Lassa Vaccine

Abstract

The present invention relates to recombinant measles viruses expressing Lassa virus polypeptides, particularly immunogenic LASV particles expressed by measles viruses and / or virus-like particles (VLPs) containing Lassa virus proteins. These particles are recombinant infectious particles capable of replicating in a host after administration. The present invention provides means, particularly nucleic acid constructs, vectors, cells, and rescue systems for producing these infectious particles. The present invention also relates to the use of these recombinant infectious particles, particularly in the form of compositions, more particularly in the form of vaccines, for the treatment or prevention of infection with Lassa virus.

Description

[Technical Field]

[0001] This application generally relates to recombinant gene constructs comprising recombinant measles virus and expressing at least one Lassa virus polypeptide, protein, antigen, or antigenic fragment thereof. This application also relates to the use of gene constructs or viruses, and more particularly to their application to induce protection against Lassa virus (LASV) and / or measles virus (MV or MeV).

[0002] The means of the present invention provide, more specifically, recombinant nucleic acid constructs that enable the expression of at least one of the following polypeptides of LASV, or truncated versions thereof, or antigenic fragments thereof: nucleoprotein (NP), glycoprotein precursor (GPC), zinc-binding protein (Z), or mutant versions of the native NP protein in which the exonuclease activity of the NP protein is knocked down (mutant NP or mNP).

[0003] The present invention also relates to recombinant MeV-LASV viruses expressing the aforementioned LASV polypeptide, its antigenic fragment or antigen, or its truncated version, i.e., at least one of NP, mNP, GCP, and / or Z. The present invention also relates to immunogenic particles expressed by measles virus, comprising LASV polypeptide, particularly at least GCP polypeptide or protein, or its antigenic fragment, and / or infectious virus-like particles (VLPs) comprising at least Z polypeptide, or protein, or its antigenic fragment, the immunogenic particles and / or VLPs being able to induce cellular and / or humoral responses to LASV, particularly T cell responses, particularly CD4+ and / or CD8+ T cell responses.

[0004] In particular, the present invention relates to the use of recombinant viral infectious particles, VLPs, such as gene constructs, recombinant nucleic acid constructs, and expression vector-like plasmid vectors, for inducing immunogenic or antigenic responses in a host. [Background technology]

[0005] Lassa virus (LASV) is an Old World arenavirus belonging to the family Arenaviridae. LASV is an enveloped, single-stranded, two-segmented ambient RNA virus. Its genome contains two RNA segments, each encoding four viral proteins: two proteins per strand. The larger segment (approximately 7 kb) encodes zinc-binding protein Z. The Z protein regulates replication and transcription. The larger segment also encodes RNA polymerase L. The smaller segment (approximately 3.4 kb) encodes nucleoprotein (NP) and glycoprotein precursors (GCPs), which are post-translation cleaved into envelope glycoproteins GP1 and GP2 and the stable signal peptide SSP. These two glycoproteins mediate host cell entry. The synthetic capacity of arenaviruses is contained within the L polymerase protein. This protein uses a viral RNA template composed of genomic RNA encapsulated by the NP protein and viral ribonucleoprotein. During infection, the virus is delivered to the cytoplasm of the host cell, and the L polymerase protein initiates transcription from the genomic promoter located at the 3' end of each genomic RNA segment. Primary transcription results in the synthesis of mRNA for the viral genes encoded in the antigenomic direction, namely the NP and L genes. Transcription terminates at the distal end of a stem-loop structure within the intergenomic region. The L polymerase then moves beyond the intergenomic region, generating complementary antigenomic RNA. This RNA contributes as a template for the synthesis of mRNA for the viral genes GPC and Z, as well as for the synthesis of full-length genomic RNA.

[0006] LASV is the pathogen that causes Lassa fever, a severe hemorrhagic fever, in humans. The virus's natural host is the African rodent Mastomys natalensis. Lassa virus is transmitted from rodents to humans, but it can also be transmitted from person to person, leading to localized outbreaks.

[0007] Between 100,000 and 300,000, and sometimes up to 500,000, Lassa fever cases are reported annually in endemic areas of West Africa, particularly Guinea, Liberia, Nigeria, and Sierra Leone. Therefore, Lassa fever is a significant public health concern in these regions. The severity of the disease varies from asymptomatic infection to severe complications leading to fatal hemorrhagic fever. Clinical signs and symptoms include fever, cough, chest pain, urinary dysfunction, headache, vomiting, diarrhea, pharyngitis, conjunctivitis, hemorrhage, and facial edema. The mortality rate for patients infected with LASV is high, approximately 10% in some areas where LASV is prevalent. The mortality rate is even higher, at 50%, in young children. Furthermore, approximately 20% of survivors exhibit long-term complications, including hearing impairment. Therefore, Lassa fever has a serious impact on populations in these regions and is a major health problem. Recently, the distribution of Lassa fever infection appears to be spreading to other West African countries, as cases have been reported in Mali, Ghana, Côte d'Ivoire, and Burkina Faso in the last decade.

[0008] Despite its discovery in Nigeria in 1969, there is currently no preventative or preventative measure against Lassa fever. Most patients are treated with ribavirin, an antiviral drug. Unfortunately, drug treatment appears to be most effective only when administered early in the course of the disease, and is not entirely effective. Treatment is also completed with supportive care such as maintaining blood pressure and oxygenation, fluid and electrolyte balance, and treating any other infections. Therefore, effectively treating patients in endemic areas of West Africa where Lassa fever has spread can be difficult.

[0009] Whether an infection leads to serious illness or death appears to depend on the host immune response. Most severe cases involve a deficient cellular response, where dendritic cells and macrophages release large amounts of LASV but are not activated and therefore do not produce sufficient cytokines. Disease severity, as well as the evolution and spread of the virus to new geographical areas, are serious public health issues that need to be addressed.

[0010] In this context, the development of preventive measures such as preventive vaccines is a critical priority to meet the needs of these populations. Therefore, there is a need for fully effective measures that can treat or prevent LASV infection, including preventing the outcome of primary LASV infection, particularly Lassa fever.

[0011] One of the most promising treatments for preventing LASV infection is a preventive vaccine, but such a vaccine is not currently available. Prevention is the simplest and safest way to control LASV infection and protect local populations. Despite this urgent need, no vaccine candidates have yet successfully progressed to clinical trials.

[0012] Therefore, there is a need for vaccines and products, including active ingredients for preparing the vaccine, as well as methods for producing these products and vaccines. The vaccine candidate must be safe and effective without significant adverse effects when immunizing those who need it, and must induce the production of antibodies that neutralize LASV, and possibly T cell-like T helper cells and / or cytotoxic T cells. In other words, the vaccine should induce a strong cellular and / or humoral response. Advantageously, the vaccine must provide bactericidal immunity after a single immunization. For this reason, there is a need for a vaccine that will enable the in vivo production of LASV proteins and / or LASV VLPs in infected cells, particularly infected host cells, and thus will induce effective, persistent immunity, especially lifelong immunity after a single or two-dose course.

[0013] Another need is to facilitate vaccination for populations that have difficulty accessing medical centers. Vaccine candidates that induce immunity against two disease factors could enhance the overall health of these populations. A single dose of vaccination could therefore enable immunity against several disease factors present in the aforementioned areas. In particular, to completely eradicate measles virus (MeV), vaccine immunity against both MeV and LASV could reliably protect these populations against these two critical threats, especially in West Africa.

[0014] The measles virus was isolated in 1954 (Enders, JF, and TC Peebles. 1954. Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soc. Exp. Biol. Med. 86: pp. 277-286). The measles virus belongs to the order mononegavirus, meaning it is a virus with a non-segmented minus-strand RNA genome. The non-segmented genome of MeV exhibits anti-message polarity, resulting in genomic RNA that is not translated in vivo or in vitro, or, when purified, is not infectious. The transcription and replication of non-segmented (-)-strand RNA viruses, as well as their assembly into viral particles, have been studied and reported, particularly in Fields virology (3rd edition, vol. 1, 1996, Lippincott - Raven publishers - Fields BN et al.). Transcription and replication of the measles virus occur in the cytoplasm of the host cell rather than in the nucleus of the infected cell, just like LASV. The MeV genome contains genes encoding six major structural proteins named N, P, M, F, H, and L, as well as two further non-structural proteins derived from the P gene, C, and V. The order of the genes is as follows: from the 3' end of the genomic RNA; N, P (including C and V), M, F, H, and L large polymerase at the 5' end. The genome further contains a non-coding region in the intergenetic region M / F. This non-coding region contains approximately 100 nucleotides of uncoding RNA. The genes mentioned each encode the viral nucleocapsid protein or nucleoprotein (N), phosphoprotein (P), and macroprotein (L), respectively, which assemble around the genomic RNA to provide the nucleocapsid, hemagglutinin (H), fusion protein (F), and matrix protein (M).

[0015] Attenuated viruses, derived from the MeV virus, provide vaccine strains, particularly the Schwarz strain. The Schwarz measles vaccine is currently a safe and effective vaccine available for preventing measles. In addition to providing a vaccine, attenuated strains of the measles virus, such as the Schwarz strain, have been shown to be stable and suitable for designing effective delivery vectors for immunity against other viruses, such as the Zika virus or Chikungunya virus. Measles vaccines have been administered to hundreds of millions of children over the past 30 years, demonstrating their efficacy and safety. They are produced on a large scale in many countries and distributed at low cost. [Prior art documents] [Patent Documents]

[0016] [Patent Document 1] International Publication No. 97 / 06270 [Patent Document 2] International Publication No. 2004 / 000876 [Patent Document 3] International Publication No. 2008 / 078198 [Non-patent literature]

[0017] [Non-Patent Document 1] Enders, JF, and TC Peebles. 1954. Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soc. Exp. Biol. Med.86:277~286 [Non-Patent Document 2] Fields Virology (3rd edition, vol. 1, 1996, Lippincott-Raven publishers - Fields BN et al.) [Non-Patent Document 3] Horikami S.M. and Moyer S.A. (Curr. Top. Microbiol. Immunol. (1995), 191, pp. 35 - 50) [Non - Patent Document 4] Combredet C. et al. (Journal of Virology, Nov 2003, pp. 11546 - 11554) for the Schwarz vaccination strain of the virus or for broadly considered negative - sense RNA viruses [Non - Patent Document 5] Neumann G. et al. (Journal of General Virology (2002), 83, pp. 2635 - 2662) [Non - Patent Document 6] Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus (Andersen Kristian G et al.; 2015; Cell. Aug 13, 2015; 162(4): 738 - 750. doi:10.1016 / j.cell.2015.07.020) [Non - Patent Document 7] Cattaneo R et al., Cell. Mar 10, 1989;56(5):759 - 64 [Non - Patent Document 8] Radecke, F. et al., Rescue of measles viruses from cloned DNA. Embo J, 1995. 14(23): 5773 - 84 [Summary of the Invention] [Means for Solving the Problems]

[0018] To address, at least in part, the shortcomings of the prior art, the inventors have achieved the production of active ingredients (or components) for vaccines based on recombinant nucleic acid constructs, particularly those containing cloned polynucleotides encoding Lassa virus polypeptides, proteins, or antigens, or antigenic fragments thereof, within infectious, replicable measles virus. The vaccine can be recovered when the recombinant measles virus replicates in the host after administration. Accordingly, the present invention relates to LASV vaccines, particularly pediatric vaccines, and to active ingredients based on attenuated measles virus strains, such as known commercial vaccine strains, particularly the widely used Schwarz measles vaccine. For all these reasons, the inventors use attenuated measles virus to produce recombinant measles virus particles, particularly their immunogenic particles and / or VLPs, that stably express the structural antigens of LASV. The measles method of the present invention meets all relevant criteria for future LASV vaccines.

[0019] One object of the present invention is to provide a gene construct, particularly a recombinant gene construct, particularly a nucleic acid construct, for recovering infectious viruses, especially measles viruses expressing LASV particles, and optionally LASV virus-like particles (VLPs) from nucleic acid constructs.

[0020] Accordingly, the present invention relates to nucleic acid constructs comprising a full-length antigenomic (+) RNA strand of measles virus and a first heterologous polynucleotide, or at least one protein, or at least one antigen, or at least one antigenic fragment thereof, selected from the group consisting of nucleoprotein (NP), mutant nucleoprotein (mNP), zinc-binding protein (Z), and glycoprotein precursor (GPC). The first heterologous polynucleotide is operably cloned within an additional transcription unit (ATU) inserted into the cDNA of the MeV antigenomic (+) RNA strand. Specific nucleic acid constructs described in this embodiment are shown in Figures 1 and 31 to 36 and in the Examples.

[0021] The above expression “coding” encompasses the ability of cDNA to enable transcription of full-length antigenomic (+)RNA, the cDNA particularly contributing as a template for transcription and appropriate translation for product expression in cells or cell lines. Thus, if the cDNA is a double-stranded molecule, one strand has the same nucleotide sequence as the measles virus antigenomic (+)RNA from which the first heterologous polynucleotide was cloned, except for the “U” nucleotide which is substituted by a “T” nucleotide in the cDNA. The nucleic acid constructs of the present invention may include regulatory elements that control the transcription of the coding sequence, in particular promoters and transcription termination sequences, possibly enhancers and other cis-activating elements. These regulatory elements may be heterologous with respect to heterologous polynucleotides arising from or derived from the LASV gene, and in particular may be regulatory elements of measles virus strains.

[0022] The expression “operatably cloned,” which may be replaced by the expression “operatably bound,” refers to the functional cloning or insertion of heterologous polynucleotides into the nucleic acid constructs of the present invention such that the polynucleotides and nucleic acid constructs are effectively or fully transcribed and appropriately translated in cells, cell lines, or host cells used as part of a rescue system for the production of recombinant infectious MeV particles or MeV expressing at least one polypeptide, or at least one protein, or at least one antigen, or at least one antigenic fragment of LASV. In other words, the nucleic acid constructs of the present invention enable the production of infectious antigenomic (+) RNA that, when placed under appropriate conditions, can produce at least one polypeptide, or at least one protein, or at least one antigen, or at least one antigenic fragment of LASV.

[0023] In certain embodiments of the present invention, a nucleic acid construct comprising cDNA encoding nucleotides of the full-length infectious antigenomic (+) RNA strand of MeV, but without manipulably cloned heterologous polynucleotides, follows the six rules of the measles virus genome. In other words, the cDNA encoding nucleotides of the full-length infectious antigenomic (+) RNA strand of MeV is a polyhexameric cDNA.

[0024] The genome structure of the measles virus, as well as its replication and transcription methods, have been fully identified in prior art, particularly in Horikami SM and Moyer SA (Curr. Top. Microbiol. Immunol. (1995) pp. 191, 35-50) or Combredet C. et al. (Journal of Virology, Nov 2003, pp. 11546-11554) for the Schwarz vaccination strain of the virus or for broadly considered negative-sense RNA viruses, and Neumann G. et al. (Journal of General Virology (2002) pp. 83, 2635-2662).

[0025] The "Rule of 6" is expressed as the fact that the total number of nucleotides present in a nucleic acid representing the MeV(+) strand RNA genome or in a nucleic acid construct containing the same is a multiple of 6. The "Rule of 6" is recognized in the art as a requirement for the total number of nucleotides in the measles virus genome that enables effective or optimal replication of MeV genomic RNA. In embodiments of the present invention that define nucleic acid constructs satisfying Rule of 6, the Rule is applied to a nucleic acid construct that identifies a cDNA encoding a full-length MV(+) strand RNA genome. In this case, Rule of 6 is applied individually to the cDNA encoding the nucleotide sequence of the full-length infectious antigenomic(+) RNA strand of the measles virus, but not necessarily to the polynucleotides cloned into the cDNA that encode at least one polypeptide of LASV.

[0026] The nucleic acid constructs of the present invention are, in particular, purified DNA molecules obtained or obtainable by the recombination of at least one polynucleotide of MeV and at least one or more polynucleotides of LASV that are operably cloned or linked to one another.

[0027] According to the present invention, nucleic acid constructs are prepared by cloning at least one polypeptide or protein, or an antigen or antigenic fragment thereof, selected from the group consisting of the GPC protein, NP protein, mNP protein, and Z protein of LASV, or one polynucleotide or several polynucleotides encoding the antigenic fragment thereof, into a cDNA encoding the full-length antigenome (+) RNA of measles virus. The MeV genome is shown in Figure 1A, while some nucleic acid constructs described in the present invention are shown in Figures 1B and 31 to 36. Alternatively, the nucleic acid constructs of the present invention may be prepared using steps of nucleic acid fragment synthesis or polymerization from a template, including PCR. The polynucleotides and nucleic acid constructs of the present invention may rather be prepared, in particular cloned, obtained, or synthesized according to any method known in the art, in particular by polymerization using the PCR method.

[0028] Heterogeneous polynucleotides can arise from the fusion of several other polynucleotides that each encode a specific polypeptide of LASV, or a specific protein, antigen, or its antigenic fragment. For example, heterogeneous polynucleotides may arise from the fusion of polynucleotides that each encode a single protein, such as a GPC protein and an NP protein or mNP protein, and these two polynucleotides are linked together in a nucleic acid construct by a linker sequence. The linker sequence is well known in the art and may be a short nucleotide sequence that is included in or composed of the regulatory sequence of the measles virus.

[0029] Therefore, heterologous polynucleotides can encode a single polypeptide, two different polypeptides, two identical polypeptides, three different polypeptides, two identical polypeptides and another polypeptide, four different polypeptides, two identical polypeptides and two other identical polypeptides, two identical polypeptides and two other different polypeptides, or three identical polypeptides and another different polypeptide. Any one of these polynucleotides encoding at least two (identical or different) polypeptides may arise from the fusion of several polynucleotides or be prepared using a nucleic acid fragment synthesis or polymerization process from a template, including PCR. Alternatively, any one of these polynucleotides may be a cDNA resulting from the genomic RNA of Lassa virus after reverse transcription, the cDNA being either whole-genome cDNA or a fragment thereof, encoding a polypeptide of LASV.

