WO2016079560A1 - A live and attenuated japanese encephalitis virus comprising a mutated m protein - Google Patents

A live and attenuated japanese encephalitis virus comprising a mutated m protein Download PDF

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WO2016079560A1
WO2016079560A1 PCT/IB2014/002917 IB2014002917W WO2016079560A1 WO 2016079560 A1 WO2016079560 A1 WO 2016079560A1 IB 2014002917 W IB2014002917 W IB 2014002917W WO 2016079560 A1 WO2016079560 A1 WO 2016079560A1
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jev
cell
sequence
protein
application
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PCT/IB2014/002917
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Mélissanne DE WISPELAERE
Nathalie PARDIGON
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Institut Pasteur
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24161—Methods of inactivation or attenuation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the application relates to the attenuation of Japanese Encephalitis Virus (JEV).
  • JEV Japanese Encephalitis Virus
  • the application notably provides a live and attenuated JEV comprising a mutated M protein.
  • Said mutated M protein comprises or consists of a sequence, wherein the amino acid at position 36 in said sequence is mutated.
  • the amino acid at position 5 may also be mutated.
  • the application also provides means deriving from said live and attenuated JEV, such as nucleic acids, expression vectors, cells, cDNA clones, immunogenic compositions as well as uses and methods. BACKGROUND OF THE INVENTION
  • Flaviviruses such as Japanese Encephalitis Virus (JEV), Dengue Virus (DV), West Nile Virus (WNV) and Yellow Fever Virus (YFV) viruses, are arthropod-borne pathogens (arboviruses) that are transmitted through the bite of an infected mosquito and may cause serious human diseases worldwide ⁇ Lindenbach BD et al., Adv Virus Research, 2003, 59, 23-61). To date, very few vaccines against flaviviruses are commercially available. The first one was the live-attenuated vaccine 17D against YFV (Barrett, ADT Yellow Fever Vaccines Biologicals 1997).
  • live-attenuated and inactivated vaccines against JEV such as the live-attenuated virus vaccine SAu-14-2 (Yun SI, Lee YM. Hum Vaccin Immunother 2014 Feb, 10(2): 263- 279) and inactivated vaccines against tick-borne encephalitis virus (Lani R et al., Ticks Tick Borne Dis. 2014 Sep, 5(5): 457-465). Determination of the attenuation factors of these viruses can help in the development of new molecular vaccines.
  • structural proteins capsid C, membrane M and envelope E
  • flaviviruses Kerman RM et al. J Virol.
  • JEV contains a positive single-stranded RNA genome encoding a single polyprotein that is processed into three structural proteins, the capsid (C), the precursor of membrane (prM) and the envelope (E) proteins, and seven nonstructural proteins (NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5).
  • the membrane protein is synthesized as a precursor prM. It is cleaved in the trans-Golgi during viral particles secretion into pr and M (Li L et al. Science. 2008, 319(5871): 1830-1834). This cleavage is mandatory to produce infectious particles (Randolph VB et al., Virology, 1990, 174(2): 450- 458).
  • the resulting M protein is composed of an ectodomain (ectoM) consisting of 40 amino acids and 2 transmembrane domains (Zhang et al., EMBO J. 2003, 22(11): 2604-2613).
  • prM protein acts as a chaperone for the E protein folding (Konishi et al., J Virol. 1993, 67(3): 1672-1675) and prevents fusion within the infected cells (Yu et al., J Virol. 2009, 83(23): 12101-12107). It has also been disclosed that the C-terminal helical domain of DENV ectoM is involved in virus assembly (Pryor et ai, J Gen Virol, 2004, 85(Pt 12): 3627-3636; Hsieh et ai, J Virol.
  • patent US 7,785,604 describes that a nonapeptide (ApoptoM) from flavivirus ectoM is able to modulate specifically the apoptotic activity of diverse flaviviruses, and that the proapoptotic properties of ectoM are conserved among apoptosis-inducing flaviviruses, i.e. JEV, DV and YFV.
  • ApoptoM nonapeptide
  • McElroy et al. have demonstrated that the replacement of the leucine at position 36 of YFV strain Asibi ectoM into a phenylalanine (YFV-17D vaccine strain) reduce the mean dissemination of YFV in mosquitoes.
  • a higher mean dissemination was obtained when the sequences encoding the full M-E proteins or the E protein domain III of YFV-17D vaccine strain were incorporated to replace the same proteins of YFV strain Asibi (McElroy et al., J. Gen Virol., 2006, 87, 2993-3001).
  • the application provides a live and attenuated JEV comprising a mutated M protein.
  • Said mutated M protein comprises or consists of a sequence, wherein the amino acid at position 36 in said sequence is mutated, more particularly replaced by another amino acid, more particularly by the amino acid phenylalanine.
  • the amino acid at position 5 may also be mutated, more particularly replaced by another amino acid, more particularly by the amino acid proline.
  • the application also provides means deriving from said live and attenuated JEV, such as nucleic acids, more particularly RNA and cDNA, proteins and polypeptides, expression vectors, more particularly recombinant expression vectors, cells, more particularly recombinant cDNA clones as well as immunogenic compositions and vaccines.
  • JEV live and attenuated JEV
  • the application also provides as uses and methods, more particularly uses and methods to prevent a JEV infection in a mammalian host, especially in a human or an animal host.
  • FIGS 1A and 1 B M-I36F virus life cycle is not impaired in mosquito cells, while it is impaired in SK-N-SH cells.
  • C6/36 mosquito cells and mammalian SK-N-SH cells were infected at an MOI of 5 and were harvested at 24h and 48h post-infection.
  • Figure 1A Supernatants from C6/36 infected cells collected at 24h and 48h post-infection were titrated on C6/36 mosquito cells.
  • Figure 1 B Supernatants from SK-N-SH infected cells collected at 24h and 48h post-infection were titrated on C6/36 mosquito cells.
  • FIGs 2A and 2B M-I36F virus is attenuated and induces production of antibodies in C57BL/6 mice.
  • Figure 2A Three-week-old C57BL/6 female mice were injected intraperitoneally with 10 3 ffu of RP9 virus, or with different doses from 10 3 to 10 6 ffu of M-I36F mutant virus. Survival percentages were calculated ( **** : P ⁇ 0.00001 ).
  • Figure 2B Sera were collected 27 days after inoculation from the mice that survived and were diluted. Dilutions were analyzed by ELISA, using purified and inactivated JEV particles as target. Dilution to 1/100 of serum from mice injected with DPBS was used as negative control ( *** : P ⁇ 0.001 ).
  • Figures 3A and 3B The replacement by phenylalanine of the amino acid, which is at position 36 in protein M of a Flavivirus other than JEV, i.e., in the Yellow Fever Virus, does not lead to attenuation [cf. example 2 below].
  • Figure 3A schematic representation of a while-type infectious Yellow Fever Virus [YF-Asibi], which has been modified by replacement of the amino acid at position 36 in protein M by the amino acid phenylalanine (L36F) and by replacement of the amino acid at position 95 in protein NS4B by the amino acid Met (I95M) [YF-Asibi mut ].
