected cynomolgus macaques against a lethal challenge with LASV strain Josiah after a single immunization. However, MeV-NP.sub.ExoN+GPC.sub.LASV conferred the best protection with a robust T cell response and a nearly-sterilizing immunity in all vaccinated monkeys. Thus, this vector is a candidate of choice for advance to clinical trials in humans. The immunogenicity of this vector prior challenge could certainly be improved by a prime/boost strategy. Nonetheless, we here bring the proof of principle that a single immunization could protect 100% of challenge animals. In addition, these vectors should protect monkeys against measles and could thus be used, in addition to emergency vaccine, as a bivalent vaccine in endemic countries where both LASV and MeV are major public health issues.

[0260] SEQ ID NO: 1

[0261] SEQ ID No: 1 corresponds to a recombinant GPC protein of the Lassa Virus strain Josiah encoded by the codon-optimised sequence of SEQ ID No: 2.

TABLE-US-00002 MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLV TFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHY IMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFH LSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVL QTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSR PSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTR WMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAE AQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTT TGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQ GKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLN HMGICSCGLYKQPGVPVKWKR*

[0262] SEQ ID NO: 2

[0263] SEQ ID No: 2 corresponds to a codon-optimized nucleotide sequence encoding the GPC protein of SEQ ID No. 1.

TABLE-US-00003 1ATGGGCCAGATTGTCACATTCTTTCAGGAAGTGCCACACGTCATTGAGGAGGTCATGAAC 61ATCGTGCTGATTGCTCTGTCAGTGCTGGCAGTGCTGAAAGGACTGTACAACTTCGCTACC 121TGTGGACTGGTGGGACTGGTCACATTCCTGCTGCTGTGCGGCAGAAGTTGCACTACCTCA 181CTGTACAAAGGAGTGTACGAGCTGCAGACTCTGGAACTGAACATGGAGACACTGAATATG 241ACAATGCCTCTGAGCTGCACCAAGAATAATAGCCACCACTATATCATGGTCGGGAACGAA 301ACCGGCCTGGAACTGACCCTGACAAACACCAGCATCATTAACCACAAGTTCTGCAATCTG 361AGCGACGCTCACAAGAAGAACCTGTATGACCACGCTCTGATGTCCATCATCAGTACCTTT 421CACCTGTCCATCCCCAATTTCAACCAGTACGAGGCAATGTCATGCGACTTCAACGGGGGC 481AAGATCAGTGTCCAGTACAACCTGAGCCACTCCTACGCCGGCGACGCAGCCAACCACTGC 541GGAACTGTCGCCAATGGCGTGCTGCAGACATTCATGAGGATGGCATGGGGGGGATCTTAC 601ATCGCACTGGATAGCGGCAGGGGCAATTGGGATTGCATCATGACTTCCTATCAGTATCTG 661ATTATCCAGAATACTACATGGGAGGATCATTGCCAGTTCAGTCGGCCCAGCCCTATTGGA 721TATCTGGGGCTGCTGTCACAGAGAACACGGGATATCTATATTTCAAGACGCCTGCTGGGC 781ACATTCACTTGGACACTGTCAGACAGTGAGGGCAAGGATACTCCAGGGGGCTACTGCCTG 841ACACGATGGATGCTGATCGAAGCAGAGCTGAAATGCTTCGGCAATACCGCAGTGGCCAAG 901TGCAACGAGAAACACGACGAGGAGTTCTGCGACATGCTGAGGCTGTTCGACTTCAACAAA 961CAGGCTATCCAGAGACTGAAGGCAGAAGCCCAGATGTCAATCCAGCTGATCAACAAGGCA 1021GTGAACGCCCTGATCAACGACCAGCTGATCATGAAGAACCACCTGAGAGACATTATGGGC 1081ATCCCCTACTGTAATTACAGCAAGTATTGGTACCTGAACCACACTACAACCGGGAGAACA 1141TCCCTGCCCAAGTGCTGGCTGGTCAGCAATGGGAGTTATCTGAATGAAACCCATTTCAGC 1201GACGATATCGAACAGCAGGCTGACAACATGATCACAGAGATGCTGCAGAAAGAGTACATG 1261GAAAGACAGGGCAAGACACCACTGGGACTGGTCGATCTGTTCGTCTTCTCCACTAGCTTC 1321TATCTGATTTCCATCTTCCTGCACCTGGTGAAGATCCCCACTCATAGGCACATTGTCGGC 1381AAGAGTTGCCCTAAACCCCATAGGCTGAATCACATGGGGATTTGTAGTTGCGGCCTGTAT 1441AAGCAGCCTGGCGTGCCTGTGAAATGGAAGAGATGA

[0264] SEQ ID NO: 3

[0265] SEQ ID No: 3 corresponds to a recombinant NP protein of the Lassa Virus strain Josiah encoded by the codon-optimised sequence of SEQ ID No: 4.