[0030] Heterogeneous polynucleotides, particularly LASV genes, are cloned within additional transcription units (ATUs) inserted into the MeV cDNA. ATU sequences are known to those skilled in the art and, for use in the process of cloning into MeV cDNA, include cis-acting sequences necessary for MeV-dependent expression of the transgene, such as a promoter of a gene preceding the insert, represented by polynucleotides encoding the LASV polypeptide inserted into multiple cloning site cassettes in the MeV cDNA. ATUs can be further defined as disclosed by Billeter et al. in International Publication No. 97 / 06270. Three ATUs are shown in Figure 1A. ATUs can also be defined as multiple cloning cassettes inserted within the MeV cDNA, particularly between NP intergeneric regions of the MeV genome and / or between intergeneric HL regions of the MeV genome. ATUs may contain cis-acting sequences necessary for the transcription of the MeV P gene. Different ATUs, particularly ATU1 and ATU2, can be identified with respect to their nucleic acid sequences. The ATU is generally located between two CTT genes, each corresponding to the start and stop codons of a polymerase. The ATU may further include ATG and TAG codons, each corresponding to the start and stop codons for the translation of a heterologous polynucleotide cloned within the ATU. Alternatively, the ATU is located between ATG and TAG genes, each corresponding to the start and stop codons for the translation of a heterologous polynucleotide cloned within the ATU. The ATU may further include ATG and TAG codons, each corresponding to the start and stop codons for the translation of a heterologous polynucleotide cloned within the ATU. In a preferred embodiment of the present invention, the ATU is a polynucleotide comprising or consisting of SEQ ID NO: 16.

[0031] Sequence ID 16 Sequence ID 16 is the ATU sequence located within the cDNA molecule encoding the full-length antigenome (+) RNA strand of the measles virus. The CTT codon, corresponding to the polymerase start and stop codons, is shown in bold. The ATG and TAG codons, corresponding to the start and stop codons for the translation of heterologous polynucleotides cloned within the ATU, are underlined. CTTAGGAACCAGGTCCACACAGCCGCCAGCCCATCAacgcgtacg ATG * TAGgcgcgcagcgcttagacgtctcgcga TCG ATACTAGTACAACCTAAATCCATTATAAAAAACTT Here, * This corresponds to a heterogeneous codon-optimized sequence polynucleotide that encodes at least one LASV polypeptide.

[0032] An ATU containing a heterogeneous polynucleotide encoding a GPC polypeptide is located, for example, between amino acid residues 3487 and 5071 in SEQ ID NO: 9. SEQ ID NO: 17 corresponds to an ATU containing a codon-optimized heterogeneous polynucleotide encoding a GPC protein as a cloned insert.

[0033] The ATU (known as reference ATU2) is located between the P and M genes of MeV. Another ATU (known as reference ATU1) is located upstream of the N gene of MeV. Yet another ATU (known as reference ATU3) is located between the H and L genes of MeV. The transcription of MeV viral RNA has been observed to follow a gradient from the 5' end to the 3' end. This explains why the expression level of heterologous polynucleotides changes depending on where they are inserted, and why they are somewhat effective when inserted within ATU1, ATU2, or ATU3.

[0034] The term “polypeptide” is used interchangeably with the terms “antigen,” “protein,” or “antigenic fragment,” and defines a molecule resulting from a sequence of amino acid residues. In particular, the polypeptides disclosed in this application are based on LASV and are antigens, proteins, structural proteins, or antigenic fragments thereof, which may be identical to native proteins or derivatives thereof by mutations including substitution (particularly by conserved amino acid residues), addition of amino acid residues, or post-translational secondary modifications, or deletion of a portion of a native protein resulting in a fragment having a shortened size relative to the native protein of reference. Fragments are included within the present invention insofar as they have native protein epitopes suitable for inducing an immune response in a human host, including a host, particularly a child host, preferably a response that enables protection against LASV infection or LASV-related disease. Epitopes are, in particular, types of T epitopes involved in inducing cellular immune responses (CMI responses). T epitopes are involved in stimulating T cells by presenting some portion of a T cell epitope that can bind to MHC class II molecules, resulting in T cell activation. Alternatively, the epitope may be of type B, involved in the activation of antibody production in a host to which the protein is administered or expressed after administration of the infectious, replicable particles of the present invention. The fragment may have a size greater than 50% of the amino acid sequence size of the native protein of LASV strain Josiah, preferably at least 90% or 95%. The polypeptide may have at least 50% identity with the native protein of LASV strain Josiah, preferably at least 60%, preferably at least 70%, preferably at least 85%, or at least 95%.

[0035] In certain embodiments of the present invention, each polynucleotide operably cloned within the cDNA of an antigenomic (+) RNA encodes a polypeptide containing an epitope located within one of the LASV polypeptides. In this embodiment, the epitope sequence shares 100% identity with the epitope sequence of the native LASV protein. Such epitopes are listed in the Immunoepitope Database and Analysis Resource (www.iedb.org). Within the LASV polypeptide encoded by the polynucleotide and having an epitope sequence as defined herein, amino acid residues that do not belong to any epitope may differ from the sequence of the native LASV protein.

[0036] "Lasv polypeptide" means "polypeptide" as defined herein (polypeptide, antigen, protein, or antigenic fragment thereof), and its amino acid sequence is identical to that of a strain of Lasv, particularly a counterpart of the Lasv strain Josiah, which includes a polypeptide that is the spontaneous maturation or precursor of the Lasv protein, or, as defined herein, an antigenic fragment or variant thereof, having at least 50%, at least 80%, particularly advantageously at least 90%, or preferably at least 95% amino acid sequence identity with the spontaneously occurring Lasv GPC, NP, or Z protein. Amino acid sequence identity can be determined by alignment by those skilled in the art using manual alignment or using one of the many available alignment programs (e.g., BLASTP - http: / / blast.ncbi.nlm.nih.gov / ). The fragments or variants of the Lasv polypeptide of the present invention may be defined with respect to the specific amino acid sequences shown herein, particularly from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7. In certain embodiments of the present invention, the polypeptide shares at least 50%, at least 80%, particularly favorably at least 90%, or preferably at least 95% amino acid sequence identity with those native proteins of the LASV strain Josiah, or with the polypeptide of SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; or SEQ ID NO: 7.

[0037] In one aspect of the present invention, a polynucleotide encoding at least one polypeptide of LASV arises from or originates from the genome of an isolated and purified wild-type strain of LASV, including any Lassa strain whose genome has been fully or partially sequenced. At least some of these sequences can be found in the NCBI nucleotide database. In particular, the polynucleotide encoding at least one polypeptide of LASV arises from or originates from any Lassa strain that has been sequenced and referenced in Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus (Andersen Kristian G et al.; 2015; Cell. August 13, 2015; 162(4): pp. 738-750. doi:10.1016 / j.cell.2015.07.020), in particular, any Lassa strain referenced in the supplementary data of the paper in which the corresponding entrusted references for the strain name and sequence are enumerated. Preferably, the polynucleotides are derived from or originate from the Josiah strain, whose genomic sequences of two RNA segments can be found under GenBank accession number J04324.1 for the short genomic RNA encoding the NP and GPC proteins, and under European Nucleotide Archive accession number U73034.2 for the long genomic RNA encoding the Z and L proteins. The native proteins of the LASV strain Josiah can be defined as having sequences derived from RNA segments found under GenBank accession number J04324.1 for the short genomic RNA encoding the NP and GPC proteins, and under European Nucleotide Archive accession number U73034.2 for the long genomic RNA. The term “derive” as found in the definition of polynucleotides merely indicates that the sequence of the polynucleotide may be identical to the corresponding sequence of the LASV strain, or may be different insofar as it encodes a polypeptide, antigen, protein, or fragment thereof of LASV that satisfies the definition of “polypeptide” as described in the present invention. In particular, polynucleotides are derived from the nucleic acids of the LASV strain if they are codon-optimized with respect to such sequences.Therefore, the term does not restrict the mode of polynucleotide production.

[0038] Alternatively, the fragment may be a short polypeptide having at least 10 amino acid residues that carries an epitope of a native protein listed in the Immunoepitope Database and Analysis Resource (www.iedb.org). In this regard, the fragment also includes polypeptides.

[0039] In another embodiment of the present invention, the nucleic acid construct further comprises a second heterologous polynucleotide encoding at least one polypeptide of LASV, or an antigenic fragment thereof, selected from the group consisting of GPC protein, NP protein, mNP protein, and Z protein, wherein the second heterologous nucleotide is operably cloned into another ATU at a different position from the position of the first cloned heterologous polynucleotide, preferably upstream of the N gene of MeV, and in particular, the other ATU is ATU1 inserted upstream of the N gene of MeV. The second heterologous polynucleotide or its antigenic fragment encodes at least one polypeptide or its antigenic fragment that is different from the polypeptide encoded by the first heterologous polynucleotide.

[0040] In this embodiment, another ATU (known as reference ATU1) is advantageously located upstream of the N gene of MeV, at the N-terminal sequence of the cDNA molecule encoding the full-length (+) RNA strand of the MeV antigenome, while another ATU (ATU2) is preferably located between the P and M genes of the virus. Because the transcription of MeV viral RNA follows a gradient from the 5' end to the 3' end, we have found that cloning two polynucleotides at different positions within the cDNA encoding the full-length antigenome (+) RNA of the measles virus can result in a higher yield of antigenic particles and / or LASV virus-like particles (VLPs) than when the Z protein is encoded by at least one heterologous polynucleotide, although this production may not be significant when the polynucleotides are all cloned within a single and identical position. Furthermore, cloning heterologous polynucleotides at different positions can reduce the reduction in the expression of the encoded polypeptide. Indeed, if several genes are cloned within a single ATU, it can result in a reduction in the expression of the encoded polypeptide. Specific nucleic acid constructs described in this embodiment are shown in Figure 1b and the examples.

[0041] Within other ATUs, the second polynucleotide may encode one of the LASV polypeptides listed above, or its antigenic fragment. Thus, a second polynucleotide located within another ATU may encode the same polypeptide as the first polynucleotide inserted within the first ATU, and the second polynucleotide may encode at least one polypeptide common to the polypeptide encoded by the first polynucleotide. In preferred embodiments of the present invention, the first and second polynucleotides encode at least one different polypeptide or its antigenic fragment.

[0042] The cDNA molecule encoding the full-length antigenome (+) RNA strand of MeV is specific to or derived from attenuated strains of MeV. An "attenuated strain" of MeV is defined as a strain that is non-toxic or less toxic than the parent strain in the same host, but retains adjuvant activity, such as induction of T cell costimulatory proteins or the cytokine IL-12, when administered to a host to preserve immunogenicity and immunodominant T and B cell epitopes.

[0043] A measles virus attenuated strain therefore refers to a strain with a stable genome that does not undergo reversion to pathogenicity or integration into the host chromosome, which is then sequentially passaged in selected cells and adapted to other cells, possibly to produce seed strains suitable for the preparation of human vaccine strains. A strain approved for vaccine use as a specific “attenuated strain” is a suitable attenuated strain for the present invention if it meets the criteria defined by the FDA (U.S. Food and Drug Administration); that is, after rigorous review of laboratory and clinical data, it meets the safety, efficacy, quality, and reproducibility criteria (www.fda.gov / cber / vaccine / vacappr.htm).

[0044] In particular, cDNA molecules encoding the full-length antigenome (+) RNA strand of MeV are obtained from attenuated virus strains selected from the group including or comprising the Schwarz strain, Zagreb strain, AIK-C strain, Moraten strain, Philips strain, Beckenham 4A strain, Beckenham 16 strain, Edmonston seed A strain, Edmonston seed B strain, CAM-70 strain, TD 97 strain, Leningrad-16 strain, Shanghai 191 strain, and Belgrade strain. All of these strains are described in the prior art. The present invention particularly uses specific strains that enable use as a commercially available vaccine. In particular, cDNA molecules encoding the full-length antigenome (+) RNA strand of MeV are obtained from the Schwarz strain.

[0045] In certain embodiments of the present invention, the cDNA molecule is placed under the control of a heterologous expression control sequence.

[0046] Inserting such a control for cDNA expression is preferable when the expression of this cDNA is sought in cell types in which complete transcription of the cDNA is not possible with its native control sequence.

[0047] In certain embodiments of the present invention, the heterologous expression control sequence comprises a T7 promoter and a T7 terminator sequence. These sequences are located at the 5' and 3' ends of the coding sequence of the full-length antigenome(+)RNA strand of MeV, respectively, and are derived from sequences adjacent to this coding sequence.

[0048] In certain embodiments of the present invention, the cDNA molecule as defined herein above is modified, i.e., includes further nucleotide sequences or motifs.

[0049] In a preferred embodiment, the cDNA molecule used in accordance with the present invention further comprises a hammerhead ribozyme sequence following a GGG motif at its 5' end, adjacent to the first nucleotide of the nucleotide sequence encoding the full-length antigenomic (+) RNA strand of the MeV-approved vaccine strain, and a ribozyme sequence at its 3' end, adjacent to the last nucleotide of the nucleotide sequence encoding the full-length antigenomic (+) RNA strand. Hepatitis delta virus ribozyme (δ) is suitable for carrying out this preferred embodiment.

[0050] A GGG motif placed at the 5' end, adjacent to the first nucleotide of the coding sequence described above, improves the efficiency of transcription of the cDNA coding sequence. A requirement for the accurate assembly of measles virus particles is that the cDNA encoding the antigenomic (+) RNA follows rule 6 when the GGG motif is added, and a ribozyme is also added to the 5' end of the cDNA coding sequence and the 3' end of the GGG motif to allow the transcript to be cleaved at the first coding nucleotide of the full-length antigenomic (+) RNA strand of MeV.

[0051] To prepare the nucleic acid constructs of the present invention, the preparation of cDNA molecules encoding full-length antigenomic (+) RNA of measles virus as disclosed in the prior art is achieved by known methods. The resulting cDNA provides a basis for genomic vectors involved in the rescue of recombinant measles virus particles, particularly when inserted into vectors such as plasmids.

[0052] Specific cDNA molecules suitable for the preparation of nucleic acid constructs of the present invention are obtained using the Schwarz strain of measles virus. Plasmid pTM-MVSchw, which contains infectious MeV cDNA corresponding to the antigenome of the Schwarz MV vaccine strain and is used for the preparation of the heterologous polynucleotide-containing recombinant vector of the present invention, is described elsewhere (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): pp. 11546-54). Accordingly, the cDNA used in the present invention can be obtained as disclosed in International Publication No. 2004 / 000876, or from plasmid pTM-MVSchw deposited at the Pasteur Institute on June 12, 2002, under CNCM number 1-2889, the sequence of which is disclosed in International Publication No. 2004 / 000876, incorporated herein by reference. Plasmid pTM-MVSchw is obtained from the Bluescript plasmid and contains a polynucleotide encoding the full-length measles virus (+) RNA strand of the Schawarz strain, under the control of a T7 RNA polymerase promoter. It has 18,967 nucleotides, and its sequence is shown as Sequence ID No. 15. cDNA molecules from other MeV strains (also referred to for convenience as measles virus cDNA or MeV cDNA) can be obtained starting from nucleic acids purified from attenuated MeV virus particles, such as those described herein. Additional transcription units (ATUs) may be a cassette of multiple cloning sites pre-inserted into the vector, as described by Combredet et al., 2003. The ATU may contain cis-acting sequences necessary for the transcription of the inserted LASV gene. Heterogeneous polynucleotides are cloned or inserted into additional transcription units (ATUs) as defined herein above.

[0053] Heterogeneous polynucleotides may also be cloned or inserted into another ATU. For example, a third ATU may be inserted between the H and L genes of MeV, and a first or second heterogeneous polynucleotide may be cloned or inserted into this third ATU. In certain embodiments of the present invention, the nucleic acid construct may include a first heterogeneous polynucleotide inserted into the first ATU, a second heterogeneous polynucleotide sequence inserted into the second ATU at a different position from the first ATU, and a third heterogeneous polynucleotide inserted into the third ATU at a different position from the first and second ATUs.

[0054] In preferred embodiments, the nucleic acid construct comprises a specific mutant NP protein or a heterologous polynucleotide encoding its antigenic fragment. The native NP protein can be mutated to knock down its exonuclease activity. The NP protein with knocked-down exonuclease activity can be determined by a luciferase assay using a reporter luciferase gene under the control of an IRF3-dependent promoter, as described in PMID:21085117, and is shown in Figure 29. NP is involved in the virus-inducible inhibition of type I IFN signaling (Martinez-Sobrido, 2006). This activity is linked to the C-terminal domain of the native NP protein. Functional analysis has confirmed the exonuclease activity of LASV NP, which has been found to be a critical step in its type I IFN counteracting function (Qi, 2010). Therefore, we introduced two mutations into the exonuclease domain of the NP protein. The exonuclease domain of the NP protein is located within the C-terminal domain of the NP protein, particularly between amino acid residues 341 and 569 of SEQ ID NO: 3. Therefore, mutant NP proteins arise from native NP proteins that have been mutated (by deletion and / or addition and / or substitution of any amino acid residue) within the exonuclease domain, particularly as defined herein above, and whose exonuclease activity is knocked down according to the luciferase assay described above. In particular, substitutions of at least one amino acid residue D389, E391, D466, D533 and H528 of SEQ ID NO: 3 can result in mutant NP proteins that lack exonuclease activity. Specifically, the amino acid residue at position 389 of SEQ ID NO: 3 can be mutated, for example, by substitution of aspartic acid with alanine. Alternatively, the amino acid residue at position 392 of SEQ ID NO: 3 can be mutated, for example, by substitution of glycine with alanine. In a preferred embodiment, both amino acid residues at positions 389 and 392 of SEQ ID NO: 3 are mutated by substitution. In a more preferred embodiment, the mNP protein has the sequence of SEQ ID NO: 5.