  • Figure 3B titration results of this mutated YFV (YF-Asibi mut ) and of the wild-type parent (i.e., non mutated) YFV (YF-Asibi), showing that mutation does not lead to attenuation in YFV.
  • Figure 4 shows the results of the titration of the viral particles produced by the wild-type (infectious) JEV and by the I36A mutated JEV in BHK21 cells and in C6/36 cells.
  • WT wild-type (i.e. , infectious) JEV
  • I36A wild-type JEV modified by I36A mutation in protein M. DETAILED DESCRIPTION OF THE INVENTION
  • the inventors introduced a mutation into the M protein of a JEV infectious clone, in particular a point mutation into the M protein of a JEV infectious clone and showed that infection of mammalian cells resulted in a reduced number of secreted viral particles relative to the wild-type virus.
  • mosquito cells were infected, the inventors did not observe any difference between the wild-type and the mutant viruses infectious cycles.
  • the inventors designed several reporter systems that uncoupled the entry, replication and assembly of JEV. Using these systems, the inventors showed that the mutation in the M protein strongly impacted the assembly of genuine viral particles in mammalian cells.
  • the mutant virus was severely attenuated in vivo in a mouse model of viral encephalitis, when compared to the wild-type virus.
  • the inventors thus identified an amino acid residue at position 36 in the M protein of JEV that plays a major role in the assembly of JEV particles in mammalian cells. More particularly, the inventors found that the replacement of the amino acid which is at position 36 in protein M of JEV by an amino acid other than isoleucine (I), more particularly by the amino acid phenylalanine (F), leads to attenuation.
  • an amino acid other than isoleucine (I) more particularly by the amino acid phenylalanine (F)
  • the M protein of JEV in particular the ectoM of JEV, contained viral determinants for viral attenuation.
  • the amino acid at position 5 in protein M of JEV may further be mutated, more particularly replaced by an amino acid other than glutamine (Q), more particularly replaced by the amino acid proline (P).
  • Q amino acid other than glutamine
  • P amino acid proline
  • said mutation comprises or consists of the replacement of the amino acid at position 36 in the sequence of said endogenous M protein
  • a live and attenuated JEV is intended in accordance with its ordinary meaning in the field. More particularly, the expression “a live and attenuated JEV” designates a JEV that has a reduced pathogenic phenotype compared to a wild-type and/or infectious and/or virulent JEV.
  • a live and attenuated JEV does not cause encephalitis (e.g., because of reduced replication capacity and/or because of reduced and/or restricted tissue tropism, and/or because of default or defect in assembly of the JEV viral particles, more particularly by default or defect in assembly of the JEV viral particles), but is able to stimulate an immune response when administered to a host, especially a human, more particularly a protecting immune response protecting against JEV infection (seroneutralization).
  • immuno response includes one or several from antibody production, seroneutralization, induction of cell-mediated immunity, complement activation, development of immunological tolerance, alteration of cytokine production and alteration of chemokine production, more particularly (neutralizing) antibody production or seroneutralization, more particularly seroneutralization.
  • a live and attenuated JEV of the application shows a default or defect in assembly of the JEV viral particles (such as a reduced or inhibited rate of viral particle production), more particularly a default or defect in assembly of the JEV viral particles (such as a reduced or inhibited rate of viral particle production), which is sufficient to not cause encephalitis.
  • a live and attenuated JEV of the application stimulates said immune response (more particularly said anti-JEV (neutralizing) antibody production or JEV seroneutralization, more particularly said JEV seroneutralization).
  • a live and attenuated JEV of the application stimulates said immune response (more particularly said anti-JEV antibody production or JEV seroneutralization, more particularly said JEV seroneutralization), but shows a default in assembly of the JEV viral particles (such as a reduced or inhibited rate of viral particle production), more particularly a default in assembly of the JEV viral particles (such as a reduced or inhibited rate of viral particle production), which is sufficient to not cause encephalitis.
  • mice which have received the live and attenuated JEV of the application (36F mutated M protein), - the production of anti-JEV (neutralizing) antibodies, or JEV seroneutralization, and which further demonstrates that
  • the wild-type and/or infectious and/or virulent JEV to be mutated for attenuation can e.g., be a JEV of genotype 3 (G3), such as the strain RP9 (GENBANK® accession number KF907505).
  • nucleotide sequence of the polynucleotide encoding the endogenous M protein of JEV strain RP9 (infectious or virulent JEV), as well as the amino acid sequence of the endogenous M protein of JEV strain RP9 may for example consist of the sequences disclosed as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.
  • SEQ ID NO: 1 (cDNA sequence of the endogenous protein M of JEV RP9): tccgtgtc ggtccaaaca catggggaga
  • SEQ ID NO: 2 endogenous protein M of JEV RP9
  • the live and attenuated JEV of the application can e.g., be a JEV of genotype 3 (G3).
  • the application generally relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) M protein, wherein the amino acid sequence of said (mutated JEV) M protein comprises or consists of a sequence, which:
  • (a) consists of 75 amino acids and is at least 93%, or at least 94%, or at least 96% or at least 97% or at least 98%, more particularly at least 96% or at least 97% or at least 98%, more particularly at least 97% or at least 98% identical to the sequence of SEQ ID NO: 2, and
  • the application more particularly relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) M protein, wherein the amino acid sequence of said (mutated JEV) M protein comprises or consists of a sequence, which:
  • (a) consists of 75 amino acids and is at least 98% identical to the sequence of SEQ ID NO: 2, and
  • the application also relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) M protein, wherein the amino acid sequence of said (mutated JEV) M protein comprises or consists of a sequence, which:
  • (a) consists of 75 amino acids and is at least 93%, or at least 94%, or at least 96% or at least 97%, more particularly at least 96% or at least 97%, more particularly at least 97% identical to the sequence of SEQ ID NO: 2, and
  • amino acid at position 5 in said sequence of 75 amino acids is the amino acid proline
  • amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine
  • said mutated M protein replaces an endogenous M protein, more particularly the endogenous M protein of a wild-type and/or infectious and/or virulent JEV, more particularly the endogenous M protein, the sequence of which is SEQ ID NO: 2.
  • the live and attenuated JEV does advantageously not comprise (nor codes for) the endogenous M protein of an infectious and/or virulent JEV, more particularly the M protein of SEQ ID NO: 2.
  • a particular nucleotide sequence of the polynucleotide encoding said mutated JEV M protein as well as a particular amino acid sequence of said mutated JEV M protein are the sequences disclosed as SEQ ID NO: 3 and SEQ ID NO: 4 respectively.
  • SEQ ID NO: 3 (cDNA sequence coding for a mutated JEV M protein of the application):
  • nnn a codon coding for phenylalanine (TTT or TTC).
  • SEQ ID NO: 4 (mutated JEV M protein of the application):
  • the live and attenuated JEV of the application can e.g., be a JEV, which comprises or codes for a (mutated JEV ) M protein, wherein said (mutated JEV) protein M comprises or consists of the protein of SEQ ID NO: 4.
  • nucleotide sequence of the polynucleotide encoding a mutated JEV M protein of the application as well as another particular amino acid sequence of said mutated M protein are the sequences disclosed as SEQ ID NO: 9 and SEQ ID NO: 10 respectively.