TABLE-US-00004 MSASKEIKSFLWTQSLRRELSGYCSNIKLQVVKDAQALLHGLDFSEV SNVQRLMRKERRDDNDLKRLRDLNQAVNNLVELKSTQQKSILRVGTL TSDDLLILAADLEKLKSKVIRTERPLSAGVYMGNLSSQQLDQRRALL NMIGMSGGNQGARAGRDGVVRVWDVKNAELLNNQFGTMPSLTLACLT KQGQVDLNDAVQALTDLGLIYTAKYPNTSDLDRLTQSHPILNMIDTK KSSLNISGYNFSLGAAVKAGACMLDGGNMLETIKVSPQTMDGILKSI LKVKKALGMFISDTPGERNPYENILYKICLSGDGWPYIASRTSITGR AWENTVVDLESDGKPQKADSNNSSKSLQSAGFTAGLTYSQLMTLKDA MLQLDPNAKTWMDIEGRPEDPVEIALYQPSSGCYIHFFREPTDLKQF KQDAKYSHGIDVTDLFATQPGLTSAVIDALPRNMVITCQGSDDIRKL LESQGRKDIKLIDIALSKTDSRKYENAVWDQYKDLCHMHTGVVVEKK KRGGKEEITPHCALMDCIMFDAAVSGGLNTSVLRAVLPRDMVFRTST PRVVL*

[0266] SEQ ID NO: 4

[0267] SEQ ID No: 4 corresponds to a codon-optimized nucleotide sequence encoding the NP protein of SEQ ID No. 3.

TABLE-US-00005 1ATGAGTGCCAGCAAAGAAATCAAGAGCTTCCTGTGGACCCAGAGTCTGCGGAGGGAACTG 61AGCGGATACTGTAGCAACATCAAACTGCAGGTGGTCAAGGACGCTCAGGCACTGCTGCAT 121GGGCTGGACTTCTCCGAGGTGTCTAATGTGCAGCGGCTGATGCGGAAAGAACGGAGGGAC 181GATAATGACCTGAAGCGACTGCGCGACCTGAACCAGGCAGTGAACAATCTGGTCGAGCTG 241AAGAGCACCCAGCAGAAATCAATCCTGCGGGTCGGGACACTGACATCTGACGACCTGCTG 301ATCCTGGCTGCAGACCTGGAGAAGCTGAAATCGAAAGTGATCCGCACCGAAAGGCCACTG 361TCCGCCGGGGTCTACATGGGCAATCTGTCTTCCCAGCAGCTGGACCAGAGGCGGGCTCTG 421CTGAACATGATTGGGATGTCCGGAGGAAATCAGGGAGCTAGAGCCGGGAGGGACGGAGTC 481GTGCGGGTCTGGGACGTGAAGAATGCCGAACTGCTGAACAACCAGTTCGGGACCATGCCA 541AGTCTGACACTGGCATGCCTGACTAAACAGGGCCAGGTGGATCTGAATGATGCAGTCCAG 601GCTCTGACCGACCTGGGCCTGATCTACACCGCCAAGTACCCCAATACTAGCGACCTGGAT 661AGACTGACCCAGAGCCACCCCATCCTGAACATGATCGACACTAAGAAGTCCTCACTGAAC 721ATCAGTGGCTATAATTTCTCCCTGGGGGCAGCAGTCAAGGCTGGCGCATGCATGCTGGAC 781GGCGGGAATATGCTGGAAACCATCAAAGTGTCTCCCCAGACCATGGATGGCATCCTGAAA 841TCTATTCTGAAAGTCAAGAAGGCCCTGGGAATGTTTATTTCAGACACCCCCGGCGAGAGG 901AATCCATATGAGAACATTCTGTATAAGATTTGCCTGAGTGGCGACGGGTGGCCATACATT 961GCAAGCCGGACATCAATTACCGGAAGAGCTTGGGAGAATACAGTCGTGGACCTGGAAAGC 1021GACGGCAAGCCCCAGAAGGCCGACTCAAACAACTCCTCAAAGAGTCTGCAGTCAGCTGGC 1081TTCACAGCAGGGCTGACTTACTCCCAGCTGATGACACTGAAGGACGCAATGCTGCAGCTG 1141GACCCAAACGCTAAGACATGGATGGACATCGAGGGACGGCCAGAAGATCCAGTGGAAATC 1201GCACTGTATCAGCCATCATCCGGATGCTATATCCATTTCTTCCGGGAACCAACTGATCTG 1261AAGCAGTTCAAGCAGGATGCAAAGTACTCCCACGGAATCGATGTCACCGATCTGTTCGCA 1321ACCCAGCCAGGACTGACATCAGCCGTCATCGATGCCCTGCCTAGGAACATGGTCATTACT 1381TGCCAGGGCTCCGACGATATTAGGAAGCTGCTGGAGAGCCAGGGACGGAAGGATATCAAA 1441CTGATCGATATTGCCCTGTCTAAGACTGATAGCCGGAAATATGAGAATGCAGTCTGGGAT 1501CAGTACAAGGACCTGTGCCATATGCATACCGGAGTGGTCGTCGAGAAGAAGAAGAGGGGC 1561GGAAAGGAAGAGATCACACCCCACTGTGCCCTGATGGATTGCATCATGTTCGACGCAGCC 1621GTGTCCGGGGGCCTGAACACCTCAGTCCTGAGGGCTGTCCTGCCAAGAGATATGGTGTTT 1681AGAACTTCAACCCCAAGAGTCGTCCTGTAA