[0055] In this embodiment, the mNP polypeptide encoded by the polynucleotide induces type I IFN. In other words, the immunosuppressive function of the innate NP is shut down in the mNP. Thus, it provides a nucleic acid construct containing a polynucleotide that increases the efficiency of the immunogenicity of chimeric recombinant MeV-LASV infectious particles.

[0056] In preferred embodiments, the present invention also relates to modifications, particularly the optimization of polynucleotides, that enable the effective expression of LASV polypeptides, proteins, antigens, or fragments thereof in host cells.

[0057] Therefore, the optimization of polynucleotide sequences can be manipulated while avoiding the cis-acting domains of nucleic acid molecules: internal TATA boxes, chi sites and ribosome entry sites; AT-rich or GC-rich sequence stretches; ARE, INS, CRS sequence elements; repeat sequences and RNA secondary structures; potential splice donor and acceptor sites and branching points.

[0058] The optimized polynucleotide may be codon-optimized for expression in specific cell types, and in particular, may be modified for the use of Maccaca codons or human codons. This optimization allows for increased efficiency of chimeric infectious particle production in cells without affecting the amino acid composition of the expressed protein.

[0059] In particular, the optimization of the polynucleotide encoding the LASV polypeptide can be carried out by modifying the unstable position of the codon without affecting the identity of the amino acid residues translated from the codon with respect to the original.

[0060] Optimization is performed while avoiding measles virus-derived editing sequences. Editing of measles virus transcripts is a method that occurs particularly in transcripts encoded by the measles virus P gene. This editing involves the insertion of an extra G residue at a specific site within the P transcript, resulting in a new, truncated protein compared to the P protein. The addition of only a single G residue results in the expression of a V protein containing a distinctive carboxyl terminus (Cattaneo R et al., Cell. 1989 Mar 10;56(5):759-64).

[0061] In the polynucleotides described in this particular embodiment of the present invention, the following edit-like sequences derived from the measles virus may be mutated: AAAGGG, AAAAGG, GGGAAA, GGGGAA, and their complementary sequences: TTTCCC, TTTTCC, CCCTTT, CCCCTT. For example, AAAGGG may be mutated to AAAGGC, AAAAGG to AGAAGG or TAAAGG or GAAAGG, and GGGAAA to GCGAAA.

[0062] Therefore, heterogeneous polynucleotides may contain one or more of the following sequences, or at least two of the following sequences, or at least three of the following sequences, or four of the following sequences: - Sequence ID 2 encoding the GPC protein; and / or - Sequence ID No. 4 encoding the NP protein; and / or - Sequence ID 6 encoding the mNP protein; and / or - Sequence ID 8, which codes for the Z protein.

[0063] Within heterogeneous polynucleotides, each sequence as defined herein may exist once or multiple times. In preferred embodiments of the present invention, each sequence may exist once within a single heterogeneous polynucleotide, or together once within a heterogeneous nucleotide.

[0064] One of the specific embodiments of the present invention provides a nucleic acid construct comprising polynucleotides that increases the efficiency of chimeric recombinant MeV-LASV infectious particle production.

[0065] Alternatively, or complementaryly, a heterologous polynucleotide may encode one of the following polypeptides, or an antigenic fragment thereof, or at least two of the following polypeptides, or at least three of the following polypeptides, or four of the following polypeptides: - The GPC protein of SEQ ID NO: 1 or its antigenic fragment; and / or - The NP protein of SEQ ID NO: 3 or its antigenic fragment; and / or - The mNP protein of SEQ ID NO: 5 or its antigenic fragment; and / or - The Z protein of sequence number 7 or its antigenic fragment.

[0066] It should be noted that polynucleotides may encode single or multiple polypeptides as defined above herein. In preferred embodiments, each polypeptide is single-encoded within a single heterologous polynucleotide, and more preferably, each polypeptide is single-encoded within multiple polypeptides. In certain embodiments of the present invention, several polypeptides, each encoding at least one LASV protein, are combined or fused to form a polynucleotide encoding several proteins of LASV. These polynucleotides may be distinguished from one another by the fact that they encode proteins of various strains of LASV, or different proteins of a LASV strain.

[0067] In a particular embodiment, the nucleic acid construct of the present invention has the following polynucleotides from the 5' end to the 3' end: (a) Polynucleotide encoding the MeV N protein; (b) Polynucleotides encoding the MeV P protein; (c) A first heterologous polynucleotide encoding at least one polypeptide selected from the group consisting of the GPC protein, NP protein, mNP protein, and Z protein of LASV, or an antigenic fragment thereof, particularly encoding a single polypeptide that is the GPC protein or an antigenic fragment thereof, or encoding at least two polypeptides that are either the GPC protein or an antigenic fragment thereof and either the NP protein or the mNP protein or an antigenic fragment thereof, particularly a first polynucleotide encoding the GPC protein and the mNP protein, which is particularly cloned operably within ATU, particularly within ATU2; (d) A polynucleotide encoding the M protein of MeV; (e) A polynucleotide encoding the F protein of MeV; (f) A polynucleotide encoding the H protein of MeV; (g) A polynucleotide encoding the L protein of MeV comprising, wherein said polynucleotide is operably linked within a nucleic acid construct and is under the control of viral replication and transcription control elements, such as the MeV leader and trailer sequences.

[0068] Some examples of this embodiment obtained according to the present invention are schematically shown in FIG. 1B: MeV-GPC LASV ; MeV-NP+GPC LASV ; MeV-NP ExoN +GPC LASV Constructs named as such belong to this particular embodiment, but other constructs not shown in FIG. 1B are also possible, such as MeV-GPC LASV +NP; MeV-GPC LASV +NP ExoN ; MeV-Z+GPC LASV ; MeV-GPC LASV +Z; MeV-Z+NP; MeV-Z+NP ExoN ; MeV-NP+Z; MeV-NP ExoN +Z; MeV-Z+GPC LASV +NP; MeV-Z+GPC LASV +NP ExoN; MeV-Z-NP-GPC LASV ; MeV-Z-NP ExoN -GPC LASV MeV-GPC LASV +NP+Z; MeV-GPC LASV +NP ExoN +Z; MeV-GPC LASV +Z+NP; MeV-GPC LASV +Z+NP ExoN ; MeV-NP+GPC LASV +Z; MeV-NP-Z+GPC LASV ; MeV-NP ExoN +GPC LASV +Z; MeV-NP ExoN- Z+GPC LASV It is included like this: Here, MeV corresponds to the cDNA molecule encoding the full-length antigenome (+) RNA strand of the measles virus (MeV); GPC LASV This corresponds to the polypeptide of the GPC protein, or a polynucleotide encoding its antigenic fragment; NP corresponds to the polypeptide of the NP protein, or the polynucleotide encoding its antigenic fragment; NP ExoN This corresponds to the polypeptide of the mNP protein, or a polynucleotide encoding its antigenic fragment; Z corresponds to the polypeptide of the Z protein, or a polynucleotide that codes for its antigenic fragment.

[0069] The expressions “N protein,” “P protein,” “M protein,” “F protein,” “H protein,” and “L protein” refer to the measles virus nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), and RNA polymerase large protein (L), respectively, and include references to their respective polypeptides or antigenic fragments. These components have been identified in the prior art and are disclosed in particular in Fields, Virology (Knipe & Howley, 2001).

[0070] In another specific embodiment of the present invention, the nucleic acid construct has the following polynucleotides from the 5' end to the 3' end: (a) A second heterologous polynucleotide encoding at least one polypeptide selected from the group consisting of the GPC protein, NP protein, mNP protein, and Z protein of LASV, or an antigenic fragment thereof, particularly encoding the Z protein or an antigenic fragment thereof, which is operably cloned within an ATU located upstream of the N gene of MeV, particularly within ATU1; (b) Polynucleotide encoding the MeV N protein; (c) Polynucleotide encoding the MeV P protein; (d) A first heterologous polynucleotide encoding at least one polypeptide selected from the group consisting of the GPC protein, NP protein, mNP protein, and Z protein of LASV, or antigenic fragments thereof, particularly a single polypeptide which is the GPC protein or its antigenic fragment, or at least two polypeptides which are either the GPC protein or its antigenic fragment, and the NP protein or mNP protein or their antigenic fragment, particularly a first heterologous polynucleotide encoding the GPC protein and the mNP protein, which is operably cloned in particular within an ATU, particularly within ATU2; (e) Polynucleotides encoding the MeV M protein; (f) A polynucleotide encoding the F protein of MeV; (g) A polynucleotide encoding the H protein of MeV; (h)MeV L protein encoding polynucleotide This includes, where the polynucleotide is operably bound within a nucleic acid construct and under the control of viral replication and transcriptional regulatory elements, such as MeV reader and trailer sequences.

[0071] Some examples of this embodiment are schematically shown in Figure 1B: Z-MeV-GPC LASVZ-MeV-NP+GPC LASV ; Z-MeV-NP ExoN +GPC LASV The construct named Z-MeV-GPC is included within this particular embodiment. When the LASV protein is named before MeV, the protein is cloned within an additional transcription unit located upstream of the N gene of MeV. It should be noted that several unseen constructs are also included by this embodiment. For example, heterologous polynucleotides may be cloned within a third ATU. LASV (ATU2)+NP(ATU3), or Z-MeV-NP(ATU2)-GPC LASV (ATU3), or Z-MeV-GPC LASV (ATU2)+mNP(ATU3), or Z-MeV-mNP(ATU2)-GPC LASV Nucleic acid constructs corresponding to (ATU3) are also included in the present invention. It should be noted that heterologous polynucleotides encoding at least one or any one of Z polypeptides, GPC polypeptides, and / or NP or mNP polypeptides may be inserted into ATU3. Various terms used herein have the same meanings as those used in the aforementioned specific embodiments.

[0072] In certain embodiments of the present invention, the nucleic acid construct comprises a nucleic acid encoding an NP protein, preferably the NP protein of SEQ ID NO: 3, or an antigenic fragment thereof, within a first heterologous polynucleotide; and a nucleic acid encoding a GPC protein, preferably the GPC protein of SEQ ID NO: 1. In preferred embodiments, this first heterologous polynucleotide is cloned between the P and M genes of MeV, preferably within ATU2 as defined herein above.

[0073] In certain embodiments of the present invention, the nucleic acid construct comprises, within a first heterologous polynucleotide, the nucleic acid of SEQ ID NO: 4 encoding an NP protein and the nucleic acid of SEQ ID NO: 2 encoding a GPC protein, preferably, these two nucleic acids being separated by a linker sequence. In preferred embodiments, the nucleic acid of SEQ ID NO: 4 is located upstream of the nucleic acid of SEQ ID NO: 2. This is, for example, MeV-NP+GPC LASV The structure, named [name of structure], is shown in Figure 1B.

[0074] In certain embodiments of the present invention, the nucleic acid construct comprises, within a first heterologous polynucleotide, a nucleic acid encoding an mNP protein, preferably the mNP protein of SEQ ID NO: 5, or an antigenic fragment thereof; and a nucleic acid encoding a GPC protein, preferably the GPC protein of SEQ ID NO: 1.

[0075] In certain embodiments of the present invention, the nucleic acid construct comprises, within a first heterologous polynucleotide, the nucleic acid of SEQ ID NO: 6 encoding an mNP protein and the nucleic acid of SEQ ID NO: 2 encoding a GPC protein, preferably, these two nucleic acids are separated by a linker sequence. In preferred embodiments, MeV-NP ExoN +GPC LASV As shown in Figure 1B by the construct named , the nucleic acid of SEQ ID NO: 6 is located upstream of the nucleic acid of SEQ ID NO: 2 (towards the 5' end of the construct). In a preferred embodiment, this first heterologous polynucleotide is cloned between the P and M genes of MeV, preferably within ATU2 as defined herein above.

[0076] In a particular embodiment of the present invention, the nucleic acid construct comprises a first heterologous polynucleotide and a second heterologous polynucleotide: - The second heterologous polynucleotide comprises a nucleic acid encoding the Z protein or its antigenic fragment, preferably the Z protein of Sequence ID No. 7; the second heterologous polynucleotide is preferably cloned into ATU1 as defined herein above, and - The first heterologous polynucleotide comprises a GPC protein or an antigenic fragment thereof, preferably a nucleic acid encoding the GPC protein of Sequence ID No. 1, and the first heterologous polynucleotide is preferably cloned into ATU2 as defined herein above.

[0077] In a particular embodiment of the present invention, the nucleic acid construct comprises a first heterologous polynucleotide and a second heterologous polynucleotide: - The second heterologous polynucleotide comprises the nucleic acid of Sequence ID No. 8 encoding the Z protein, and the second heterologous polynucleotide is preferably cloned into ATU1 as defined herein above, and - The first heterologous polynucleotide comprises the nucleic acid of Sequence ID No. 2 encoding the GPC protein, and the first heterologous polynucleotide is preferably cloned into ATU2 as defined herein above.

[0078] In a particular embodiment of the present invention, the nucleic acid construct has a sequence that - Sequence ID 9 (MeV-GPC); - Sequence ID 10 (MeV-NP-GPC); - Sequence ID 11 (MeV-mNP-GPC); - Sequence ID 12 (Z-MeV-GPC); - Sequence ID 13 (Z-MeV-NP-GPC); and - Sequence ID 14 (Z-MeV-mNP-GPC), It includes recombinant cDNA selected from the group consisting of the following sequences: Sequence ID 9 Sequence ID 9 is the sequence of a nucleic acid construct described in a particular embodiment of the present invention, wherein the construct contains a pTM1-MVSchwarz vector, in which the sequence encoding the GPC protein of LASV strain Josiah is cloned within additional transcription unit 2. Sequence ID 10 Sequence ID 10 is the sequence of a nucleic acid construct described in another specific embodiment of the present invention, wherein the construct contains a pTM1-MVSchwarz vector, wherein the sequence encoding the GPC protein of LASV strain Josiah is cloned into additional transcription unit 2, and the sequence encoding the NP protein of LASV strain Josiah is cloned into additional transcription unit 2. Sequence ID 11 Sequence ID 11 is the sequence of a nucleic acid construct described in another specific embodiment of the present invention, wherein the construct contains a pTM1-MVSchwarz vector, wherein the sequence encoding the GPC protein of LASV strain Josiah is cloned into additional transcription unit 2, and the sequence encoding the mutant NP protein of LASV strain Josiah is cloned into additional transcription unit 2. Sequence ID 12 Sequence ID 12 is the sequence of a nucleic acid construct described in another specific embodiment of the present invention, wherein the construct contains a pTM1-MVSchwarz vector, wherein the sequence encoding the GPC protein of LASV strain Josiah is cloned into additional transcription unit 2, and the sequence encoding the Z protein of LASV strain Josiah is cloned into additional transcription unit 1. Sequence ID 13 Sequence ID 13 is the sequence of a nucleic acid construct described in another specific embodiment of the present invention, wherein the construct contains a pTM1-MVSchwarz vector, wherein the sequence encoding the GPC protein of LASV strain Josiah is cloned into additional transcription unit 2, the sequence encoding the NP protein of LASV strain Josiah is cloned into additional transcription unit 2, and the sequence encoding the Z protein of LASV strain Josiah is cloned into additional transcription unit 1. Sequence ID 14 Sequence ID 14 is the sequence of a nucleic acid construct described in another specific embodiment of the present invention, wherein the construct contains a pTM1-MVSchwarz vector, wherein the sequence encoding the GPC protein of LASV strain Josiah is cloned into additional transcription unit 2, the sequence encoding the mutant NP protein of LASV strain Josiah is cloned into additional transcription unit 2, and the sequence encoding the Z protein of LASV strain Josiah is cloned into additional transcription unit 1.

[0079] The present invention also relates to transduction vectors, which can be used for the preparation of recombinant MeV-LASV particles when rescued from helper cells or producing cells. Several transduction vectors are shown in Figures 31 to 36. In a preferred embodiment of the present invention, the transduction vector is a transduction vector plasmid suitable for transfection of helper cells or producing cells and comprises the nucleic acid construct described in the present invention. The transduction vector plasmid can be obtained from a Bluescript plasmid and can be obtained by cloning the heterologous polynucleotide of the present invention into the pTM-MVSchw plasmid described herein. In a particular embodiment of the present invention, the transduction plasmid vector has the sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0080] The present invention also relates to the use of transducible plasmid vectors or nucleic acid constructs described in the present invention, which transform cells suitable for rescuing recombinant viral MeV-LASV particles, and in particular to the use of transfecting or transfecting such cells with plasmids or viral vectors each having the nucleic acid construct of the present invention, wherein the cells are selected based on their ability to express measles virus proteins necessary for proper replication, transcription and capsid formation of the recombinant genome of the virus corresponding to the nucleic acid construct of the present invention in recombinant, infectious, and replicable recombinant MeV-LASV particles.