  • SEQ ID NO: 9 (cDNA sequence coding for a mutated JEV M protein of the application):
  • SEQ ID NO: 10 (mutated JEV M protein of the application):
  • the live and attenuated JEV of the application can e.g., be a JEV, which comprises or codes for a (mutated) M protein, wherein said (mutated) protein M comprises or consists of the protein of SEQ ID NO: 10.
  • the live and attenuated JEV of the application comprises the RNA version of the (cDNA) nucleotide sequence of SEQ ID NO: 7 or of SEQ ID NO: 1 1 (the sequence of SEQ ID NO: 7 or of SEQ ID NO: 1 1 codes for a mutated ectoM of the application; cf. below).
  • the live and attenuated JEV of the application comprises the RNA version of the (cDNA) nucleotide sequence of SEQ ID NO: 3 or of SEQ ID NO: 9 (the sequence of SEQ ID NO: 3 or of SEQ ID NO: 9 codes for a mutated M of the application; cf. above).
  • the live and attenuated JEV of the application comprises the RNA version of the (cDNA) nucleotide sequence insert carried by the plasmid stbl2 / pBR322-JEV-RP9 (M-I36F), which has been deposited under the terms of the Budapest Treaty at the Collection Nationale de Culture de Microorganismes (CNCM) under deposit number I- 4902, on November 4, 2014.
  • CNCM Collection Nationale de Culture de
  • RNA version of a (cDNA) nucleotide sequence means the (RNA) sequence, which results from the replacement of each nucleotide T of said (cDNA) nucleotide sequence by the nucleotide U.
  • the live and attenuated JEV of the application shows a default or detect in the assembly of the viral particles (e.g. , a reduced production rate of (correctly) assembled viral particles).
  • the live and attenuated JEV of the application shows said default or defect in a mammalian cell, but not in a mosquito cell.
  • an infectious JEV (such as the JEV strain RP9) does not show this defect in a mosquito cell and does neither show it in a mammalian cell.
  • Said mammalian cell can e.g. , be a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g. , a cell of the Vero cell line [ATCC® CCL-81TM]) or a human cell (e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216TM] or of the SK-N-SH cell line [ATCC® HTB-1 1TM]). More particularly, said mammalian cell can e.g. , be a human cell (e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216TM] and/or of the SK-N-SH cell line [ATCC® HTB-1 1TM]).
  • Said mosquito cell can e.g. , be an Aedes cell, an Aedes albopictus cell or a cell of the C6/36 cell line [ATCC® CRL-1660TM].
  • the live and attenuated JEV of the application shows said default or defect in a human cell (e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216TM] and/or of the SK-N-SH cell line [ATCC® HTB-1 1TM]), but not in a mosquito cell (e.g. , be an Aedes cell, an Aedes frugipreda cell, an Aedes albopictus, or a cell of the C6/36 cell line [ATCC® CRL-1660TM]).
  • the live and attenuated JEV of the application shows said default or defect in a cell of the HEK293T cell line [ATCC® CRL-3216TM] and/or of the SK-N-SH cell line [ATCC® HTB-1 1TM]), but not in a cell of the C6/36 cell line [ATCC® CRL-1660TM].
  • the live and attenuated JEV of the application induces JEV neutralizing antibodies, more particularly JEV sero- neutralization, more particularly in a mammalian host (such as a rodent, a monkey or a human).
  • JEV neutralizing antibodies more particularly JEV sero- neutralization
  • a mammalian host such as a rodent, a monkey or a human.
  • the application also relates to the (mutated JEV) M protein of the live and attenuated JEV of the application.
  • the application thus relates to a (mutated JEV M) protein, wherein the amino acid sequence of said (mutated JEV M) protein comprises or consists of a sequence, which:
  • (a) consists of 75 amino acids and is at least 93%, or at least 94%, or at least
  • amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine.
  • amino acid phenylalanine is the amino acid phenylalanine.
  • An example of such a (mutated JEV M) protein is the protein of SEQ ID NO: 4 or of SEQ ID NO: 10 (cf. above).
  • the application relates to a (mutated JEV
  • amino acid sequence of said (mutated JEV M) protein comprises or consists of a sequence, which:
  • (a) consists of 75 amino acids and is at least 97% or at least 98%, more particularly at least 98% identical to the sequence of SEQ ID NO: 2, and (b) wherein the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine.
  • An example of such a (mutated JEV M) protein is the protein of SEQ ID NO: 4 or of SEQ ID NO: 10, more particularly of SEQ ID NO: 4 (cf. above).
  • the application relates to a (mutated JEV
  • amino acid sequence of said (mutated JEV M) protein comprises or consists of a sequence, which:
  • (a) consists of 75 amino acids and is at least 93%, or at least 94%, or at least 96% or at least 97%, more particularly at least 96% or at least 97%, more particularly at least 97% identical to the sequence of SEQ ID NO: 2, and
  • amino acid at position 5 in said sequence of 75 amino acids is the amino acid proline
  • amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine.
  • amino acid phenylalanine is the protein of SEQ ID NO: 10 (cf. above).
  • the application also relates to a nucleic acid, more particularly a cDNA, DNA or RNA nucleic acid, coding for said (mutated JEV M protein) of the application, as well as to expression vectors comprising this nucleic acid inserted in an expression cassette, and cells, more particularly recombinant cells, comprising or transformed or transfected or infected by such an expression vector.
  • nucleic acid coding for said (mutated JEV M protein) of the application examples include the nucleic acid of SEQ ID NO: 3 or of SEQ ID NO: 9.
  • Examples of such (recombinant) cells include:
  • bacterial cell such as an E. coli cell
  • yeast cell such as a S. cerevisiae cell
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660TM]),
  • a mammal cell especially a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g., a cell of the Vero cell line [ATCC® CCL-81 I M ]) or a human cell (e.g., a cell of the HEK293T cell line [ATCC® CRL-3216TM] or of the SK-N-SH cell line [ATCC® HTB-1 1TM]),
  • a rodent cell such as a mouse cell
  • a monkey cell e.g., a cell of the Vero cell line [ATCC® CCL-81 I M ]
  • a human cell e.g., a cell of the HEK293T cell line [ATCC® CRL-3216TM] or of the SK-N-SH cell line [ATCC® HTB-1 1TM]
  • bacterial cell such as an E. coli cell, or
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660TM]),
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660TM]).
  • a Drosophila cell e.g., a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660TM
  • the cell can be in isolated form.
  • the cell of the application can be contained in a culture medium, more particularly a non-naturally occurring culture medium, e.g., an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
  • a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
  • the application also relates to a polypeptide, which is a fragment of the (mutated JEV M) protein of the application, more particularly a fragment of said mutated JEV M protein, which comprises or consists of the ectodomain of said (mutated JEV) M protein [mutated ectoM].
  • the nucleotide sequence of the polynucleotide encoding the endogenous ectoM of the M protein of JEV strain RP9 (infectious or virulent JEV) as well as the amino acid sequence of the endogenous ectoM of the M protein of JEV strain RP9 are the sequences disclosed as SEQ ID NO: 5 and SEQ ID NO: 6 respectively.