[0268] SEQ ID NO: 5

[0269] SEQ ID No: 5 corresponds to a recombinant mutated NP protein of the Lassa Virus strain Josiah encoded by the codon-optimised sequence of SEQ ID No: 6, and wherein the exonuclease activity of the NP protein has been knocked down. Amino acid 388 and amino acid 391 have been mutated (M388D and E391 G).

TABLE-US-00006 MSASKEIKSFLWTQSLRRELSGYCSNIKLQVVKDAQALLHGLDFSEV SNVQRLMRKERRDDNDLKRLRDLNQAVNNLVELKSTQQKSILRVGTL TSDDLLILAADLEKLKSKVIRTERPLSAGVYMGNLSSQQLDQRRALL NMIGMSGGNQGARAGRDGVVRVWDVKNAELLNNQFGTMPSLTLACLT KQGQVDLNDAVQALTDLGLIYTAKYPNTSDLDRLTQSHPILNMIDTK KSSLNISGYNFSLGAAVKAGACMLDGGNMLETIKVSPQTMDGILKSI LKVKKALGMFISDTPGERNPYENILYKICLSGDGWPYIASRTSITGR AWENTVVDLESDGKPQKADSNNSSKSLQSAGFTAGLTYSQLMTLKDA MLQLDPNAKTWMAIEARPEDPVEIALYQPSSGCYIHFFREPTDLKQF KQDAKYSHGIDVTDLFATQPGLTSAVIDALPRNMVITCQGSDDIRKL LESQGRKDIKLIDIALSKTDSRKYENAVWDQYKDLCHMHTGVVVEKK KRGGKEEITPHCALMDCIMFDAAVSGGLNTSVLRAVLPRDMVFRTST PRVVL*

[0270] SEQ ID NO: 6

[0271] SEQ ID No: 6 corresponds to a codon-optimized nucleotide sequence encoding the mutated NP protein of SEQ ID No. 5. Nucleotides 11661 1175 and 1176 has been mutated (C1166A, C1175G and C1176A).