[0081] The nucleic acid constructs and transplasmid vectors of the present invention are suitable and suitable for the preparation of recombinant infectious, replicable recombinant measles-lassa virus (MeV-LASV), and therefore the nucleic acid constructs and transplasmid vectors are suitable for insertion into transgenome vectors, and as a result, when the Z protein is encoded by at least one heterologous polynucleotide, the pTM-MVSchw plasmid contains a cDNA molecule of measles virus, particularly the Schwarz strain, for the production of the recombinant MeV-LASV virus and the expression of the LASV polypeptide, possibly as a LASV VLP. The pTM-MVSchw plasmid is suitable for the preparation of transvestments necessary for the expression of the LASV polypeptide, protein, antigen, or antigenic fragment thereof by insertion of heterologous polynucleotides as described herein. As used herein, the term “virus-like particle” (VLP) refers to a structure that has at least one property similar to a virus but has not been demonstrated to be infectious in itself. The virus-like particles according to the present invention do not have genetic information that codes for the virus-like particle protein, and generally, virus-like particles lack a viral genome and are therefore non-infectious and non-replicable. According to the present invention, virus-like particles can be produced in large quantities and expressed together with MeV-LASV recombinant particles.

[0082] The present invention also relates to cells or cell lines transformed by the introduction vector of the present invention, and further by polynucleotides that provide helper functions and proteins. Thus, the polynucleotides present in the cells encode proteins, in particular measles virus N, P, and L proteins (i.e., the native MeV protein or its functional variants capable of forming a ribonucleoprotein (RNP) complex), preferably as proteins stably expressed with at least the N and P proteins functional for transcription and replication of recombinant viral MeV-LASV particles. The N and P proteins may be expressed in cells from plasmids containing their coding sequences, or from DNA molecules inserted into the cell's genome. The L protein may be expressed from a different plasmid. It may be expressed transiently. Helper cells may also express an RNA polymerase suitable for enabling the synthesis of recombinant RNA from the nucleic acid construct of the present invention, possibly as a stably expressed RNA polymerase. The RNA polymerase may be T7 phage polymerase or its nuclear form (nlsT7).

[0083] In one embodiment, the measles virus cDNA clone is derived from the same measles virus strain as the N protein and / or P protein and / or L protein. In another embodiment, the measles virus cDNA clone is derived from a different virus strain than the N protein and / or P protein and / or L protein.

[0084] Cells transformed or transfected with the nucleic acid constructs described in the present invention can produce recombinant measles virus and / or LASV VLPs if the Z protein is encoded by at least one heterologous polynucleotide. Thus, recombinant measles virus can contain the nucleic acid constructs of the present invention in its genome and express at least one polypeptide, protein, or antigenic fragment of LASV. Thus, the measles virus of the present invention can express GPC protein, or GPC polypeptide, or antigenic fragment thereof; and / or NP protein, or NP polypeptide, or antigenic fragment thereof; and / or mNP protein, or mNP polypeptide, or antigenic fragment thereof; and / or Z protein, or Z polypeptide, or antigenic fragment thereof. LASV VLPs may contain at least Z protein, or antigenic fragment thereof, and may further contain one of the other polypeptides of LASV; GPC protein, GPC protein-like GP1 or GP2 fragment, NP protein, and / or mNP protein. In a preferred embodiment, the LASV VLP comprises a Z protein or its antigenic fragment, and a GPC protein or its antigenic fragment, or a GPC protein-like GP1 and / or GP2 fragment. In another preferred embodiment, the LASV VLP comprises a Z protein or its antigenic fragment, a GPC protein or its antigenic fragment, and an mNP protein or NP protein or its antigenic fragment.

[0085] In a preferred embodiment of the present invention, recombinant measles virus expresses the GPC protein and mNP protein of LASV. In another preferred embodiment of the present invention, recombinant measles virus expresses the GPC protein and Z protein of LASV.

[0086] Furthermore, according to some embodiments of the present invention, recombinant measles virus also expresses at least one polypeptide or protein of measles virus, or an antigenic fragment thereof. In other words, recombinant measles virus expresses at least one of the following polypeptides: MeV's N protein, P protein, M protein, F protein, H protein, and L protein.

[0087] In this embodiment, the recombinant virus expresses recombinant antigenic particles of measles virus and lassa virus, enabling the induction of a cellular response, or a humoral response, or both cellular and humoral responses, to the polypeptides of LASV and MeV. In certain embodiments of the present invention, the induction of a cellular response includes the induction of a T cell response, particularly a CD4+ and / or CD8+ T cell response.

[0088] Therefore, the present invention is: (a) Transfecting cells, particularly helper cells, particularly HEK293 helper cells, with a nucleic acid construct or an introduction plasmid vector described in the present invention to stably express T7 RNA polymerase and measles N and P proteins; (b) A step of maintaining transfected cells under conditions suitable for the production of recombinant measles virus and / or LASV VLP; (c) A step of infecting cells and enabling the reproduction of recombinant measles virus and / or LASV VLP by co-culturing them with the transfected cells of step (b); (d) Recovering LASV VLPs expressing at least one LASV protein, preferably at least a GPC protein and optionally an NP protein, mNP protein and / or a Z protein, preferably a recombinant measles virus expressing a GPC protein and an mNP protein, and / or at least a Z protein and possibly another LASV protein selected from the group consisting of a GPC protein, an NP protein and / or an mNP protein. This invention relates to a method for preparing recombinant infectious measles virus particles, including [specific component].

[0089] In certain embodiments, the present invention: a) A step of introducing an introduction vector containing the nucleic acid construct of the present invention or a protein necessary for transcription, replication, and encapsulation of the MeV antigenomic (+) RNA sequence derived from its cDNA into a helper cell line, under conditions that enable the assembly of viral particles, particularly a step of transfecting, and b) A step of recovering recombinant infectious MeV-LASV virus expressing at least one polypeptide or protein of LASV, or an antigenic fragment thereof. This invention relates to a method for preparing recombinant infectious measles virus particles, including [specific component].

[0090] In a particular embodiment of the present invention, the method is: a) A step of transfecting helper cells with a nucleic acid construct according to the present invention via an introduced plasmid vector, wherein the helper cells are capable of expressing RNA polymerase and expressing the N, P, and L proteins of the MeV virus; b) A step of co-culturing the transfect helper cells from step a) with subculturing cells suitable for subculturing a MeV attenuated strain from which cDNA is generated; c) A step of recovering recombinant infectious MeV-LASV virus expressing at least one polypeptide of LASV. Includes.

[0091] Another specific embodiment of the present invention provides a method for producing recombinant infectious MeV-LASV: a) A step of recombining cells or cell cultures that stably produce RNA polymerase, measles virus nucleoprotein (N), and measles virus polymerase cofactor phosphoprotein (P) with the nucleic acid construct of the present invention and a vector containing nucleic acid encoding measles virus RNA polymerase large protein (L), and b) A step of recovering infectious MeV-LASV virus from the recombinant cells or cultures of recombinant cells. Includes.

[0092] In certain embodiments of the method, recombinant MeV is produced expressing a LASV protein containing at least a GPC protein and / or a LASV VLP containing at least a Z protein, where the recombinant MeV and / or VLP may express at least one other LASV protein or antigen or antigenic fragment thereof, e.g., the GPC of LASV or a fragment thereof, particularly GP1 and / or GP2, and optionally mNPs. In other embodiments, the LASV VLP comprises the Z protein or a fragment thereof, and optionally the GPC protein, and possibly GP1 and / or GP2. In preferred embodiments of the present invention, the LASV VLP comprises the Z protein or an antigenic fragment thereof, and the GPC protein or an antigenic fragment thereof. A method for rescuing recombinant MeV expressing a LASV protein, particularly a LASV VLP, as shown in the drawings, is: 1) The process of cotransfecting helper cells, particularly HEK293 helper cells, that stably express T7 RNA polymerase and measles N and P proteins with (i) an introduction vector, particularly a plasmid, containing cDNA encoding full-length antigenome (+) RNA of measles virus recombinant with at least one polynucleotide encoding at least one LASV protein, such as GPC protein, NP protein, mNP protein and / or Z protein, and (ii) a vector, particularly a plasmid, encoding MeV L polymerase cDNA; 2) A step of culturing the cotransfected helper cells under conditions that enable the production of MV-LASV recombinant virus; 3) A step of growing the recombinant virus thus produced by co-culturing the helper cells from step 2) with cells that enable proliferation, such as Vero cells; 4) Steps to recover the replicated MeV-LASV recombinant virus and LASV protein, particularly LASV virus-like particles. This includes the following steps.

[0093] As used herein, “recombinate” means introducing at least one polynucleotide into a cell, for example in the form of a vector, to which the polynucleotide integrates (in whole or in part) or does not integrate into the cell. In certain embodiments, recombination is obtained by a first polynucleotide and is a nucleic acid construct of the present invention. Recombination may also or may encompass the step of introducing a polynucleotide and is a vector encoding the measles virus RNA polymerase large protein (L), the definition, properties and expression stability of which are described herein.

[0094] In accordance with the present invention, cells, cell lines, or cell cultures that stably express RNA polymerase, measles virus nucleoprotein (N), and measles virus polymerase cofactor phosphoprotein (P) are the cells or cell lines, or cell cultures, as defined herein, that is, recombinant cells insofar as they are transformed by the introduction of one or more polynucleotides as described above. In certain embodiments of the present invention, cells, cell lines, or cell cultures that stably produce RNA polymerase, N, and P proteins do not produce measles virus L protein, or do not stably produce measles virus L protein, but allow for transient expression or production thereof, for example. Production of recombinant MeV-LASV virus in the present invention may include the introduction of cells transformed as described herein. As used herein, “introduction” refers to the step of plated recombinant cells onto different types of cells, in particular onto monolayers of different types of cells. These latter cells are capable of both replication and production of infectious recombinant MeV-LASV virus, i.e., maintaining the release of these infectious viruses extracellularly by intracellular infectious virus formation and possibly LASV immunogenic particles and / or LASV VLPs, respectively. This introduction results in co-culture of recombinant cells of the present invention with receptive cells as defined in the preamble. The above introduction may be an additional, i.e., optional step, if the recombinant cells are not in an effective virus-producing culture, i.e., infectious recombinant MeV-LASV virus cannot be effectively recovered from these recombinant cells. This step is performed after further recombination of the recombinant cells of the present invention by introduction with any nucleic acid construct of the present invention and, optionally, a vector containing nucleic acid encoding measles virus RNA polymerase large protein (L).

[0095] In certain embodiments of the present invention, the introduction step is necessary because recombinant cells, which are typically selected for their ability to be easily recombined, are not sufficiently effective for maintaining and producing recombinant infectious MeV-LASV virus. In the embodiments described above, the cell or cell line or cell culture in step 1) of the method described above is the culture of recombinant cells or cell lines or recombinant cells as described in the present invention.

[0096] Suitable cells for preparing recombinant cells of the present invention are prokaryotic or eukaryotic cells, particularly animal or plant cells, and more specifically mammalian cells, such as human cells or non-human mammalian cells, or bird cells or yeast cells. In certain embodiments, cells are isolated from either a primary culture or a cell line before their genome is recombined. The cells of the present invention may be dividing cells or non-dividing cells.

[0097] In a preferred embodiment, the helper cells are derived from human embryonic kidney cell line 293, which is deposited under ATCC number CRL-1573. The specific cell line 293 is the cell line disclosed in International Publication No. 2008 / 078198, which is referred to as 293T7 / N / P in the following embodiments. Accordingly, the present invention also relates to host cells, particularly bird cells or mammalian cells, that are transfected or transformed with the nucleic acid constructs described in any embodiment of the present invention, or that are transfected with the transplasm vector. Preferred cells are VERO NK cells (African green monkey kidney cells) and MRC5 cells (Medical Research Council cell line 5). In another aspect of this method, cells suitable for passage are CEF cells (chicken embryonic fibroblasts). CEF cells can be prepared from EARL Morizeau (8 rue Moulin, 28190 Dangers, France) or from fertilized chicken eggs obtained from any other producer of fertilized chicken eggs.

[0098] The methods disclosed in accordance with the present invention are advantageously used for the production of infectious, replicable recombinant MeV-LASV viruses suitable for use as immunizing compositions. Accordingly, the present invention relates to compositions, in particular antigenic compositions, the active ingredient comprising infectious, replicable recombinant MeV-LASV viruses rescued from nucleic acid constructs of the present invention, and obtained by the methods particularly disclosed. The compositions may be vaccine compositions for administration to humans, in particular children, who require it. The compositions may be used for treatment of LASV infection. The compositions may be used for protection against LASV. Accordingly, the compositions may be immunogenic or antigenic compositions for protective or prophylactic treatment against LASV infection. In particular, the active ingredient or active ingredient in the composition comprises recombinant MeV-LASV particles, the recombinant MeV-LASV particles rescued from an introduction plasmid vector described in the present invention, and optionally associate with a VLP containing the Z protein and optionally other proteins of LASV, or antigenic fragments thereof. In the context of the present invention, the term “associated” or “associated” refers to the presence of both MeV-LASV recombinant virus particles and LASV polypeptides or proteins within a single composition, particularly as VLPs, usually as physically separate entities. In certain embodiments of the present invention, the composition is a vaccine.

[0099] The present invention also relates to recombinant MeV-LASV infectious replicating virus particles, possibly associated with LASV polypeptides or proteins, or antigenic fragments thereof, for use in the treatment or prevention of Lassa virus infection in subjects, particularly human subjects, particularly children, or any composition described in the present invention.

[0100] The present invention also relates to recombinant MeV-LASV infectious, replicable virus and associated LASV polypeptides or proteins, or antigenic fragments thereof, as well as potentially associated LASV VLPs, for use in administration schemes and in particular in human subjects, especially children, in administration regimens that induce an immune response, advantageously a protective immune response, against LASV virus infection or induced disease.

[0101] In certain embodiments of the present invention, a composition or use of a composition can induce immunization in a subject, particularly a human subject, particularly a child, after a single injection. In other words, a composition or use of a composition may require a single dose of a selected dose of recombinant MeV-LASV infectious replicable virus, or it may require multiple doses in a priming-boosting regimen. Priming and boosting may be achieved by the same active ingredient comprising recombinant MeV-LASV infectious, replicable virus and associated LASV polypeptides and proteins, or their antigenic fragments, and / or LASV VLPs.

[0102] The present invention also relates to assemblies of different active ingredients, one of which is a recombinant MeV-LASV infectious, replicable virus and associated LASV polypeptide or protein, and / or LASV VLP. The assemblies of active ingredients are advantageous for use in immunization of hosts, particularly human hosts.

[0103] The inventors have shown that administration of recombinant MeV-LASV infectious, replicable virus induces an immune response, particularly the production of neutralizing antibodies against LASV-related polypeptides. Therefore, administration of the active ingredient described in the present invention has been shown to induce host immunity. The vaccine described in the present invention is safe and elicits an immune response within the host, which includes particularly CD4+ and CD8+ T cell responses. As shown in the examples, the vaccine described in the present invention induces an antigen-specific T cell response. Immune monkey hosts have also been shown to survive a lethal dose of LASV.

[0104] Following host immunization and LASV loading, levels of liver enzymes (ALT and AST), lactate dehydrogenase (LDH), C-reactive protein (CRP), and albumin remain normal or slightly increase in immunized monkey hosts, while these levels increase several-fold in non-immunized hosts.

[0105] The compositions described in the present invention can induce the production of recombinant LASV-specific immunoglobulins, particularly IgM and IgG, as well as neutralizing antibodies. The compositions described in the present invention are safe vaccines that are immunogenic and effective in the host. The compositions and their use confer at least a T-cell response and immunity to Lassa virus infection in vaccinated hosts.

[0106] The compositions described in the present invention can also induce the production of MeV-specific immunoglobulins, particularly IgM and IgG, as well as neutralizing antibodies. The compositions described in the present invention are safe vaccines that are immunogenic and effective in the host. The compositions and their use can confer at least a T-cell response and immunity to measles virus infection in vaccinated hosts.

[0107] The compositions described in the present invention also relate to recombinant MeV-LASV infectious, replicable virus and associated LASV polypeptides or proteins, or antigenic fragments thereof, as well as potentially associated LASV VLPs, for use in administration schemes and in particular in human subjects, especially children, in administration regimens that induce an immune response, advantageously a protective immune response, against measles virus infection or induced disease.

[0108] The present invention also relates to LASV polypeptides or proteins, or antigenic fragments thereof, and / or LASV VLPs, or recombinant MeV-LASV infectious replicating virus particles that associate with any composition described in the present invention, for use in the treatment or prevention of measles virus infection in subjects, particularly human subjects, particularly children.

[0109] The present invention also relates to heterogeneous polynucleotides comprising any one of the codon-optimized sequences encoding a GPC protein, a Z protein, an NP protein, and / or an mNP protein. Accordingly, the present invention also relates to codon-optimized polynucleotides comprising or comprising SEQ ID NO: 2; SEQ ID NO: 4, SEQ ID NO: 6, and / or SEQ ID NO: 8.