  • SEQ ID NO: 5 cDNA coding for the ectodomain of the M protein of the JEV wild-type strain RP9:
  • SEQ ID NO: 6 ectodomain of the M protein of JEV RP9:
  • a particular nucleotide sequence of the polynucleotide encoding a mutated ectoM of the application as well as a particular amino acid sequence of said mutated ectoM are the sequences disclosed as SEQ ID NO: 7 and SEQ ID NO: 8 respectively.
  • SEQ ID NO: 7 (cDNA coding for a mutated M ectodomain of the application): tccgtgtc ggtccaaaca catggggaga
  • nnn a codon coding for phenylalanine (TTT or TTC).
  • SEQ ID NO: 8 (mutated M ectodomain):
  • nucleotide sequence of the polynucleotide encoding a mutated ectoM of the application as well as another particular amino acid sequence of said mutated ectoM are the sequences disclosed as SEQ ID NO: 1 1 and SEQ ID NO: 12 respectively.
  • SEQ ID NO: 1 1 (cDNA coding for a mutated M ectodomain of the application):
  • the application thus relates to a polypeptide or to a mutated ectodomain (ectoM) of a JEV M protein, wherein the amino acid sequence of said polypeptide or mutated ectoM of a JEV M protein comprises or consists of a sequence, which:
  • (a) consists of 40 amino acids and is at least 92%, or at least 95%, more particularly at least 95% or at least 97%, more particularly at least 97% identical to the sequence of SEQ ID NO: 6, and
  • amino acid at position 36 in said sequence of 40 amino acids is the amino acid phenylalanine.
  • amino acid phenylalanine is the amino acid phenylalanine.
  • the application more particularly relates to a polypeptide or to a mutated ectodomain (ectoM) of a JEV M protein, wherein the amino acid sequence of said polypeptide or mutated ectoM of a JEV M protein comprises or consists of a sequence, which:
  • (a) consists of 40 amino acids and is at least 95% or at least 97%, more particularly at least 97% identical to the sequence of SEQ ID NO: 6, and (b) wherein the amino acid at position 36 in said sequence of 40 amino acids is the amino acid phenylalanine.
  • An example of such a polypeptide of the application is the polypeptide of SEQ ID NO: 8 or of SEQ ID NO: 12, more particularly of SEQ ID NO: 8.
  • the application more particularly relates to a polypeptide or to a mutated ectodomain (ectoM) of a JEV M protein, wherein the amino acid sequence of said polypeptide or mutated ectoM of a JEV M protein comprises or consists of a sequence, which:
  • (a) consists of 40 amino acids and is at least 95% identical to the sequence of SEQ ID NO: 6, and
  • amino acid at position 5 in said sequence of 40 amino acids is the amino acid proline
  • amino acid at position 36 in said sequence of 40 amino acids is the amino acid phenylalanine.
  • amino acid at position 5 in said sequence of 40 amino acids is the amino acid proline
  • amino acid at position 36 in said sequence of 40 amino acids is the amino acid phenylalanine.
  • the application also relates to a nucleic acid, more particularly a cDNA, DNA or RNA nucleic acid, coding for said polypeptide or mutated ectoM of the application, as well as to expression vectors comprising this nucleic acid inserted in an expression cassette, and cells, more particularly recombinant cells, comprising or transformed or transfected or infected by such an expression vector.
  • nucleic acid coding for said mutated ectoM of the application examples include the nucleic acid of SEQ ID NO: 7 or of SEQ ID NO: 1 1 .
  • Examples of such (recombinant) cells include:
  • bacterial cell such as an E. coli cell
  • yeast cell such as a S. cerevisiae cell
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660TM]),
  • a mammal cell especially a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g., a cell of the Vero cell line [ATCC® CCL-81TM]) or a human cell (e.g., a cell of the HEK293T cell line [ATCC® CRL-3216TM] or of the SK-N-SH cell line [ATCC® HTB-1 1TM]),
  • a rodent cell such as a mouse cell
  • a monkey cell e.g., a cell of the Vero cell line [ATCC® CCL-81TM]
  • a human cell e.g., a cell of the HEK293T cell line [ATCC® CRL-3216TM] or of the SK-N-SH cell line [ATCC® HTB-1 1TM]
  • bacterial cell such as an E. coli cell, or
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660TM]),
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660TM]).
  • the cell can be in isolated form.
  • the cell of the application can be contained in a culture medium, more particularly a non-naturally occurring culture medium, e.g., an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
  • a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
  • the application generally relates to the JEV mutated M protein or JEV mutated ectoM of the application, as a protein or polypeptide inducing a default or defect in JEV viral assembly, more particularly as a protein or polypeptide inducing a default or defect in JEV viral assembly that achieves JEV attenuation.
  • the application also relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) protein M, wherein the amino acid sequence of said (mutated JEV) protein M comprises the polypeptide or mutated ectoM of the application.
  • the application also relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) protein M ectodomain, wherein the sequence of said (mutated JEV) protein M ectodomain comprises or consists of the polypeptide or mutated ectoM of the application.
  • the live and attenuated JEV of the application comprises the RNA version of the (cDNA) nucleotide sequence of SEQ ID NO: 7 or 1 1 , more particularly the RNA version of the (cDNA) nucleotide sequence of SEQ ID NO: 3 or 9.
  • said polypeptide or mutated ectoM of the application replaces an endogenous ectoM, more particularly the endogenous ectoM of a wild-type and/or infectious and/or virulent JEV, more particularly the ectoM of SEQ ID NO: 6.
  • the live and attenuated JEV of the application does advantageously not comprise (nor codes for) the endogenous ectoM of an infectious and/or virulent JEV, more particularly the ectoM of SEQ ID NO: 6.
  • the JEV structural proteins other than protein M such as the JEV protein E and the JEV protein C, more particularly the JEV protein E, can be the JEV structural proteins of an infectious JEV (such as JEV strain RP9).
  • the JEV nonstructural proteins such as the JEV proteins NS1 , NS2A, NS2B, NS3, NS4A, NSA4 and NS5
  • the JEV nonstructural proteins NS1 , NS2A, NS2B, NS3, NS4A, NSA4 and NS5 can be the JEV non-structural proteins of an infectious JEV (such as JEV strain RP9).
  • JEV M protein as described in the application is sufficient to obtain a default or defect in JEV viral particles assembly that achieves JEV attenuation.
  • mice which have received the live and attenuated JEV of the application (36F mutated M protein),
  • the JEV proteins other than protein M more particularly the JEV protein E, can be non-mutated compared to a wild-type and/or infectious and/or virulent JEV.
  • the application also relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) polyprotein, wherein the amino acid sequence of said (mutated JEV) polyprotein comprises the (mutated JEV) protein M of the application, more particularly the polypeptide or mutated ectoM of the application.
  • the application thus relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) polyprotein, wherein the amino acid sequence of said (mutated JEV) polyprotein comprises or consists of the protein of SEQ ID NO: 13 (JEV RP9 polyprotein, wherein protein M is I36F mutated), or the protein of SEQ ID NO: 14 (JEV RP9 polyprotein, wherein protein M is Q5P and I36F mutated).