TABLE-US-00007 1ATGAGTGCCAGCAAAGAAATCAAGAGCTTCCTGTGGACCCAGAGTCTGCGGAGGGAACTG 61AGCGGATACTGTAGCAACATCAAACTGCAGGTGGTCAAGGACGCTCAGGCACTGCTGCAT 121GGGCTGGACTTCTCCGAGGTGTCTAATGTGCAGCGGCTGATGCGGAAAGAACGGAGGGAC 181GATAATGACCTGAAGCGACTGCGCGACCTGAACCAGGCAGTGAACAATCTGGTCGAGCTG 241AAGAGCACCCAGCAGAAATCAATCCTGCGGGTCGGGACACTGACATCTGACGACCTGCTG 301ATCCTGGCTGCAGACCTGGAGAAGCTGAAATCGAAAGTGATCCGCACCGAAAGGCCACTG 361TCCGCCGGGGTCTACATGGGCAATCTGTCTTCCCAGCAGCTGGACCAGAGGCGGGCTCTG 421CTGAACATGATTGGGATGTCCGGAGGAAATCAGGGAGCTAGAGCCGGGAGGGACGGAGTC 481GTGCGGGTCTGGGACGTGAAGAATGCCGAACTGCTGAACAACCAGTTCGGGACCATGCCA 541AGTCTGACACTGGCATGCCTGACTAAACAGGGCCAGGTGGATCTGAATGATGCAGTCCAG 601GCTCTGACCGACCTGGGCCTGATCTACACCGCCAAGTACCCCAATACTAGCGACCTGGAT 661AGACTGACCCAGAGCCACCCCATCCTGAACATGATCGACACTAAGAAGTCCTCACTGAAC 721ATCAGTGGCTATAATTTCTCCCTGGGGGCAGCAGTCAAGGCTGGCGCATGCATGCTGGAC 781GGCGGGAATATGCTGGAAACCATCAAAGTGTCTCCCCAGACCATGGATGGCATCCTGAAA 841TCTATTCTGAAAGTCAAGAAGGCCCTGGGAATGTTTATTTCAGACACCCCCGGCGAGAGG 901AATCCATATGAGAACATTCTGTATAAGATTTGCCTGAGTGGCGACGGGTGGCCATACATT 961GCAAGCCGGACATCAATTACCGGAAGAGCTTGGGAGAATACAGTCGTGGACCTGGAAAGC 1021GACGGCAAGCCCCAGAAGGCCGACTCAAACAACTCCTCAAAGAGTCTGCAGTCAGCTGGC 1081TTCACAGCAGGGCTGACTTACTCCCAGCTGATGACACTGAAGGACGCAATGCTGCAGCTG 1141GACCCAAACGCTAAGACATGGATGGCCATCGAGGCCCGGCCAGAAGATCCAGTGGAAATC 1201GCACTGTATCAGCCATCATCCGGATGCTATATCCATTTCTTCCGGGAACCAACTGATCTG 1261AAGCAGTTCAAGCAGGATGCAAAGTACTCCCACGGAATCGATGTCACCGATCTGTTCGCA 1321ACCCAGCCAGGACTGACATCAGCCGTCATCGATGCCCTGCCTAGGAACATGGTCATTACT 1381TGCCAGGGCTCCGACGATATTAGGAAGCTGCTGGAGAGCCAGGGACGGAAGGATATCAAA 1441CTGATCGATATTGCCCTGTCTAAGACTGATAGCCGGAAATATGAGAATGCAGTCTGGGAT 1501CAGTACAAGGACCTGTGCCATATGCATACCGGAGTGGTCGTCGAGAAGAAGAAGAGGGGC 1561GGAAAGGAAGAGATCACACCCCACTGTGCCCTGATGGATTGCATCATGTTCGACGCAGCC 1621GTGTCCGGGGGCCTGAACACCTCAGTCCTGAGGGCTGTCCTGCCAAGAGATATGGTGTTT 1681AGAACTTCAACCCCAAGAGTCGTCCTGTAA

[0272] SEQ ID NO: 7

[0273] SEQ ID No: 7 corresponds to a recombinant Z protein of the Lassa Virus strain Josiah encoded by the codon-optimised sequence of SEQ ID No: 8.

TABLE-US-00008 MGNKQAKAPESKDSPRASLIPDATHLGPQFCKSCWFENKGLVECNNH YLCLNCLTLLLSVSNRCPICKMPLPTKLRPSAAPTAPPTGAADSIRP PPYSP*

[0274] SEQ ID NO: 8

[0275] SEQ ID No: 8 corresponds to a codon-optimized nucleotide sequence encoding the Z protein of SEQ ID No. 7.

TABLE-US-00009 1ATGGGCAATAAGCAGGCAAAGGCACCCGAAAGCAAGGATTCACCTAGAGCATCACTGATT 61CCCGACGCAACTCATCTGGGGCCACAGTTCTGCAAATCCTGTTGGTTCGAGAACAAAGGC 121CTGGTGGAGTGCAATAACCACTACCTGTGCCTGAACTGTCTGACACTGCTGCTGAGTGTG 181AGCAACAGATGCCCAATCTGCAAGATGCCTCTGCCAACAAAGCTGAGGCCTTCTGCTGCA 241CCCACCGCACCACCAACTGGAGCCGCAGACAGCATTAGACCCCCCCCATACTCACCATAA

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