[0110] Some of the drawings referenced in this application are in color. The submitted application includes color printouts of the drawings and can therefore be accessed by searching the application file at the Patent Office. [Brief explanation of the drawing]

[0111] [Figure 1] Figure 1 is a schematic diagram of a nucleic acid construct. A: MeV vector. B: Nucleic acid construct according to the present invention. The MeV gene is shown in gray, and the LASV gene is shown in green, blue, and red. For the MV gene: N (nucleoprotein); P / V / C (phosphoprotein and V / C protein); M (matrix); F (fusion protein); H (hemagglutinin); L (polymerase). For the LASV gene: NP (nucleoprotein); NPExoN (also referred to as NPKO in some figures; a mutant sequence encoding a mutant NP with its exonuclease activity knocked down); GPC (glycoprotein precursor); Z (zinc-binding protein). The ATU is indicated by a black arrow. ATU1 is located on the left, upstream of the N gene of MeV, while ATU2 is located in the center, between the P MeV gene and the M MeV gene. [Figure 2]Figure 2 shows the viral replication kinetics in Vero E6 cells. MeV-GFP corresponds to a construct in which the polynucleotide encoding the green fluorescent protein is inserted into ATU2. MeV-GFPLASV corresponds to a construct in which the polynucleotide encoding the GPC protein of LASV is inserted into ATU2. MeV-NP+GPCLASV corresponds to a construct in which the genes encoding the GPC and NP proteins of LASV are inserted into ATU2. MeV-NPExoN+GPCLASV corresponds to a construct in which the polynucleotide encoding the GPC protein and the mutant NP protein (with knocked-down exonuclease activity) of LASV are inserted into ATU2. MeV-Z+GPCLASV corresponds to a construct in which the polynucleotide encoding the GPC protein is inserted into ATU2, and the polynucleotide encoding the Z protein is inserted into ATU1. Titers were obtained in typical experiments and measured by TCID50 from three independent experiments. Mean and standard error are shown. [Figure 3] Figure 3 shows the expression of LASV proteins (GPC, NP, and Z) and MeV protein (F) in infected Vero E6 cells and the supernatant of infected Vero E6 cells. The effect of each construct was evaluated by Western blotting as detailed in Materials and Methods. NI: Uninfected cells. ns: Non-specific. [Figure 4] Figure 4 shows the entry and replication of MeV-GFP in immune antigen-presenting cells derived from human peripheral blood mononuclear cells. [Figure 5] Figure 5 shows the expression of type I IFN in human primary macrophages infected with different MeV-LASV vectors. Quantitative RNA analysis of the type I IFN response by qPCR (quantitative expression of IFNa1, IFNa2, and IFNb) is performed. Expression is shown 24 hours after infection. All results are normalized to the GAPDH gene and expressed as induction ratios compared to GAPDH. [Figure 6]Figure 6 shows the cell surface expression of clusters of different markers CD80, CD86, and CD83-CD40 in macrophages infected with different MeV-LASV vectors. Flow cytometry of cell surface expression of co-activated molecules 48 hours after infection. [Figure 7] Figure 7 shows the expression of type I IFN in human primary dendritic cells infected with different MeV-LASV vectors. Quantitative RNA analysis of the type I IFN response by qPCR (quantitative expression of IFNa1, IFNa2, and IFNb) is shown. Expression is measured 24 hours after infection. All results are normalized to GAPDH and expressed as induction ratios compared to GAPDH. [Figure 8] Figure 8 shows the cell surface expression of clusters of different markers CD80, CD86, CD83, and CD40 in human primary dendritic cells infected with different MeV-LASV vectors. Flow cytometry of cell surface expression of co-activated molecules 48 hours after infection. [Figure 9] Figure 9 shows the body temperature of cynomolgus monkeys (Macaca fascicularis) during the 30-day post-immunization period. Monkeys 3, 4, and 4 were subcutaneously immunized with recombinant MeV strain Schwarz vaccine, recombinant MeV-NPExoN-GPC vaccine, and recombinant Z-MeV-GPC vaccine with a 50% tissue culture titer (TCID50) of 2.106, respectively. [Figure 10] Figure 10 shows the LASV antigen-specific CD4 and CD8 T cell responses in vaccinated cynomolgus monkeys (Macaca fascicularis). Flow cytometry was performed after stimulation of whole blood with overlap peptides specific to GPC, NP, and Z. [Figure 11]Figure 11 shows the clinical scores of cynomolgus monkeys (Macaca fascicularis) after being administered a lethal dose of the LASV strain Josiah. The clinical score is based on body temperature, body weight, ability to eat and hydrate normally, behavior, and clinical signs. A score of 15 is the endpoint for euthanasia. The lethal dose of the LASV strain consists of 1,500 FFU of the LASV strain Josiah injected subcutaneously into the animal. [Figure 12] Figure 12 shows the body temperature of cynomolgus monkeys (Macaca fascicularis) that were exposed to a lethal dose of the LASV strain Josiah. [Figure 13] Figure 13 shows the levels of liver enzymes (AST and ALT) in the plasma of immunized cynomolgus monkeys. [Figure 14] Figure 14 shows the plasma levels of LDH (A), CRP (B), and albumin (C) in cynomolgus monkeys (Macaca fascicularis) that were ingested with a lethal dose of the LASV strain Josiah. [Figure 15] Figure 15 shows viremia (RNA (A) and titer (B)) in cynomolgus monkeys infected with a lethal dose of the LASV strain Josiah. RNA quantification by qPCR. Titer measurement by a method known in the art. [Figure 16] Figure 16 shows the quantification of viral RNA in nasal secretions (A), oral secretions (B), and urine (C) of cynomolgus monkeys loaded with a lethal dose of LASV strain Josiah. RNA quantification was performed by qPCR. [Figure 17] Figure 17 shows the LASV RNA levels detected in the organs of loaded cynomolgus monkeys pre-immunized with different MeV-LASVs. [Figure 18] Figure 18 shows the LASV infectivity titers detected in the organs of loaded cynomolgus monkeys pre-immunized with different MeV-LASVs. [Figure 19]Figure 19 shows the IgM and IgG responses to LASV in cynomolgus monkeys treated with a lethal dose of the LASV strain Josiah. A. IgM LASV-specific. B: IgG LASV-specific. Immunoglobulin levels measured by ELISA. Optical concentration calculated by absorbance at 450 nM. [Figure 20] Figure 20 shows the LASV GP- and NP-specific CD8+ and CD4+ T cell responses after immunization. The percentage of CD8+ and CD4+ T cells producing IFNg, TNFα, and / or IL-2 after stimulation with overlapping peptides covering the entire LASV GP and NP was determined using flow cytometry. [Figure 21] Figure 21 shows the LASV antigen-specific CD4 and CD8 T cell responses of immunized cynomolgus monkeys loaded with a lethal dose of the LASV strain Josiah. Flow cytometry after stimulation of whole blood with GPC and NP-specific overlap peptides. [Figure 22] Figure 22 shows the proliferation and activation of CD4 and CD8 T cells in immunized cynomolgus monkeys treated with a lethal dose of the LASV strain Josiah. CD8 proliferation (A) is assessed by Ki67 staining. CD4 (B) and CD8 (C) activation are assessed by quantification of granzyme B expression. [Figure 23] Figure 23 shows the LASV GP- and NP-specific CD8+ T cell responses after LASV loading. Following LASV loading with overlapping peptides covering both LASV GP (23A) and NP (23B), the percentage of CD8+ T cells producing IFNg, TNFα, and / or IL-2 was determined by flow cytometry. The proportions of different subpopulations of responsive T cells are represented using pie charts. [Figure 24]Figure 24 shows the LASV GP- and NP-specific CD4+ T cell response after LASV loading. Following LASV loading with overlapping peptides covering both LASV GP(23A) and NP(23B), the percentage of CD4+ T cells producing IFNg, TNFα, and / or IL-2 was determined by flow cytometry. The proportions of different subpopulations of responsive T cells are represented using pie charts. [Figure 25] Figure 25 shows the KEGG pathway analysis performed on transcriptome data obtained from cynomolgus monkey PBMCs collected at different time points after immunization with MeV-NPExoN-GPCLASV. [Figure 26] Figure 26 shows the KEGG pathway analysis performed on transcriptome data obtained from cynomolgus monkey PBMCs collected at different time points after immunization with MeV-Z-GPCLASV. [Figure 27] Figure 27 shows the quantification of cytokines in the plasma of monkeys immunized after LASV loading. Different cytokines were quantified in the plasma of cynomolgus monkeys immunized with MeV-, MeV-NPExoN-GPCLASV, and MeV-Z+GPCLASV after LASV loading. Significant differences (p<0.05) are shown between the different conditions: nc (MeV-NPExoN-GPCLASV and MeV), nz (MeV-NPExoN-GPCLASV and MeV-Z+GPCLASV), and n-cz (MeV-NPExoN-GPCLASV and MeV; MeV-NPExoN-GPCLASV and MeV-Z+GPCLASV). [Figure 28] Figure 28 shows the IgM and IgG responses to MeV in cynomolgus monkeys immunized with a lethal dose of the LASV strain Josiah. A: IgM MeV specific. B: IgG MeV specific. IgG and IGM MeV specificity was not measured on days 7 and 14 of monkeys immunized with the MeV construct. [Figure 29]Figure 29 shows the determination of exonuclease activity of native and mutant NP proteins. Induction ratio of virus-induced luciferase activity and immunostimulatory RNA-induced interferon-beta activation. CT: control. NPLASV: native NP protein. NPExoNLASV: mutant NP protein of sequence number 5. SeV: Sendai virus with moi=1. [Figure 30] Figure 30 shows the analysis of MeV-NPExoN-GPCLASV tropism. CHO cell lines were infected with LASV GPC or any mopeavirus pseudotyped with MeV-NPExoN-GPCLASV. GPC expression was analyzed by staining with anti-GP1 antibody. The nucleus was blue, while the stained anti-GP1 was green. [Figure 31] Figure 31 is a schematic diagram of the introduction vector plasmid described in the first embodiment of the present invention. The introduction vector has the sequence of Sequence ID No. 9. The measles gene encoding the N protein is located between nucleotides 189 and 1767. The measles gene encoding the P protein is located between nucleotides 1889 and 3412. The codon-optimized heterologous polynucleotide of Sequence ID No. 2 encoding GPC is located between nucleotides 3532 and 5007. ATU2 is located between nucleotides 3487 and 5071 minus heterologous polynucleotide insert. The measles gene encoding the M protein is located between nucleotides 5104 and 6111. [Figure 32]Figure 32 is a schematic diagram of the introduction vector plasmid described in the second embodiment of the present invention. The introduction vector has the sequence of Sequence ID No. 10. The measles gene encoding the N protein is located between nucleotides 190 and 1767. The measles gene encoding the P protein is located between nucleotides 1889 and 3412. The codon-optimized heterologous polynucleotide of Sequence ID No. 4 encoding the NP protein is located between nucleotides 3532 and 5241. The codon-optimized heterologous polynucleotide of Sequence ID No. 2 encoding the GPC is located between nucleotides 5386 and 6861. The linker sequence containing the measles virus regulatory sequence is located between nucleotides 5242 and 5385. ATU2 is located between nucleotides 3487 and 6925 minus the heterologous polynucleotide insert and the linker sequence. The measles gene encoding the M protein is located between nucleotides 6958 and 7965. [Figure 33] Figure 33 is a schematic diagram of the introduction vector plasmid described in the third embodiment of the present invention. The introduction vector has the sequence of Sequence ID No. 11. The measles gene encoding the N protein is located between nucleotides 190 and 1767. The measles gene encoding the P protein is located between nucleotides 1889 and 3412. The codon-optimized heterologous polynucleotide of Sequence ID No. 6 encoding the mutant NP protein is located between nucleotides 3532 and 5241. The codon-optimized heterologous polynucleotide of Sequence ID No. 2 encoding the GPC is located between nucleotides 5386 and 6861. The linker sequence containing the measles virus regulatory sequence is located between nucleotides 5242 and 5385. ATU2 is located between the heterologous polynucleotide insert and the linker sequence minus nucleotides 3487 and 6925. The measles gene encoding the M protein is located between nucleotides 6958 and 7965. [Figure 34]Figure 34 is a schematic diagram of an introduction vector plasmid described in a fourth embodiment of the present invention. The introduction vector has the sequence of Sequence ID No. 12. The codon-optimized heterogeneous polynucleotide of Sequence ID No. 8 encoding the Z protein is located between nucleotides 193 and 504. The measles gene encoding the N protein is located between nucleotides 646 and 2223. The measles gene encoding the P protein is located between nucleotides 2345 and 3868. The codon-optimized heterogeneous polynucleotide of Sequence ID No. 2 encoding the GPC is located between nucleotides 3988 and 5463. The measles gene encoding the M protein is located between nucleotides 5560 and 6567. [Figure 35] Figure 35 is a schematic diagram of the introduction vector plasmid described in the fifth embodiment of the present invention. The introduction vector has the sequence of Sequence ID No. 13. The codon-optimized heterogeneous polynucleotide of Sequence ID No. 8 encoding the Z protein is located between nucleotides 193 and 504. The measles gene encoding the N protein is located between nucleotides 646 and 2223. The measles gene encoding the P protein is located between nucleotides 2345 and 3868. The codon-optimized heterogeneous polynucleotide of Sequence ID No. 4 encoding the NP protein is located between nucleotides 3988 and 5697. The codon-optimized heterogeneous polynucleotide of Sequence ID No. 2 encoding the GPC is located between nucleotides 5842 and 7317. The measles gene encoding the M protein is located between nucleotides 7414 and 8421. [Figure 36]Figure 36 is a schematic diagram of the introduction vector plasmid described in the sixth embodiment of the present invention. The introduction vector has the sequence of Sequence ID No. 14. The codon-optimized heterogeneous polynucleotide of Sequence ID No. 8 encoding the Z protein is located between nucleotides 193 and 504. The measles gene encoding the N protein is located between nucleotides 646 and 2223. The measles gene encoding the P protein is located between nucleotides 2345 and 3868. The codon-optimized heterogeneous polynucleotide of Sequence ID No. 6 encoding the mutant NP protein is located between nucleotides 3988 and 5697. The codon-optimized heterogeneous polynucleotide of Sequence ID No. 2 encoding the GPC is located between nucleotides 5842 and 7317. The measles gene encoding the M protein is located between nucleotides 7414 and 8421. [Modes for carrying out the invention] [Examples]

[0112] Materials and methods Cells and viruses 293T7 / N / P cells, which stably express T7 polymerase and measles N and P proteins, were used to rescue recombinant measles virus and maintained as described above {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 Etablissement Francais du Sang (EFS, Lyon, France). Mononuclear cells were purified by Ficol density gradient centrifugation (GE Healthcare). Monocytes were first isolated from peripheral blood mononuclear cells by centrifugation on a cushion of 50% Percoll in PBS (GE Healthcare, Velizy, France), and then purified using 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 supplemented with 50 ng / mL M-CSF, 10% SVF, and 10% autologous serum. M-CSF was added every 2 days, and 40% of the culture medium was replaced.

[0113] Plasmid construct Codon-optimized ORFs for LASV GPC, NP, and Z (LASV strain Josiah) were cloned into the pTM1-MVSchwarz vector at additional transcription units (ATUs) located upstream of the nucleoprotein (N) (ATU1 for Z) or between the phosphoprotein (P) and matrix (M) genes of the Schwarz MV genome (ATU2, GPC alone or NP+GPC), as described above (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): 11546~54). All plasmid constructs were confirmed by sequencing.

[0114] Western blot and antibody Recombinant MeV-GFP, MeV-GPC LASV MeV-NP+GPC LASV MeV-NP ExoN +GPC LASV Or MeV-Z + GPC LASV Vero NK cells infected with [the virus] 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 membranes. The membranes were stained with primary antibodies against GP1 (in-house mouse monoclonal product), NP (mouse anti-LASV serum), Z (in-house rabbit polyclonal product), or F (rabbit polyclonal Fcyt, a kind gift from R. Cattaneo). Cell lysates were also stained with anti-actin antibodies conjugated to horseradish peroxidase (HRP). After staining with secondary antibodies conjugated to HRP, the membranes were confirmed using West Dura substrate (Pierce) and imaged using an LAS4000 imager (GE Healthcare).

[0115] Virus rescue and titer measurement Recombinant measles viruses expressing the LASV antigen were rescued as described above (Combredet, 2003; Radecke, F. et al., Rescue of measles viruses from cloned DNA. Embo J, 1995. 14(23): pp. 5773-5784; International Publication No. 2008 / 078198). Briefly, 293T7 / N / P cells were transfected with plasmids encoding measles L polymerase and the antigenic fragment of the desired MeV vector. Clonal syncytium was collected and used to infect Vero NK cells in a 6-well plate. When the syncytium reached approximately 50% of the well area, the cells were detached and placed on Vero NK cells in a 10 cm dish to produce a passage 1 (P1) stock. To prepare high-passage viral stocks, Vero NK cells were infected with an infection multiplicity (MOI) of 0.01 and then incubated at 32°C for 2–3 days. To recover the virus, the cells were scraped into Opti-MEM I low-serum medium and subjected to two freeze-thaw cycles. Titer was determined by the 50% tissue culture infectivity titer (TCID50) in Vero NK cells.

[0116] Quantitative RNA analysis For RT-qPCR experiments, total RNA was isolated from mock or infected cells using the Rneasy Mini Kit (Qiagen, Courtaboeuf, France) and a supplemental DNase step added using the Turbo DNA free kit Ambion (Thermo Fisher Scientific), following the manufacturer's instructions. cDNA synthesis was performed using SuperScript III, and amplification was carried out using the Gene Expression Master Mix kit (Applied Biosystems, Thermo Fisher Scientific). For type I IFN, primer / probe mixes were developed in-house. qPCR assay runs were performed on a LightCycler 480 (Roche Diagnostics, Meylan, France). All gene expression was standardized to that of the GAPDH gene and expressed as an induction ratio compared to GAPDH. For viral RNA quantification, RNA probes from the 771-934bp region of the NP ORF were cloned into a pGEM vector (Promega) to generate T7 promoter-driven transcripts. RNA probes were treated with DNAse, purified, and quantified (Dropsense96, Trinean, Gent, Belgium). Quantitative PCR of viral RNA was performed using LASV-specific primers with EuroBioGreen qPCR Mix Lo-ROX (Eurobio, Les Ulis, France).