  • sequence of SEQ ID NO: 13 is:
  • JEV M protein more particularly the replacement by phenylalanine of the amino acid, which is at position 36 within JEV M protein, is sufficient to achieve a default or defect is JEV viral particle assembly that achieves attenuation
  • the person of ordinary skill in the art may choose to mutate JEV proteins other than JEV protein M, for example: - to increase the genetic stability of the live and attenuated JEV of the application, and/or
  • the person of ordinary skill in the art may thus choose to mutate one or several of proteins E, C, NS1 , NS2A, NS2B, NS3, NS4A, NSA4 and NS5, more particularly to mutate JEV protein E and/or JEV protein C, more particularly to mutate JEV protein E.
  • Said mutated protein(s) then have a sequence, which differ from the one (the respective ones) of JEV strain RP9, more generally from the one (the respective ones) of an infectious JEV.
  • Said mutation may comprise or consist of one or several (amino acid) point mutations.
  • the application also relates to the viral particles or virions of said live attenuated JEV of the application.
  • the application also relates to a RNA nucleic acid, which is the RNA genomic nucleic acid of the live and attenuated JEV of the application. More particularly, the application relates to the coding sequence (CDS) of said genomic RNA.
  • CDS coding sequence
  • the application also relates to a DNA nucleic acid, more particularly to a cDNA nucleic acid, the sequence of which is the retro-transcript or cDNA sequence of the RNA genomic nucleic acid of the application, e.g., according to the universal genetic code. More particularly, the application relates to the coding sequence (CDS) of said DNA or cDNA nucleic acid.
  • CDS coding sequence
  • the application also relates to an expression vector, more particularly to a recombinant expression vector, which comprises a nucleic acid (operably) inserted in an expression cassette (for expression of said nucleic acid), wherein said inserted nucleic acid is a nucleic acid of the application, more particularly the RNA genomic nucleic acid of the application or the cDNA or retro-transcript thereof, more particularly the RNA CDS or the cDNA CDS of the application.
  • the (recombinant) expression vector can e.g., be a prokaryotic expression vector or an eukaryotic expression vector.
  • Examples of (recombinant) prokaryotic expression vector include (recombinant) expression vectors for expression in E. coli.
  • Examples of (recombinant) eukaryotic expression vectors include:
  • yeast cell such as a S. cerevisiae cell
  • a - (recombinant) expression vectors for expression in an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the
  • a Spodoptera frugiperda cell e.g. , a cell of the Sf9 cell line
  • a mosquito cell e.g., an Aedes cell, an Aedes albopictus cell (e.g. , a cell of the C6/36 cell line [ATCC® CRL-1660TM])
  • a mammal cell especially a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g. , a cell of the Vero cell line [ATCC® CCL-81TM]) or a human cell (e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216TM] or of the SK-N-SH cell line [ATCC® HTB-1 1TM]).
  • a rodent cell such as a mouse cell
  • a monkey cell e.g., a cell of the Vero cell line [ATCC® CCL-81TM]
  • a human cell e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216TM] or of the SK-N-SH cell line [ATCC® HTB-1 1TM].
  • Examples of said (recombinant) prokaryotic expression vectors include (recombinant) plasmids for expression in E. coli, such as the pBR322 plasmid.
  • Examples of said (recombinant) eukaryotic expression vectors include plasmids, more particularly plasmids for expression in a yeast cell (such the 2-microns plasmid), Baculovirus vectors (e.g. , for expression in an insect cell), and Simian Virus 40 (SV40) vectors (e.g. , for expression in a mammal cell, such as a monkey cell).
  • plasmids more particularly plasmids for expression in a yeast cell (such the 2-microns plasmid)
  • Baculovirus vectors e.g. , for expression in an insect cell
  • Simian Virus 40 (SV40) vectors e.g. , for expression in a mammal cell, such as a monkey cell.
  • a (recombinant) vector generally comprises a cloning site, more particularly a multiple cloning site.
  • the (cloned) nucleic acid can be inserted in said cloning site.
  • a (recombinant) expression vector comprises an expression cassette wherein said nucleic acid can be inserted.
  • Said expression cassette generally comprises a promoter and a transcriptional terminator.
  • Suitable promoters include a CMV inducible promoter (such as the CMV inducible TET-ON® promoter, available from CLONTECH LABORATORIES, 1290 Terra Bella Ave., Mountain View, CA 94043 U.S.A.), the promoter of the lac operon, the T7 promoter, the AOX1 promoter, the PGK promoter, the pPolh promoter, the CaMV 35S promoter, the CMV promoter, the CAG promoter, the SV40 promoter, more particularly a CMV inducible promoter (such as the CMV inducible TET-ON® promoter).
  • a CMV inducible promoter such as the CMV inducible TET-ON® promoter, available from CLONTECH LABORATORIES, 1290 Terra Bella Ave., Mountain View, CA 94043 U.S.A.
  • the promoter of the lac operon the T7 promoter, the
  • the application also relates to a cell, more particularly a host and/or recombinant cell.
  • the cell of the application comprises the live and attenuated JEV of the application, or the mutated M protein of the application, or the mutated ectoM of the application, or the RNA nucleic acid of the application, or the DNA or cDNA nucleic acid of the application, or the expression vector of the application.
  • the cell of the application can e.g., be a cell, which has been infected, transfected or transformed by the live and attenuated JEV of the application, or the mutated M protein of the application, or the mutated ectoM of the application, or the RNA nucleic acid of the application, or the DNA or cDNA nucleic acid of the application, or the expression vector of the application.
  • the cell of the application can be infected, transfected or transformed by methods well known to the person skilled in the art, e.g., by chemical transfection (calcium phospate, lipofectamine), lipid-based techniques (liposome), electroporation, photoporation. Said infection, transfection or transformation can be transient or permanent.
  • Examples of a cell of the application include:
  • bacterial cell such as an E. coli cell
  • yeast cell such as a S. cerevisiae cell
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660TM]),
  • a mammal cell especially a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g., a cell of the Vero cell line [ATCC® CCL-81TM]) or a human cell (e.g., a cell of the HEK293T cell line [ATCC® CRL-3216TM] or of the SK-N-SH cell line [ATCC® HTB-1 1TM]), more particularly
  • bacterial cell such as an E. coli cell, or
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g. , a cell of the C6/36 cell line [ATCC® CRL- 1660TM]),
  • an insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g. , a cell of the C6/36 cell line [ATCC® CRL- 1660TM]).
  • a Drosophila cell e.g. , a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g. , a cell of the C6/36 cell line [ATCC
  • the cell of the application can be in isolated form.
  • the cell of the application can be contained in a culture medium, more particularly a non- naturally occurring culture medium, e.g. , an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
  • a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
  • a particular embodiment of the application is a nucleic acid clone, more particularly a RNA or cDNA clone, more particularly a cDNA clone of the live and attenuated JEV of the application.
  • a nucleic acid clone of the application can e.g. , be under the form of a (recombinant) cell, more particularly a (recombinant) insect cell [such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g.
  • a (recombinant) insect cell such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g.