[0117] Flow cytometry for MP activation, T cell activation, and proliferation. Mock and MOI 1-infected MPs were detached 48 hours after infection, saturated with human IgG, and surface-stained with antibodies against 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 analyzed from fresh whole blood. Cells were incubated at 37°C for 6 hours with a pool of GPC, NP, or Z overlapping peptides in the presence of CD28 and CD49d antibodies (2 μg / ml) and Brefeldin A (10 μg / ml). SEA (1 μg / ml) or PBS were used as positive or negative controls for activation, respectively. The peptides were 15-mer amino acid lengths (1 μg / ml each) with 11 residue overlaps, spanning the complete GPC, NP, or Z ORF of the LASV strain Josiah. PBS-EDTA 20 mM was added to the samples before cell surface staining for CD3, CD4, and CD8 (BD Biosciences). Red blood cells were then lysed using PharmLyse (BD Biosciences). The cells were then fixed and permeabilized for intracellular staining with an antibody against IFNγ (Biolegend). For proliferation and activation, wells were stained with antibodies against Ki67 or Granzyme B. Cells were analyzed by flow cytometry using an LSR Fortessa cytometer (BD Biosciences) or a 10-color Gallios cytometer (Beckman Coulter). Data were analyzed using Kaluza software (Beckman Coulter).

[0118] LASV-based cynomolgus monkey load A group of four male crab-eating macaques (Macaca Fascicularis, 32-39 months old, 3-4 kg) each had a ratio of 2.10 6 TCID50 MeV-NP ExoN +GPC LASV Or MeV-Z + GPC LASVThe monkeys were immunized by subcutaneous injection at Facility A2 (SILABE, France). A separate control group of three monkeys was immunized with the MeV vaccine strain Schwarz. Blood, oral, and nasal swabs, as well as urine samples, were taken every 2-3 days for the first two weeks, and then weekly until day 37, to assess vaccine replication and excretion, IgM and IgG responses, and T-cell responses to LASV GPC, NP, or Z. After 37 days, the monkeys were transferred to Facility BSL-4 (Laboratoire P4-Inserm Jean Merieux), where they were subcutaneously loaded with 1,500 FFU of the LASV strain Josiah. The animals were tracked for clinical signs of disease and euthanized based on a scoring system developed based on body temperature, weight, diet, hydration, behavior, and clinical signs. The experimental endpoint was set 28 days after loading, and all animals that survived to this point were euthanized according to the validated experimental procedure. Blood, oral, and nasal swab, as well as urine samples, were collected every 2-3 days for the first two weeks, and then weekly until day 28, to evaluate LASV virus replication and shedding, IgM and IgG responses to LASV GPC, NP, or Z, and T cell responses. This study was authorized by the Comite Regional d'Ethique en Matiere d'Experimentation Animale de Strasbourg (APAFIS#6543-20160826144775) and the Comite Regional d'Ethique pour l'Experimentation Animale Rhone Alpes (CECCAPP 20161110143954).

[0119] Determination of exonuclease activity of native and mutant NP proteins (Figure 29): 293T cells were co-transfected with 100 ng of a vector expressing the firefly luciferase (Fluc) reporter gene from a known functional promoter sequence of the IFN-beta gene using calcium phosphate, a variable amount of either the natural (wild-type) or mutant LASV NP vector, and 50 ng of a β-gal expression plasmid for normalization of transfection. Twenty-four hours post-transfection, cells were infected with Sendai virus (moi=1) to induce IFN-β expression. Twenty-four hours after infection, cell lysates were prepared for luciferase and β-gal assays. Fluc activity was normalized by β-gal levels. To determine whether the NP possesses exonuclease activity, its effect on suppressing immunostimulatory RNA-induced IFN production was analyzed, and HEK293 cells were transfected with pIFN-beta-LUC, a variable amount of the natural (WT) or mutant LASV NP vector, and a β-gal expression plasmid for normalization of transfection. After 18 hours, cells were transfected with either 1 μg of Poly(I:C) or 250 ng of Pichinde virion RNA using lipofectamine 2000. Luciferase activity was determined 18 hours after immunostimulatory RNA transfection and normalized by beta-gal activity. Mutant NP proteins with knocked-down exonuclease activity did not suppress immunostimulatory RNA-induced IFN production.

[0120] (Example 1) Generation of recombinant MeV virus expressing LASV antigen To determine the best combination of LASV antigens to introduce into a MeV vector for optimal immunogenicity, the inventors generated several MeV / LASV vaccine candidates to produce antigenic LASV virus-like particles (VLPs) in vivo, either by GPC alone or in combination with NP (mutated or without exonuclease domain to eliminate the immunosuppressive function contained in LASV NP), or using the Schwarz MeV vaccine platform expressing GPC and Z (Figure 1A). The inventors also constructed constructs expressing Z, GPC, and NP with or without mutations in the exonuclease domain. The GPC and NP genes were cloned between the MeV P and M genes in additional transcription unit 2 (ATU2). The Z gene was cloned upstream of the N gene in ATU1 (Figure 1B).

[0121] All viruses were rescued and grown to similar titers in Vero E6 cells as control MeV GFP expressing GFP from ATU2, except for MeV / LASV-Z+GPC which was attenuated by approximately 1 log compared to other constructs (Figure 2). Expression of different LASV antigens was controlled by Western blotting using specific antibodies against LASV GPC, NP, or Z, or against measles fusion protein F (Figure 3). GPC expression was controlled by MeV-GPC LASV MeV-NP+GPC LASV MeV-NP ExoN +GPC LASV and MeV-Z+GPC LASV Detected in infected Vero E6 cells; MeV-NP+GPC LASV , MeV-NP ExoN +GPC LASV and MeV-Z+GPC LASV NP expression in infected Vero E6 cells; MeV-Z+GPC LASV Only Z expression was detected in infected cells. MeV F expression was detected in all MeV-infected cells. As expected, GPC was also expressed along with Z in the MeV-Z-GPC group. LASVIt was detected in the supernatant of infected cells and supported GPC release along with Z under VLP morphology. All vectors were passaged 10 times without deletion of LASV antigen expression.

[0122] (Example 2) Immunogenicity of MeV virus expressing LASV antigen in human primary antigen-presenting cells To characterize the immunogenicity of different MeV vectors in human immune cells, we infected monocyte-derived macrophages and dendritic cells. As shown by GFP expression in Figure 4, MeV invades and replicates in these cells. However, the infectivity titer was only slightly detectable on post-infection day 1 and did not increase over time, suggesting that the virus did not effectively replicate in these cells, likely due to the induction of an innate antiviral response.

[0123] The inventors analyzed the immune responses of macrophages and dendritic cells to different recombinant viruses by combining flow cytometry analysis of activation markers and qPCR analysis of type 1 IFN responses. The inventors analyzed type 1 IFN responses induced by different vectors by qPCR 24 hours after infection (Figure 5). In macrophages, MeV-GPC LASV and MeV-Z+GPC LASV It induced IFN alpha-1, alpha-2, and beta at the same levels as the control MeV-GFP. However, the addition of LASV NP reduced the induction of type I IFN by almost 3 log (MeV-NP + GPC). LASV ), mutations in the ExoN domain of LASV NP (MeV-NP ExoN +GPC LASV ) induces type I IFNs with MEV-GFP and -GPC LASVThe levels recovered to a level comparable to that of the previous vector. This result demonstrates that LASV NP can control the induction of type I IFN, possibly by digesting dsRNA molecules expressed during MeV replication, through its ExoN activity (Son, 2015). We then investigated the induction of co-activated molecules by different vectors 48 hours after infection in macrophages (Figure 6). Importantly, cell surface expression of co-activated molecules is essential for the activation of the T cell response. As shown in Figure 6, all vectors induced strong cell surface expression of CD80, CD86, and CD83, particularly in MeV-NP+GPC compared to expression in macrophages infected with other vectors. LASV Expression was reduced in infected macrophages, but recovered when the ExoN domain of LASV NP was mutated.

[0124] Similar experiments were performed in dendritic cells (Figures 7 and 8). As observed in macrophages, MeV-GPC LASV and MeV-Z+GPC LASV This induced IFN alpha-1, alpha-2, and beta at the same levels as the control MeV-GFP (Figure 7). The addition of LASV NP also reduced the induction of type I IFN, but mutations in the ExoN domain of LASV NP reduced the induction of type I IFN in MEV-GFP and MEV-Z+GPC. LASV and MeV-GPC LASV The levels recovered to a level comparable to that of MEV-GFP and MEV-Z+GPC. LASV and MeV-GPC LASV The same induction was observed in dendritic cells infected with (Figure 8).

[0125] Vaccine strains of MeV-LASV induce type I IFN responses and cell surface expression of co-activated molecules; the presence of wild-type NPs strongly reduces the vaccine's ability to induce these effects, while mutations within the ExoN domain restore the vaccine's ability to induce these effects.

[0126] (Example 3) Safety, immunogenicity, and efficacy of two vaccines in cynomolgus monkeys. Based on the results obtained in human macrophages, the inventors decided to test two vaccine candidates in cynomolgus monkeys, the gold standard model for studying LASV pathogenicity. Three control animals were tested at 2.10 6 Subcutaneous immunization with the TCID50 recombinant MeV strain Schwarz vaccine resulted in two groups of four animals each showing a ratio of 2.10 6 TCID50 MeV-NP ExoN +GPC LASV and MeV-Z+GPC LASV Subcutaneous immunization was administered. The animals' health was then tracked for 37 days post-immunization (body temperature, weight, respiratory rate), and no adverse events were recorded. Clearly, the body temperature of the animals, continuously monitored thanks to the intraperitoneal device, was not altered by immunization (Figure 9).

[0127] The inventors also evaluated viremia in immunized animals every 2-3 days for two weeks after immunization, and then weekly, and could not detect any traces of viral RNA in either the plasma or PBMCs. Similarly, the inventors could not detect any viral RNA in the nasal and oral secretions or urine of vaccinated animals. Therefore, the vaccine candidate appears to be safe in monkeys and does not appear to be excreted at any point after immunization.

[0128] To evaluate the immunogenicity of the vector, the inventors performed an ELISA and detected LASV-specific IgM and IgG. LASV In immunized animals, specific IgM and IgG could not be detected. The inventors found that 37 days after immunization, four MeV-NPs were detected in four animals. ExoN +GPC LASV In three of the vaccinated animals, only low levels of LASV-specific IgG were detected. Furthermore, MeV-NP was detected in one animal. ExoN +GPC LASVVaccinated animals had neutralizing antibodies as demonstrated by plaque reduction neutralization tests (1:100 titer). The inventors also evaluated LASV-specific T cell responses by flow cytometry after stimulation of whole blood with overlapping peptides specific for GPC, NP, or Z. MeV-NP ExoN +GPC LASV In vaccinated animals, the inventors detected both CD4 and CD8 T cell responses to GP that started on day 7 post-immunization and declined by day 14 (Figure 10A and Figure 10C, orange bars). In these animals, the inventors also detected both CD4 and CD8 T cell responses to NP between days 10 and 21 post-immunization (Figure 10B and Figure 10D, orange bars). MeV-Z+GPC LASV In immunized animals, the CD4 T cell response to GPC was delayed compared to MeV-NP ExoN +GPC LASV vaccinated animals, starting by day 10 post-immunization but continuing until day 21 post-challenge (Figure 10A, green bar). The GPC-specific CD8 T cell response started at low levels on day 7 post-immunization but reached a peak on day 21 (Figure 10C, green bar). Both CD4 and CD8 Z-specific T cell responses also started on day 7 post-immunization, were detected until day 30, and reached a peak on day 21 (Figure 10B and 10D, green bars). Importantly, T cell responses to LASV antigens were not detected in MeV-vaccinated control animals. Thus, both vaccine candidates induced LASV antigen-specific T cell responses, and MeV-NP ExoN +GPC LASV induced a faster response than MeV-Z+GPC LASV .

[0129] (Example 4) Vaccine efficacy To test the efficacy of the vaccine candidates, immunized animals were challenged on day 37 post - immunization with a lethal dose (1,500 ffu, subcutaneous) of the LASV strain Josiah. The animals were then monitored for up to 30 days and assigned clinical scores based on their body temperature, body weight, ability to eat and drink normally, behavior, and clinical signs. A score of 15 was the endpoint for euthanasia. Three control animals had increasing scores starting on day 3 and had to be euthanized on days 12, 14, and 15 post - challenge, respectively (Figure 11A).

[0130] In contrast, all vaccinated animals survived the LASV infection, but the clinical outcomes differed depending on the vaccine. Indeed, MeV - NP ExoN +GPC LASV vaccinated animals showed a slight increase in clinical score by day 5, mainly due to an increase in body temperature (Figure 12, middle graph), with a maximum score of 3 (Figure 11B). In contrast, MeV - Z+GPC LASV immunized animals experienced severe symptoms such as high fever between days 3 and 12 (see Figure 12, bottom graph). Two animals recovered completely by day 12, while the other two animals showed difficulties in eating and drinking, fatigue, and one animal showed balance problems and a weight loss of more than 7.5% by day 30, reaching a score of 14 (Figure 11C).

[0131] <000J802>The inventors also followed several biological parameters in plasma over the course of infection, among other parameters, such as liver enzyme levels (ALT and AST), lactate dehydrogenase (LDH), C - reactive protein (CRP), and albumin. In control animals, liver enzyme levels started increasing on day 6 and continued to increase until the death of the animals (Figure 13, left panel). In contrast, in MeV - NP ExoN +GPC LASV vaccinated animals, liver enzyme levels remained normal at all time points (Figure 13, middle panel), and in MeV - Z+GPC LASV immunized animals, they increased only slightly between days 6 and 15 (Figure 13, right panel).

[0132] Plasma levels of LDH are a marker of tissue damage. In control animals, LDH levels began to increase on day 6 after loading and continued until the animals died (Figure 14A, left panel). MeV-NP ExoN +GPC LASV In vaccinated animals, no increase in infection was observed throughout the course of the infection (Figure 14A, center panel). However, LDH levels were elevated in MeV-Z+GPC LASV In immunized animals, levels increased between day 6 and day 15, and two animals (Figure 14A, right panel) with LDH levels similar to control animals on day 9 showed some tissue damage. CRP, an inflammation marker, also increased rapidly in control animals before death (Figure 14B, left panel). CRP levels were elevated in MeV-NP, except for one animal that showed a transient increase in CRP levels by day 6. ExoN +GPC LASV The group remained low (Figure 14B, center panel). Conversely, MeV-Z+GPC LASV All animals in the group showed increased CRP levels between day 3 and day 15 (Figure 14B, right panel). In this group, one animal showed a second wave of CRP synthesis between day 15 and day 30, indicating that the LASV virus was still replicating in this animal (Figure 14B, right panel, light green). The inventors also tracked plasma levels of albumin, renal, and hepatic dysfunction markers in immunized monkeys. In control animals, albumin levels decreased steadily starting on day 3 (Figure 14C, left panel), but these levels were associated with MeV-NP ExoN +GPC LASV In immunized animals, the levels remained constant (Figure 14C, center panel). MeV-Z+GPC LASV In the group, all animals experienced a decrease in albumin plasma levels between day 3 and day 12, but these levels eventually returned to normal by day 15 (Figure 14C, right panel).

[0133] Viremia in the loaded animals was also monitored post-loading by both qRT-PCR and titer measurement. As shown in Figure 15A, RNA levels in the blood of infected controls increased relentlessly from day 3 to day of slaughter, reaching 10 per mL in one animal on day 15. 9 The peak was reached in RNA copies (left panel). Infective titers were also detected in these animals (Figure 15B, left panel). MeV-NP ExoN +GPC LASV In vaccinated animals, viral RNA was detected only on day 6 after loading and was at a lower level compared to control animals (Figure 15A, center panel), and no associated viremia was detected (Figure 15B, center panel). Viral RNA levels were measured using MeV-Z+GPC. LASV The levels were higher in immunized animals, approximately 10 per mL on day 6. 6 RNA copy count peaked and decreased until day 15 (Figure 15A, right panel). Clearly, one animal recovered its RNA copy number in the blood by day 15 (Figure 15A, right panel, bright green), which may correlate with the recovery of CRP observed in the same animal (Figure 14B, right panel). Furthermore, infectivity titers were detected in all animals on day 6 and in animals showing elevated RNA levels in the blood up to day 15 (Figure 15B, right panel).

[0134] In addition to viremia, the inventors evaluated the presence of viral RNA in nasal and oral swabs of the infected animals. As shown in Figure 16, viral RNA levels peaked on day 9 in nasal and oral secretions of control animals (Figures 16A and 16B, left panel), decreased, but were still detectable at the time of death. Similarly, viral RNA levels were assessed in MeV-Z+GPC LASVIn these secretions from immunized animals, levels peaked on day 9 and recovered by day 15 in one or two animals (Figures 16A and 16B, right panel). Conversely, only small amounts of LASV RNA were detected in nasal swabs on day 3 in one animal and day 6 in another (Figures 16A and 16B, center panel), and were not associated with the presence of the infectious virus (data not shown). Furthermore, the inventors tracked viral RNA excretion in the urine of the loaded animals. LASV RNA levels were compared between control and MeV-Z+GPC. LASV It was detected only in immunized animals, and was initiated on day 9 or day 15 after loading, respectively (Figure 16C).

[0135] The amount of LASV RNA (Figure 17) and the LASV infectivity titer (Figure 18) are MeV and MeV-NP. ExoN -GPC LASV , and MeV-Z-GPC LASV Analysis was performed on different organs recovered during autopsy of animals immunized with MeV-Z-GPC. All MeV control animals showed detectable levels of LASV RNA and high viral titers in each organ tested except the bladder, with only one animal showing detectable levels of LASV RNA. The highest infective titers were found in the spleen, liver, and lungs. MeV-Z-GPC LASV In all animals immunized by MeV-NP, detectable levels of LASV RNA were found in the inguinal lymph nodes, mesenteric lymph nodes, and spleen. Detectable levels of LASV RNA were found in all organs, but not in any single animal within this group. Two animals showed Lassa virus infectivity titers in the inguinal lymph nodes, one animal showed Lassa virus infectivity titers in the spleen, and the other organs were free of the virus. ExoN -GPC LASV In groups of animals immunized by [the agent], detectable amounts of LASV RNA were found in lymphoid organs and in the lungs of 1-3 animals, but the presence of RNA was not associated with the presence of infectious Lassa virus.

[0136] (Example 5) Immune response to LASV To determine the immune response to infection, the inventors first measured the levels of LASV-specific immunoglobulin produced after LASV loading. The IgM response began on day 9 in all animals and peaked on day 12 (Figure 19A). Interestingly, IgM levels were obtained from the control group and from the MeV-Z+GPC group. LASV In animals from the group, the IgM response level was higher on day 12, and while it did not positively correlate with protection, it did correlate with viral load. Regarding the IgG response, the inventors found that all MeV-NPs were eliminated by day 9. ExoN +GPC LASV We noticed a strong induction of LASV-IgG in vaccinated animals, but MeV-Z+GPC LASV The IgG response in immunized animals only reached a similar level on day 15 (Figure 19B). LASV-specific IgG levels in control animals remained very low at all time points. Furthermore, serum neutralizing titers were lower than those of MeV-NP. ExoN -GPC LASV MeV-Z+GPC LASV The neutralizing titer was determined in the plasma of monkeys immunized with MeV at different time points after immunization (results are shown in Table 1). At the time of loading (i.e., 37 days post-immunization, J0 in Table 1), at least one animal per group had a neutralizing titer of 1 / 100e. MeV-NP was determined at 15 days post-loading and up to the day of necropsy. ExoN -GPC LASV and MeV-Z+GPC LASV All animals vaccinated with MeV-Z+GPC had neutralizing antibodies between 1 / 100e and 1 / 500e, the opposite of animals vaccinated with MeV. LASV Monkeys vaccinated with [the drug name] showed the highest titer on day 30. Neutralizing antibodies were detected in all animals except the control animals.

[0137] [Table 1]

[0138] Induction of LASV antigen-specific CD8+ and CD4+ T cells was also monitored postimmunolating by quantifying the percentage of T cells producing IFNg, TNFα, and / or IL-2 in response to overlap peptides covering all LASV, GP, NP, and Z proteins (Figure 20). T cells did not respond to the Z peptide (data not shown). The number of cytokine-producing T cells in response to GP and NP peptides increased only moderately compared to baseline levels (day 0) and MeV-control animals, with TNFα being the main cytokine involved in this response. Nevertheless, MeV-NP ExoN -GPC LASV Although immunization has been performed, MeV-Z+GPC LASV A not-so-significant increase in the percentage of GP-specific cytokine-producing CD8+ and CD4+ T cells was observed on day 21, separate from immunization. Furthermore, NP-specific cytokine-producing CD4+ and CD8+ T cells appeared at day 14 in immunized animals and were still present at day 22.

[0139] Similarly, the inventors tracked T cell responses to LASV GPC, NP, or Z after loading in T cell activation assays using overlap peptides. CD8 and CD4 responses to GPC and NP were compared with MeV-NP ExoN +GPC LASV The response was rapid and potent in vaccinated animals, peaking on day 9 and then slowly decreasing (Figure 21, orange bars). The CD8 and CD4 responses to GPC were observed in MeV-Z+GPC. LASV In immunized animals, the response was slow and weak, peaking on day 12 (Figure 21, green bars). These animals did not show a LASV-Z specific cellular response. Control animals experienced only very weak, transient CD8 and CD4 responses to GPC and NP between days 6 and 12 (Figure 21, red bars).

[0140] The intensity of the CD8 response correlated with the proliferation of these cells, as assessed by Ki67 staining (Figure 22A), and MeV-Z-GPC showed mild proliferation, peaking only on day 15 in control animals. LASV Compared to immunized animals, MeV-NP levels were lower up to day 9. ExoN +GPC LASV The immunized animals showed strong proliferation of CD8 T cells (orange bars are compared to red and green bars, respectively). This proliferation was observed in MeV-NP ExoN +GPC LASV Rapid and potent expression of granzyme B in immunized animals, as well as in control animals and MeV-Z+GPC LASV As demonstrated by the delayed response in immunized animals (Figures 22B and 22C, compare orange and green bars), it was also associated with cytotoxic phenotypes of CD8 and CD4 T cell responses.

[0141] We monitored the induction of LASV GP- and NP-specific T cells in animals immunized with LASV. Following LASV loading and stimulation of PBMCs with LASV Z peptide, no cytokine production was observed (data not shown). Data regarding cytokine-producing CD8+ and CD4+ T cells in immunized animals are shown in Figures 23 and 24, respectively. No significant response cells were found in animals immunized with MeV. MeV-NP ExoN +GPC LASV In animals immunized with MeV-Z+GPC, the percentage of CD8+ and CD4+ T cells producing cytokines in response to the GPC peptide rose to 2% and 0.6%, respectively, on day 12 post-loading, and then returned to basal levels by day 22. While the majority of T cells produced only IFNg, the proportion of multifunctional CD8+ and CD4+ T cells (Pf-T) producing at least two cytokines increased from day 12 to day 30. Within the CD4+ T cell group, the percentage of IFNg-producing cells decreased until day 30 post-loading, but the opposite was observed in Pf-T cells, increasing to 59%. LASVIn animals immunized with [the drug], moderate numbers of cytokine-producing CD4+ and CD8+ T cells were observed from day 15 and day 12, respectively. Most T cells produced only IFNg, and the Pf-T ratio remained at approximately 20% in CD8+ T cells but increased to 40% in CD4+ T cells. On day 30 post-loading, in all immunized animals, the prominent portion of T cells produced only TNFa.

[0142] In response to LASV NP peptides, trace amounts of cytokine-producing T cells from MeV-immunized animals were detected only on day 15 post-loading. (MeV-Z+GPC) LASV Response T cells from animals immunized with were detected on day 6 post-loading and reached a peak response on day 12. While T cell phenotypes varied after day 6, TNFα-secreting T cells were predominant on day 9. IFNg-producing T cells were dominant on day 12, but the proportion of Pf-T cells increased until day 30.

[0143] MeV-NP ExoN +GPC LASV and MeV-Z+GPC LASV The total cellular RNA content of PBMCs from monkeys immunized with MeV-NP was also extracted at different time points after immunization, and RNA sequencing was performed to analyze the different gene expression in PBMCs at different time points. To identify the pathways associated with these genes, enrichment analysis of differentially expressed genes was performed using ClusterProfiler (KEGG analysis). ExoN +GPC LASV and MeV-Z+GPC LASV The pathways differentially regulated throughout the time following immunization are shown in Figures 25 and 26, respectively. MeV-NP ExoN +GPC LASVIn animals immunized with MeV-Z+GPC, pathways involved in the immune response are activated in the first week postimmunization (D2 vs. D0, D4 vs. D0, and D7 vs. D0). Activation of hematopoietic cell lineages and the phospholipase D pathway leads to strong proliferation of immune cells during the first four days, associated with phagocytic function (FC-gamma-R mediated phagocytosis) and chemokine signaling. Increased Th1, Th2, and Th17 responses up to day 7 indicate T cell proliferation. During the second week postimmunization, pathways involved in regulating the immune response, particularly ubiquitin-mediated proteolysis, NF-κB signaling, and IL-17 signaling pathways, are activated. LASV Immunity resulting from this involves weak, transient activation of Th1, Th2, and Th17 cells on day 4 post-immunization, as well as MeV-NP. ExoN +GPC LASV Compared to results observed in animals immunized by [method / method], delayed activation of the NF-κB and IL-17 signaling pathways (day 14) appears to induce a weak immune response. Taken together, activation of different pathways supports immune activity and effective cellular responses.

[0144] Release of soluble mediators into the plasma of animals after immunization and LASV loading was observed. Among the 29 analytes quantified using the Luminex assay, no differences in soluble mediator levels were found among immunized animals (data not shown). In loaded animals (Figure 27), transient release of IFNg was detected in the plasma of all animals at levels peaking on days 6 and 9 post-infection, respectively, in immunized animals and MeV-controls. Nevertheless, lower concentrations were observed in MeV-NP. ExoN +GPC LASV Observed in the plasma of monkeys immunized with MeV-NP. Perforin concentrations increased in all animals until day 9 or 12, then decreased to low levels until day 22. Again, MeV-NP ExoN +GPC LASV The levels observed in monkeys immunized with [the drug] were low. Elevated soluble CD137 (sCD137) levels were observed in MeV and MeV-Z+GPC. LASVIn monkeys immunized by [the drug], MeV-NP was observed 9 days after infection, but only moderate concentrations of sCD137 were present. ExoN +GPC LASV It was observed in animals immunized with MeV and MeV-Z+GPC. IL-6 was detected in MeV and MeV-Z+GPC. LASV In animals vaccinated by [method], MeV-NP was found in the plasma of all animals by day 6 and was still present on day 9. ExoN +GPC LASV It was not detected in the plasma of animals immunized by MeV-Z+GPC. IL-6 levels were still elevated in the plasma of MeV-Z+GPC animals, but not in MeV-Z+GPC animals. LASV The increase was moderate in the plasma of animals immunized by [method / method]. The elevated amount of IL-8 was observed from day 6 in MeV and MeV-Z+GPC LASV It was observed in the plasma of animals immunized by [method], but only at low concentrations was MeV-NP observed. ExoN +GPC LASV In monkeys, it was detected between the 9th and 12th day. IL-18 is MeV-NP ExoN +GPC LASV Although not detected in monkey plasma, high and low levels of MeV and MeV-Z+GPC were observed, respectively. LASV It was detected in animals immunized with MeV-NP. ExoN +GPC LASV In monkeys, the levels remained at the standard level. Conversely, high concentrations were started on days 6 and 9, respectively, for MeV and MeV-Z+GPC. LASV Found in animals. Levels of IL-10 and IL-1 receptor antagonists (IL-1RAs) were measured in MeV-Z+GPC. LASV In monkeys, levels increased until day 9, then decreased, reaching low levels on day 22. IL-10 and IL-1RA levels remained elevated in MeV animals, but except for small amounts detected after day 6, IL-10 and IL-1RA were not present in MeV-NPs. ExoN +GPC LASV It was not released into the plasma of animals immunized by [the agent].

[0145] (Example 6) Immune response to MeV The levels of MeV-specific immunoglobulin produced in response to MeV-specific immunoglobulin were also evaluated by ELISA after loading (Figure 28A: IgM and Figure 28B: IgG). MeV-specific IgM and IgG were produced in all animals (MeV group, MeV-NP). ExoN +GPC LASV Group; MeV-Z+GPC LASV (group). Similar MeV-specific IgM and IgG responses are observed in MeV-NP ExoN -GPC LASV、 MeV-Z + GPC LASV , and induced by the MeV vaccine (Figure 28), MeV-NP ExoN +GPC LASV Or MeV-Z + GPC LASV Animals immunized by this method were then vaccinated against LASV (see Figure 15) and MeV (Figure 28) (Figure 28).

[0146] (Example 7) The tropism of MeV-LASV vaccine strains The tropism of MeV-LASV was analyzed. Lassa virus uses α-dehydrogenase (α-DG) as its receptor. MeV vaccine strains use CD46, SLAM, and nectin-4 as receptors. Mopeia virus was used as a control to analyze whether the introduction of Lassa antigen into the MeV vector affects the tropism of MeV vaccine strains. Mopeia virus is an arenavirus closely related to Lassa virus and uses the same receptor. Mopeia virus pseudotyped by Lassa virus GPC replicated in CHO-K1 cells expressing α-DG and in CHO-hCD46 cells expressing α-DG and human CD46, as shown in Figure 30, and staining with anti-GP1 was positive in both cell lines. Conversely, MeV-NP ExoN +GPC LASV Although it cannot replicate in CHO-K1 cells, it can replicate in the CHO-hCD46 cell line (Figure 30, photo below). Therefore, the introduction of Lassa antigen into the MeV vector does not expand the tropism of MeV.

[0147] conclusion In conclusion, MeV-NPExoN +GPC LASV and MeV-Z+GPC LASV The vaccine is safe, immunogenic, and effective in non-human primates. Both protected cynomolgus monkeys from a lethal load caused by the LASV strain Josiah after a single immunization. However, MeV-NP ExoN -GPC LASV This vector provided the best protection in all vaccinated monkeys through a strong T-cell response and near-bacterial immunity. Therefore, this vector is a candidate for advancement to clinical trials in humans. The immunogenicity of this vector before loading can certainly be improved by a prime / boost strategy. Nevertheless, we hereby demonstrate the principle that a single immunization can protect 100% of loaded animals. Furthermore, these vectors should protect monkeys against measles and therefore can be used as a bivalent vaccine in addition to emergency vaccines in endemic countries where LASV and MeV are major public health issues.

[0148] Sequence ID 1 Sequence ID 1 corresponds to the recombinant GPC protein of the Lassa virus strain Josiah, encoded by the codon-optimized sequence of Sequence ID 2. MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQYEAMSCDFNG GKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFN KQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR*

[0149] Sequence ID 2 Sequence ID 2 corresponds to the codon-optimized nucleotide sequence encoding the GPC protein of Sequence ID 1. 1 ATGGGCCAGA TTGTCACATT CTTTCAGGAA GTGCCACACG TCATTGAGGA GGTCATGAAC 61 ATCGTGCTGA TTGCTCTGTC AGTGCTGGCA GTGCTGAAAG GACTGTACAA CTTCGCTACC 121 TGTGGACTGG TGGGACTGGT CACATTCCTG CTGCTGTGCG GCAGAAGTTG CACTACCTCA 181 CTGTACAAAG GAGTGTACGA GCTGCAGACT CTGGAACTGA ACATGGAGAC ACTGAATATG 241 ACAATGCCTC TGAGCTGCAC CAAGAATAAT AGCCACCACT ATATCATGGT CGGGAACGAA 301 ACCGGCCTGG AACTGACCCT GACAAACACC AGCATCATTA ACCACAAGTT CTGCAATCTG 361 AGCGACGCTC ACAAGAAGAA CCTGTATGAC CACGCTCTGA TGTCCATCAT CAGTACCTTT 421 CACCTGTCCA TCCCCAATTT CAACCAGTAC GAGGCAATGT CATGCGACTT CAACGGGGGC 481 AAGATCAGTG TCCAGTACAA CCTGAGCCAC TCCTACGCCG GCGACGCAGC CAACCACTGC 541 GGAACTGTCG CCAATGGCGT GCTGCAGACA TTCATGAGGA TGGCATGGGG GGGATCTTAC 601 ATCGCACTGG ATAGCGGCAG GGGCAATTGG GATTGCATCA TGACTTCCTA TCAGTATCTG 661 ATTATCCAGA ATACTACATG GGAGGATCAT TGCCAGTTCA GTCGGCCCAG CCCTATTGGA 721 TATCTGGGGC TGCTGTCACA GAGAACACGG GATATCTATA TTTCAAGACG CCTGCTGGGC 781 ACATTCACTT GGACACTGTC AGACAGTGAG GGCAAGGATA CTCCAGGGGG CTACTGCCTG 841 ACACGATGGA TGCTGATCGA AGCAGAGCTG AAATGCTTCG GCAATACCGC AGTGGCCAAG 901 TGCAACGAGA AACACGACGA GGAGTTCTGC GACATGCTGA GGCTGTTCGA CTTCAACAAA 961 CAGGCTATCC AGAGACTGAA GGCAGAAGCC CAGATGTCAA TCCAGCTGAT CAACAAGGCA 1021 GTGAACGCCC TGATCAACGA CCAGCTGATC ATGAAGAACC ACCTGAGAGA CATTATGGGC 1081 ATCCCCTACT GTAATTACAG CAAGTATTGG TACCTGAACC ACACTACAAC CGGGAGAACA 1141 TCCCTGCCCA AGTGCTGGCT GGTCAGCAAT GGGAGTTATC TGAATGAAAC CCATTTCAGC 1201 GACGATATCG AACAGCAGGC TGACAACATG ATCACAGAGA TGCTGCAGAA AGAGTACATG 1261 GAAAGACAGG GCAAGACACC ACTGGGACTG GTCGATCTGT TCGTCTTCTC CACTAGCTTC 1321 TATCTGATTT CCATCTTCCT GCACCTGGTG AAGATCCCCA CTCATAGGCA CATTGTCGGC 1381 AAGAGTTGCC CTAAACCCCA TAGGCTGAAT CACATGGGGA TTTGTAGTTG CGGCCTGTAT 1441 AAGCAGCCTG GCGTGCCTGT GAAATGGAAG AGATGA

[0150] SEQ ID NO: 3 SEQ ID NO: 3 corresponds to the recombinant NP protein of the Lassa virus strain Josiah encoded by the codon-optimized sequence of SEQ ID NO: 4. MSASKEIKSFLWTQSLRRELSGYCSNIKLQVVKDAQALLHGLDFSEVSNVQRLMRKERRDDNDLKRLRDLNQAVNNLVELKSTQQKSILRVGTLTSDDLLILAADLEKLKSKVIRTERPLSAGVYMGNLSSQQLDQRRALLNMIGMSGGNQGARAGRDGVVRVWDVKNAELLNNQFGTMPSLTLACLTKQGQVDLNDAVQALTDLGLIYTAKYPNTSDLDRLTQSHPILNMIDTKKSSLNISGYNFSLGAAVKAGACMLDGGNMLETIKVSPQTMDGILKSILKVKKALGMFISDTPGERNPYENILYKICLSGDGWPYIASRTSITGRAWENTVVDLESDGKPQKADSNNSSKSLQSAGFTAGLTYSQLMTLKDAMLQLDPNAKTWMDIEGRPEDPVEIALYQPSSGCYIHFFREPTDLKQFKQDAKYSHGIDVTDLFATQPGLTSAVIDALPRNMVITCQGSDDIRKLLESQGRKDIKLIDIALSKTDSRKYENAVWDQYKDLCHMHTGVVVEKKKRGGKEEITPHCALMDCIMFDAAVSGGLNTSVLRAVLPRDMVFRTSTPRVVL*