  • An example of cDNA clone of the application is the (recombinant) cell deposited at the Collection Nationale de Culture de Microorganismes (CNCM) under 1-4902, or a (recombinant) cell (more particularly a (recombinant) insect cell) containing the plasmid of said cell, which has been deposited at the Collection Nationale de Culture de Microorganismes (CNCM) under 1-4902.
  • cDNA clone of the application is a (recombinant) cell (more particularly a (recombinant) insect cell), which comprises or has been infected, transfected or transformed by a (recombinant) expression vector carrying a nucleic acid of the application as an insert for expression thereof.
  • cDNA clone of the application is a (recombinant) cell (more particularly a (recombinant) insect cell), which comprises a (mutated JEV M) protein of the application or a (mutated JEV M protein ectomain of the application.
  • a clone or cDNA clone of the application does advantageously not comprise (nor codes for) the M protein of an infectious and/or virulent JEV (such as the JEV M protein of SEQ ID NO: 2).
  • a clone or cDNA clone of the application does advantageously not comprise (nor codes for) the M protein ectodomain of an infectious and/or virulent JEV (such as the JEV M ectodomain of SEQ ID NO: 6).
  • the clone or cDNA clone of the application shows a viral particle assembly default or defect in a mammalian cell but not in a mosquito cell, as described above or below illustrated.
  • the clone or cDNA clone of the application induces the production of JEV neutralizing antibodies, more particularly JEV sero- neutralization, more particularly in a mammalian host (such as a rodent, a monkey or a human), as described above or below illustrated.
  • a mammalian host such as a rodent, a monkey or a human
  • the clone or cDNA clone of the application is a live clone or cDNA clone, but does not cause encephalitis (i.e., is an attenuated JEV clone or cDNA).
  • the application also relates to a culture medium comprising the cell or nucleic acid clone of the application, more particularly to a culture medium comprising the cDNA clone of the application.
  • Said culture medium can e.g.
  • a non-naturally occurring culture medium e.g., an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
  • DMEM Dulbecco's Modified Eagle Medium
  • L15 L15, INVITROGEN
  • the application also relates to a non-infectious and non-replicative particle, which comprises the mutated M protein of the live and attenuated JEV of the application.
  • the application relates to a non-infectious and non-replicative particle, which comprises the mutated ectoM of the M protein of the live and attenuated JEV of the application.
  • a non-infectious and non-replicative particle of the application does not comprise the (full-length) JEV genomic RNA, nor the cDNA retro- transcript thereof.
  • a non-infectious and non-replicative particle of the application does advantageously not comprise the protein M or the protein M ectodomain of an infectious and/or virulent JEV.
  • non-infectious and non-replicative particles notably include a Virus-Like Particle (VLP).
  • VLP Virus-Like Particle
  • Said non-infectious and non-replicative particle can be produced by a cell of the application, e.g., by a cell of the application, which has been infected, transfected or transformed by an expression vector of the application, for example an insect cell (e.g., a Drosophila cell such as a S2 cell) infected or transfected by a Baculovirus vector, or a bacterial cell (e.g., a E. coli cell) transfected by a plasmid.
  • an insect cell e.g., a Drosophila cell such as a S2 cell
  • Baculovirus vector e.g., a bacterial cell
  • bacterial cell e.g., a E. coli cell
  • the application also relates to a composition, more particularly a pharmaceutical composition, more particularly an immunogenic composition, more particularly a vaccine, comprising the live and attenuated JEV of the application, or the expression vector of the application, or the cell of the application, or the clone or cDNA clone of the application (or at least one of these elements).
  • the live and attenuated JEV of the application, the expression vector of the application, the cell of the application, or the clone or cDNA clone of the application can be used as active ingredient for immunization, in particular for prophylactic immunization against a JEV infection in a mammalian host, especially in a human or an animal host.
  • the live and attenuated JEV of the application, the expression vector of the application, the cell of the application, or the clone or cDNA clone of the application can e.g., be used as active ingredient for prophylactic vaccination against JEV.
  • composition of the application is suitable for administration into a host, in particular in a mammalian host, especially in a human or an animal host.
  • composition of the application may further comprise a pharmaceutically suitable excipient or carrier and/or vehicle, when used for systemic or local administration.
  • a pharmaceutically suitable excipient or carrier and/or vehicle refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type.
  • a “pharmaceutically acceptable carrier” is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation; suitable carriers include, but are not limited to, phosphate buffered saline solutions, distilled water, emulsions such as an oil/water emulsions, various types of wetting agents sterile solutions and the like, dextrose, glycerol, saline, ethanol, and combinations thereof.
  • composition of the application may further comprise an immunogenic adjuvant, such as Freund type adjuvants, generally used in the form of an emulsion with an aqueous phase or can comprise water-insoluble inorganic salts, such as aluminium hydroxide, zinc sulphate, colloidal iron hydroxide, calcium phosphate or calcium chloride.
  • an immunogenic adjuvant such as Freund type adjuvants, generally used in the form of an emulsion with an aqueous phase or can comprise water-insoluble inorganic salts, such as aluminium hydroxide, zinc sulphate, colloidal iron hydroxide, calcium phosphate or calcium chloride.
  • said immune antibody response is a protective humoral response.
  • the protective humoral response results mainly in maturated antibodies, having a high affinity for their antigen, such as IgG.
  • the protective humoral response induces the production of neutralizing antibodies.
  • the composition of the application (in particular the live and attenuated JEV of the application) has a protective capacity against JEV infection when after challenge of immunized host with JEV, it enables the delay and/or the attenuation of the symptoms usually elicited after infection with said JEV against which protection is sought by the administration of the composition of the application, or when especially the JEV infection is delayed.
  • said composition of the application is formulated for an administration through parental route such as subcutaneous (s.c), intradermal (i.d.), intramuscular (i.m.), intraperitoneal (i.p.) or intravenous (i.v.) injection, more particularly intradermal (i.d.) injection.
  • parental route such as subcutaneous (s.c), intradermal (i.d.), intramuscular (i.m.), intraperitoneal (i.p.) or intravenous (i.v.) injection, more particularly intradermal (i.d.) injection.
  • said composition of the application is administered in one or multiple administration dose(s), in particular in a prime-boost administration regime.
  • rime-boost regimen generally encompasses a first administration step eliciting an immune response and one or several later administration step(s) boosting the immune reaction.
  • an efficient prime-boost system can be used for iterative administration, enabling successively priming and boosting the immune response in a host, especially after injections in a host in need thereof.
  • iterative means that the active principle is administered twice or more to the host.
  • the priming and boosting immunization can be administered to the host at different or identical doses, and injections can be administered at intervals of several weeks, in particular at intervals of four weeks or more.
  • the quantity to be administered depends on the subject to be treated, including the condition of the patient, the state of the individual's immune system, the route of administration and the size of the host. Suitable dosages can be adjusted by the person of average skill in the art.
  • the application also relates to a method to treat, prevent or protect, more particularly to prevent or protect against a JEV infection in a mammalian host, especially in a human or a non-human animal host, comprising administering said live and attenuated JEV of the application, or said expression vector of the application, or said cell of the application, or said clone or cDNA clone of the application or said composition of the application to said mammalian host.
  • the expression "to protect against JEV infection” refers to a method by which a Japanese encephalitis virus infection is obstructed or delayed, especially when the symptoms accompanying or following the infection are attenuated, delayed or alleviated or when the infecting virus is cleared from the host.
  • the application also relates to a method to produce a live and attenuated JEV, which comprises producing said live and attenuated JEV of the application, or said expression vector of the application, or said cell of the application, or said clone or cDNA clone of the application or said composition of the application.
  • the application also relates to a method to produce an immunogenic composition or vaccine against JEV infection, which comprises producing said live and attenuated JEV of the application, e.g., as a clone or cDNA clone in a culture medium, optionally collecting the viral particles or virions produced by said live and attenuated JEV, and formulating said cultured JEV (or said collected viral particles) in a composition suitable for administration to an animal, more particularly to a human.
  • Said culture medium can e.g., be a non-naturally occurring culture medium, e.g., an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
  • DMEM Dulbecco's Modified Eagle Medium
  • L15 L15, INVITROGEN
  • the application also relates to a method of (in vitro) attenuation of an infectious and/or virulent JEV, which comprises or consists of mutating the protein M of said infectious and/or virulent JEV, wherein said mutation comprises or consists of the replacement of the amino acid which is at position 36 within the (75 amino acid long) sequence of said protein M by the the amino acid phenylalanine.
  • the application also relates to a method of (in vitro) attenuation of an infectious and/or virulent JEV, which comprises or consists of mutating the protein M of said infectious and/or virulent JEV, wherein said mutation comprises or consists of the replacement of the amino acid which is at position 5 within the (75 amino acid long) sequence of said protein M by the the amino acid proline, and the replacement of the amino acid which is at position 36 within the (75 amino acid long) sequence of said protein M by the the amino acid phenylalanine.
  • the attenuated JEV thus produced still is a live virus.
  • the (live and) attenuated JEV thus produced shows a viral particle assembly default or defect in a mammalian cell but not in a mosquito cell, as described above or below illustrated.
  • the (live and) attenuated JEV thus produced induces the production of JEV neutralizing antibodies, more particularly JEV sero- neutralization, more particularly in a mammalian host (such as a rodent, a monkey or a human), as described above or below illustrated.
  • the application generally relates to the mutation of JEV M protein or fo JEV ectoM, more particularly to the replacement by phenylalanine of the amino acid, which is at position 36 within JEV M protein, as a JEV M or ectoM mutation, which induces a default or defect in JEV viral assembly, more particularly as a JEV M or ectoM mutation inducing a default or defect in JEV viral assembly that achieves JEV attenuation.
  • Human neuroblastoma cells SK-N-SH (ATCC® HTB-1 1TM) and human kidney cells HEK293T (ATCC® CRL-3216TM) were cultured at 37°C in Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) containing 10% of fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • Aedes albopictus cells C6/36 [ATCC® CRL-1660TM] were cultured at 28°C in Leibovitz's 15 (L15, INVITROGEN) medium containing 10% of FBS and 1 % of penicillin and streptomycin. Plasmids
  • PCR products were digested with Dpnl enzyme (NEW ENGLAND
  • BIOLABS BIOLABS
  • STBL2 Bacteria were cultured in rich medium Terrific Broth containing 100 mM of carbenicillin at 30°C during 48h.
  • VVT Wild type
  • mutant M-I36F viruses were produced by transfection of infectious clone in C6/36 cells using Lipofectamine2000 reagent (LIFE TECHNOLOGIES), according to the supplier's instructions. Supernatants were collected 7 days post-transfection. Infections
  • Virus infections were performed in 24-well-culture plaques. 10 5 SK-N- SH cells or 5.10 5 C6/36 cells were seeded. 24 hours later, they were infected with 200 L of medium containing a given number of viral particles, depending on the MOI. One hour after inoculation, inoculum was replaced by medium containing 2% of FBS.
  • mice Three-week-old female C57BL/6 mice were obtained from JANVIER LABS (France). They were inoculated intraperitoneally with 10 3 ffu of virus RP9 or doses from 10 3 to 10 6 ffu of virus M-I36F. Mice were monitored daily and mortality was evaluated. 27 days after inoculation, sera were collected from surviving mice and pooled by group.
  • Mutant M-I36F life cycle is not impaired in mosquito cells, but is impaired in mammalian cells.
  • Liang et al. have synthesized an infectious molecular clone of JEV genotype 3-strain RP9 ⁇ Chen et al., Virology, 1996, 217(1): 220-229 and Lin et al., Virus Res. 1996, 44(1): 45-56) in 2009. They demonstrated that transfection of this molecular clone into BHK21 cells induced production of JEV virions.
  • the inventors used this molecular clone to synthesize a mutant molecular clone containing mutation M-I36F. Mammalian HEK293T cells when transfected with the wt JEV produced JEV virions. However, when transfected with M-I36F mutant infectious clone, less virions were produced (2 log decrease, data not shown).
  • JEV is an arbovirus that infects both mosquitoes and mammals. Due to the limited amount of infectious particles produced in HEK293T cells, the inventors chose to produce our viruses in Aedes albopictus C6/36 cells by transfection of the two infectious clones into these cells. Viruses were produced in similar quantities (data not shown). To investigate the effect of mutation M-I36F on the virus life cycle in both cell types, Aedes albopictus cells C6/36 and human neuroblastoma cells SK-N-SH were infected at an MOI of 5 ( Figures 1A and 1 B). The cells were harvested 24h and 48h post-infection and the supernatants were collected.
  • the supernatants were titrated on C6/36 cells.
  • the inventors determined that the production of infectious viral particles was similar for both viruses in C6/36 cells at 24 and 48h ( Figure 1A). However, in mammalian SK-N-SH cells, less infectious viral particles were produced for mutant M-I36F than for WT virus at 24 and 48h ( Figure 1 B). The inventors thus demonstrated that the mutant virus M-I36F life cycle is not impaired in C6/36 mosquito cells, while it is impaired in human SK-N-SH cells.
  • Mutant virus M-I36F is attenuated and induces production of antibodies targeting JEV in mice.
  • mice three-week-old female C57BL/6 mice were injected intraperitoneal ⁇ with 10 3 ffu of WT virus, or from 10 3 to 10 6 ffu of M-I36F virus (Figure 2A).
  • Mice injected with WT virus started to die from neurological disease 9 days after inoculation, whereas mice injected with all doses of M-I36F virus survived the inoculation ( Figure 2A).
  • mice sera were collected 27 days after inoculation and ELISA was conducted on these sera to determine if mice had produced antibodies against JEV. Purified and inactivated JEV particles were used as target for the ELISA test ( Figure 2B). Mice injected with WT and M-I36F virus produced antibodies targeting JEV particles ( Figure 2B). The produced antibodies were neutralizing (data not shown). However, it seems that mice injected with M- I36F virus produced fewer antibodies than mice injected with RP9 virus, regardless of the dose injected. EXAMPLE 2: 36F mutation in protein M of a Flavivirus other than JEV
  • EXAMPLE 3 I36 mutation in protein M of JEV other than I36F
  • Parental and mutant I36A JEV plasm id constructs were transfected in 293T cells, following the methods described in example 1 above.