[0151] Sequence number 4 Sequence number 4 corresponds to the codon-optimized nucleotide sequence encoding the NP protein of sequence number 3. 1 ATGAGTGCCA GCAAAGAAAT CAAGAGCTTC CTGTGGACCC AGAGTCTGCG GAGGGAACTG 61 AGCGGATACT GTAGCAACAT CAAACTGCAG GTGGTCAAGG ACGCTCAGGC ACTGCTGCAT 121 GGGCTGGACT TCTCCGAGGT GTCTAATGTG CAGCGGCTGA TGCGGAAAGA ACGGAGGGAC 181 GATAATGACC TGAAGCGACT GCGCGACCTG AACCAGGCAG TGAACAATCT GGTCGAGCTG 241 AAGAGCACCC AGCAGAAATC AATCCTGCGG GTCGGGACAC TGACATCTGA CGACCTGCTG 301 ATCCTGGCTG CAGACCTGGA GAAGCTGAAA TCGAAAGTGA TCCGCACCGA AAGGCCACTG 361 TCCGCCGGGG TCTACATGGG CAATCTGTCT TCCCAGCAGC TGGACCAGAG GCGGGCTCTG 421 CTGAACATGA TTGGGATGTC CGGAGGAAAT CAGGGAGCTA GAGCCGGGAG GGACGGAGTC 481 GTGCGGGTCT GGGACGTGAA GAATGCCGAA CTGCTGAACA ACCAGTTCGG GACCATGCCA 541 AGTCTGACAC TGGCATGCCT GACTAAACAG GGCCAGGTGG ATCTGAATGA TGCAGTCCAG 601 GCTCTGACCG ACCTGGGCCT GATCTACACC GCCAAGTACC CCAATACTAG CGACCTGGAT 661 AGACTGACCC AGAGCCACCC CATCCTGAAC ATGATCGACA CTAAGAAGTC CTCACTGAAC 721 ATCAGTGGCT ATAATTTCTC CCTGGGGGCA GCAGTCAAGG CTGGCGCATG CATGCTGGAC 781 GGCGGGAATA TGCTGGAAAC CATCAAAGTG TCTCCCCAGA CCATGGATGG CATCCTGAAA 841 TCTATTCTGA AAGTCAAGAA GGCCCTGGGA ATGTTTATTT CAGACACCCC CGGCGAGAGG 901 AATCCATATG AGAACATTCT GTATAAGATT TGCCTGAGTG GCGACGGGTG GCCATACATT 961 GCAAGCCGGA CATCAATTAC CGGAAGAGCT TGGGAGAATA CAGTCGTGGA CCTGGAAAGC 1021 GACGGCAAGC CCCAGAAGGC CGACTCAAAC AACTCCTCAA AGAGTCTGCA GTCAGCTGGC 1081 TTCACAGCAG GGCTGACTTA CTCCCAGCTG ATGACACTGA AGGACGCAAT GCTGCAGCTG 1141 GACCCAAACG CTAAGACATG GATGGACATC GAGGGACGGC CAGAAGATCC AGTGGAAATC 1201 GCACTGTATC AGCCATCATC CGGATGCTAT ATCCATTTCT TCCGGGAACC AACTGATCTG 1261 AAGCAGTTCA AGCAGGATGC AAAGTACTCC CACGGAATCG ATGTCACCGA TCTGTTCGCA 1321 ACCCAGCCAG GACTGACATC AGCCGTCATC GATGCCCTGC CTAGGAACAT GGTCATTACT 1381 TGCCAGGGCT CCGACGATAT TAGGAAGCTG CTGGAGAGCC AGGGACGGAA GGATATCAAA 1441 CTGATCGATA TTGCCCTGTC TAAGACTGAT AGCCGGAAAT ATGAGAATGC AGTCTGGGAT 1501 CAGTACAAGG ACCTGTGCCA TATGCATACC GGAGTGGTCG TCGAGAAGAA GAAGAGGGGC 1561 GGAAAGGAAG AGATCACACC CCACTGTGCC CTGATGGATT GCATCATGTT CGACGCAGCC 1621 GTGTCCGGGG GCCTGAACAC CTCAGTCCTG AGGGCTGTCC TGCCAAGAGA TATGGTGTTT 1681 AGAACTTCAA CCCCAAGAGT CGTCCTGTAA

[0152] Sequence ID 5 Sequence ID 5 corresponds to the recombinant mutant NP protein of the Lassa virus strain Josiah, encoded by the codon-optimized sequence of Sequence ID 6, where the exonuclease activity of the NP protein is knocked down. Amino acids 388 and 391 are mutated (M388D and E391G). *

[0153] Sequence ID 6 Sequence ID 6 corresponds to the codon-optimized nucleotide sequence encoding the mutant NP protein of Sequence ID 5. Nucleotides 1166, 1175, and 1176 have been mutated (C1166A, C1175G, and C1176A). 1 ATGAGTGCCA GCAAAGAAAT CAAGAGCTTC CTGTGGACCC AGAGTCTGCG GAGGGAACTG 61 AGCGGATACT GTAGCAACAT CAAACTGCAG GTGGTCAAGG ACGCTCAGGC ACTGCTGCAT 121 GGGCTGGACT TCTCCGAGGT GTCTAATGTG CAGCGGCTGA TGCGGAAAGA ACGGAGGGAC 181 GATAATGACC TGAAGCGACT GCGCGACCTG AACCAGGCAG TGAACAATCT GGTCGAGCTG 241 AAGAGCACCC AGCAGAAATC AATCCTGCGG GTCGGGACAC TGACATCTGA CGACCTGCTG 301 ATCCTGGCTG CAGACCTGGA GAAGCTGAAA TCGAAAGTGA TCCGCACCGA AAGGCCACTG 361 TCCGCCGGGG TCTACATGGG CAATCTGTCT TCCCAGCAGC TGGACCAGAG GCGGGCTCTG 421 CTGAACATGA TTGGGATGTC CGGAGGAAAT CAGGGAGCTA GAGCCGGGAG GGACGGAGTC 481 GTGCGGGTCT GGGACGTGAA GAATGCCGAA CTGCTGAACA ACCAGTTCGG GACCATGCCA 541 AGTCTGACAC TGGCATGCCT GACTAAACAG GGCCAGGTGG ATCTGAATGA TGCAGTCCAG 601 GCTCTGACCG ACCTGGGCCT GATCTACACC GCCAAGTACC CCAACTTAG CGACCTGGAT 661 AGACTGACCC AGAGCCACCC CATCCTGAAC ATGATCGACA CTAAGAAGTC CTCACTGAAC 721 ATCAGTGGCT ATAATTTCTC CCTGGGGGCA GCAGTCAAGG CTGGCGCATG CATGCTGGAC 781 GGCGGGAATA TGCTGGAAAC CATCAAAGTG TCTCCCCAGA CCATGGATGG CATCCTGAAA 841 TCTATTCTGA AAGTCAAAGAA GGCCCTGGGA ATGTTTATTT CAGACACCCC CGGCGAGAGG 901 AATCCATATG AGAACATTCT GTATAAGATT TGCCTGAGTG GCGACGGGTG GCCATACATT 961 GCAAGCCGGA CATCAATTAC CGGAAGAGCT TGGGAGAATA CAGTCGTGGA CCTGGAAAGC 1021 GACGGCAAGC CCCAGAAGGC CGACTCAAAC AACTCCTCAA AGAGTCTGCA GTCAGCTGGC 1081 TTCACAGCAG GGCTGACTTA CTCCCAGCTG ATGACACTGA AGGACGCAAT GCTGCAGCTG 1141 GACCAAAACG CTAAGACATG GATGG C CATC GAGG CC CGGC CAGAAGATCC AGTGGAAATC 1201 GCACTGTATC AGCCATCATC CGGATGCTAT ATCCATTTCT TCCGGGAACC AACTGATCTG 1261 AAGCAGTCA AGCAGGATGC AAAGTACTCC CACGGAATCG ATGTCACCGA TCTGTTCCGCA 1321 ACCCAGCCAG GACTGACATC AGCCGTCATC GATGCCCTGC CTAGGAACAT GGTCATTACT 1381 TGCCAGGGCT CCGACGATAT TAGGAAGCTG CTGGAGAGCC AGGGACGGAA GGATATCAAA 1441 CTGATCGATA TTGCCCTGTC TAAGACTGAT AGCCGGAAAT ATGAGAATGC AGTCTGGGAT 1501 CAGTACAAGG ACCTGTGCCA TATGCATACC GGAGTGGTCG TCGAGAAGAA GAAGAGGGGC 1561 GGAAAGGAAG AGATCACACC CCACTGTGCC CTGATGGATT GCATCATGTT CGACGCAGCC 1621 GTGTCCGGGG GCCTGAACAC CTCAGTCCTG AGGGCTGTCC TGCCAAGAGA TATGGTGTTT 1681 AGAACTTCAA CCCCAAGAGT CGTCCTGTAA

[0154] SEQ ID NO: 7 SEQ ID NO: 7 corresponds to the recombinant Z protein of the Lassa virus strain Josiah encoded by the codon-optimized sequence of SEQ ID NO: 8. MGNKQAKAPESKDSPRASLIPDATHLGPQFCKSCWFENKGLVECNNHYLCLNCLTLLLSVSNRCPICKMPLPTKLRPSAAPTAPPTGAADSIRPPPYSP*

[0155] SEQ ID NO: 8 SEQ ID NO: 8 corresponds to the codon-optimized nucleotide sequence encoding the Z protein of SEQ ID NO: 7. 1 ATGGGCAATA AGCAGGCAAA GGCACCCGAA AGCAAGGATT CACCTAGAGC ATCACTGATT 61 CCCGACGCAA CTCATCTGGG GCCACAGTTC TGCAAATCCT GTTGGTTCGA GAACAAAGGC 121 CTGGTGGAGT GCAATAACCA CTACCTGTGC CTGAACTGTC TGACACTGCT GCTGAGTGTG 181 AGCAACAGAT GCCCAATCTG CAAGATGCCT CTGCCAACAA AGCTGAGGCC TTCTGCTGCA 241 CCCACCGCAC CACCAACTGG AGCCGCAGAC AGCATTAGAC CCCCCCCATA CTCACCATAA

[0156] (References) TIFF0007841847000002.tif224170TIFF0007841847000003.tif127170

Claims

1. (1) cDNA molecules encoding the full-length antigenome (+) RNA strand of measles virus (MeV); and (2) A first heterologous polynucleotide encoding at least one glycoprotein precursor (GPC) of Lassa virus (LASV) and a mutant nucleoprotein (mNP) in which the exonuclease activity of the NP protein of LASV is knocked down, or (2') comprising a first heterogeneous polynucleotide encoding at least one LASV glycoprotein precursor (GPC) and a second heterogeneous polynucleotide encoding at least one LASV zinc-binding protein (Z protein), Here, the first heterologous polynucleotide is manipulably cloned into an additional transcription unit (ATU) inserted into the cDNA of the antigenome (+) RNA. If the second heterologous polynucleotide is present, it is operably cloned into another ATU at a different location than the location of the cloned first heterologous polynucleotide. Nucleic acid constructs.

2. The nucleic acid construct according to claim 1, wherein heterologous polynucleotides encoding GPC, mNP and / or Z protein are derived from the LASV strain Josiah or from the sequences of GenBank J04324.1 and / or U73034.

2.

3. The nucleic acid construct according to claim 1 or 2, wherein the mNP has a mutated exonuclease domain, and the amino acid sequence of the encoded mNP is mutated at amino acid residues 389 and / or 392 of SEQ ID NO:

3.

4. A heterogeneous polynucleotide encoding a GPC, mNP and / or Z protein has a codon-optimized open reading frame (ORF), and the heterogeneous polynucleotide has the following sequence: - Sequence ID 2, which codes for GPC; and / or - Sequence ID 6, which codes for mNP; and / or - Sequence ID 8, which codes for the Z protein. A nucleic acid construct according to any one of claims 1 to 3, comprising at least one of the following.

5. The first heterogeneous polynucleotide encodes the GPC of SEQ ID NO: 1 and the mNP of SEQ ID NO: 5, or The first heterogeneous polynucleotide encodes the GPC of SEQ ID NO: 1, and the second heterogeneous polynucleotide encodes the Z protein of SEQ ID NO:

7. A nucleic acid construct according to any one of claims 1 to 4.

6. The nucleic acid construct according to any one of claims 1 to 5, wherein a first heterologous polynucleotide is operably cloned into an ATU located between the P and M genes of MeV.

7. The nucleic acid construct according to any one of claims 1 to 6, wherein the second heterologous polynucleotide is operably cloned into an ATU located upstream of the N gene of MeV.

8. From the 5' end to the 3' end, the following polynucleotides are present: (a) Polynucleotide encoding the MeV N protein; (b) Polynucleotides encoding the MeV P protein; (c) A first heterogeneous polynucleotide encoding at least an mNP and then a GPC, wherein the first polynucleotide is a first heterogeneous polynucleotide that is operably cloned within an ATU; (d) Polynucleotides encoding the MeV M protein; (e) Polynucleotides encoding the MeV F protein; (f) Polynucleotides encoding the MeV H protein; (g)MeV polynucleotide encoding the L protein A nucleic acid construct according to any one of claims 1 to 5, comprising, wherein the polynucleotide is operably bound within the nucleic acid construct and is under the control of viral replication and transcriptional regulatory elements.

9. From the 5' end to the 3' end, the following polynucleotides are present: (a) A second heterologous polynucleotide encoding at least the Z protein of LASV, which is manipulably cloned into an ATU located upstream of the N gene of MeV; (b) Polynucleotide encoding the N protein of MeV; (c) Polynucleotide encoding the MeV P protein; (d) A first heterologous polynucleotide that encodes at least a GPC and is operably cloned within the ATU; (e) Polynucleotides encoding the MeV M protein; (f) A polynucleotide encoding the F protein of MeV; (g) A polynucleotide encoding the H protein of MeV; (h)MeV L protein encoding polynucleotide A nucleic acid construct according to any one of claims 1 to 5, comprising, wherein the polynucleotide is operably bound within the nucleic acid construct and is under the control of viral replication and transcriptional regulatory elements.

10. The first heterologous polynucleotide extends from the 5' end to the 3' end. (a) the nucleic acid of sequence number 6 that encodes mNP, and (b) Nucleic acid of sequence number 2 encoding GPC, A nucleic acid construct according to any one of claims 1 to 9, comprising, wherein a first heterologous polynucleotide sequence is operably cloned between the P and M genes of MeV within the ATU.

11. A nucleic acid construct according to any one of claims 1 to 10, wherein the second heterologous polynucleotide encodes the Z protein of LASV, the first heterologous polynucleotide encodes the GPC of LASV, 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 1, wherein the measles virus is an attenuated virus strain selected from the group consisting of Schwarz strain, Zagreb strain, AIK-C strain, Moraten strain, Philips strain, Beckenham 4A strain, Beckenham 16 strain, Edmonston seed A strain, Edmonston seed B strain, CAM-70 strain, TD 97 strain, Leningrad-16 strain, Shanghai 191 strain, and Belgrade strain.

13. The nucleic acid construct is - Sequence ID 11 (Construction MeV-mNP-GPC); and - Sequence ID 12 (Construction Z-MeV-GPC) A nucleic acid construct according to any one of claims 1 to 12, comprising a recombinant cDNA sequence selected from the group consisting of the above, or an introduction plasmid vector comprising a nucleic acid construct according to any one of claims 1 to 12.

14. Recombinant measles virus comprising a nucleic acid construct according to any one of claims 1 to 13, or an introduced plasmid vector according to claim 13, in its genome, or the genome comprising the introduced plasmid vector according to claim 13.

15. Recombinant measles virus according to claim 14, expressing at least LASV GPC and mNP, or LASV GPC and Z protein.

16. Host cells transfected with a nucleic acid construct according to any one of claims 1 to 13, or with an introduction plasmid vector according to claim 13, or infected with recombinant measles virus according to claim 14 or 15.

17. An immunogenic composition comprising the recombinant measles virus according to claim 14 or 15, and a pharmaceutically acceptable vehicle.

18. The composition according to claim 17 for use in a host requiring the same, for use in inducing a protective immune response against Lassa virus and / or measles virus by inducing antibodies against LASV protein and / or measles virus protein, and / or in inducing a cellular response and / or humoral and cellular response to Lassa virus and / or measles virus.

19. (a) Transfecting cells that stably express T7 RNA polymerase and measles virus N and P proteins with a nucleic acid construct according to any one of claims 1 to 13 or an introduction plasmid vector according to claim 13; (b) A step of maintaining transfected cells under conditions suitable for the production of recombinant measles virus and / or LASV VLP; (c) A step of infecting cells suitable for passage with the transfected cells of step (b) to enable the reproduction of recombinant measles virus and / or LASV VLP; (d) A step of recovering recombinant measles virus expressing at least the GPC, mNP and / or Z protein of LASV. A method for rescuing recombinant Lassa virus-like particles (VLPs) and / or recombinant measles virus expressing at least the GPC, mNP and / or Z protein of LASV, including the following.

Images