  • the produced viral particles were titrated in BHK21 and C6/36 cells ( Figure 4).

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Abstract

The application relates to the attenuation of Japanese Encephalitis Virus (JEV). The application notably provides a live and attenuated JEV comprising a mutated M protein. Said mutated M protein comprises or consists of a sequence, wherein the amino acid at position 36 in said sequence is mutated. The amino acid at position 5 may also be mutated. The application also provides means deriving from said live and attenuated JEV, such as nucleic acids, expression vectors, cells, cDNA clones, immunogenic compositions as well as uses and methods.

Description

A LIVE AND ATTENUATED JAPANESE ENCEPHALITIS VIRUS COMPRISING A MUTATED M PROTEIN
FIELD OF THE INVENTION
The application relates to the attenuation of Japanese Encephalitis Virus (JEV). The application notably provides a live and attenuated JEV comprising a mutated M protein. Said mutated M protein comprises or consists of a sequence, wherein the amino acid at position 36 in said sequence is mutated. The amino acid at position 5 may also be mutated. The application also provides means deriving from said live and attenuated JEV, such as nucleic acids, expression vectors, cells, cDNA clones, immunogenic compositions as well as uses and methods. BACKGROUND OF THE INVENTION
Flaviviruses such as Japanese Encephalitis Virus (JEV), Dengue Virus (DV), West Nile Virus (WNV) and Yellow Fever Virus (YFV) viruses, are arthropod-borne pathogens (arboviruses) that are transmitted through the bite of an infected mosquito and may cause serious human diseases worldwide {Lindenbach BD et al., Adv Virus Research, 2003, 59, 23-61). To date, very few vaccines against flaviviruses are commercially available. The first one was the live-attenuated vaccine 17D against YFV (Barrett, ADT Yellow Fever Vaccines Biologicals 1997). There are also live-attenuated and inactivated vaccines against JEV such as the live-attenuated virus vaccine SAu-14-2 (Yun SI, Lee YM. Hum Vaccin Immunother 2014 Feb, 10(2): 263- 279) and inactivated vaccines against tick-borne encephalitis virus (Lani R et al., Ticks Tick Borne Dis. 2014 Sep, 5(5): 457-465). Determination of the attenuation factors of these viruses can help in the development of new molecular vaccines. Among the different proteins encoded by the virus genome, it seems that structural proteins (capsid C, membrane M and envelope E) have a role in the pathogenesis of flaviviruses (Kofler RM et al. J Virol. 2002 Apr, 76(7): 3534-3543; Zhu W et al. Virus Res. 2007 Jun; 126(1- 2): 226-232; Keelapang et al., Vaccine, 2013, 31 (44), 5134-5140; Langevin SA et al., J Gen Virol. 2011, 92(Pt 12): 2810-2820; Yun et al., A PLoS Pathogens 2014, 10(7) : e1004290; Yang D. et al., Vaccine, 2014, 32(23): 2675-2681; Guirakhoo F et al., J Virol. 2004, 78(18): 9998-10008; Arroyo J. et al., J. Virol. 2001, 75(2): 934-942; Zhao Z. et al., J Gen Virol. 2005, 86(Pt 8): 2209-2220; Mandl et al., J Virol. 2000, 74(20): 9601-9609; Holzmann H. et al. J Virol. 1990, 64(10): 5156-5159; Lee E.et al., J Virol 2008, 82(12): 6024-6033).
JEV contains a positive single-stranded RNA genome encoding a single polyprotein that is processed into three structural proteins, the capsid (C), the precursor of membrane (prM) and the envelope (E) proteins, and seven nonstructural proteins (NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5).
The membrane protein is synthesized as a precursor prM. It is cleaved in the trans-Golgi during viral particles secretion into pr and M (Li L et al. Science. 2008, 319(5871): 1830-1834). This cleavage is mandatory to produce infectious particles (Randolph VB et al., Virology, 1990, 174(2): 450- 458). The resulting M protein is composed of an ectodomain (ectoM) consisting of 40 amino acids and 2 transmembrane domains (Zhang et al., EMBO J. 2003, 22(11): 2604-2613).
To date, little is known about the role of protein M. It has been shown that prM protein acts as a chaperone for the E protein folding (Konishi et al., J Virol. 1993, 67(3): 1672-1675) and prevents fusion within the infected cells (Yu et al., J Virol. 2009, 83(23): 12101-12107). It has also been disclosed that the C-terminal helical domain of DENV ectoM is involved in virus assembly (Pryor et ai, J Gen Virol, 2004, 85(Pt 12): 3627-3636; Hsieh et ai, J Virol. 2010, 84(9): 4782-4797) and entry (Hsieh et al., J Virol. 2010, 84(9): 4782-4797), and that DENV, YFV strain Asibi and WNV ectoM induces apoptosis in mammalian cells. In this study, it has been shown that a mutation of the leucine at position 36 of YFV ectoM into a phenylalanine or a mutation of the isoleucine at position 36 of DENV ectoM into a phenylalanine reduces the induction of apoptosis (Catteau et al., J. Gen Viro 2003, 84(Pt 10): 2781-2793; US 7,785,604 patent). In particular, patent US 7,785,604 describes that a nonapeptide (ApoptoM) from flavivirus ectoM is able to modulate specifically the apoptotic activity of diverse flaviviruses, and that the proapoptotic properties of ectoM are conserved among apoptosis-inducing flaviviruses, i.e. JEV, DV and YFV.
McElroy et al. have demonstrated that the replacement of the leucine at position 36 of YFV strain Asibi ectoM into a phenylalanine (YFV-17D vaccine strain) reduce the mean dissemination of YFV in mosquitoes. A higher mean dissemination was obtained when the sequences encoding the full M-E proteins or the E protein domain III of YFV-17D vaccine strain were incorporated to replace the same proteins of YFV strain Asibi (McElroy et al., J. Gen Virol., 2006, 87, 2993-3001). SUMMARY OF THE INVENTION
The application provides a live and attenuated JEV comprising a mutated M protein. Said mutated M protein comprises or consists of a sequence, wherein the amino acid at position 36 in said sequence is mutated, more particularly replaced by another amino acid, more particularly by the amino acid phenylalanine. The amino acid at position 5 may also be mutated, more particularly replaced by another amino acid, more particularly by the amino acid proline.
The application also provides means deriving from said live and attenuated JEV, such as nucleic acids, more particularly RNA and cDNA, proteins and polypeptides, expression vectors, more particularly recombinant expression vectors, cells, more particularly recombinant cDNA clones as well as immunogenic compositions and vaccines.
The application also provides as uses and methods, more particularly uses and methods to prevent a JEV infection in a mammalian host, especially in a human or an animal host. BRIEF DESCRIPTION OF THE FIGURES
Figures 1A and 1 B. M-I36F virus life cycle is not impaired in mosquito cells, while it is impaired in SK-N-SH cells. C6/36 mosquito cells and mammalian SK-N-SH cells were infected at an MOI of 5 and were harvested at 24h and 48h post-infection. Figure 1A: Supernatants from C6/36 infected cells collected at 24h and 48h post-infection were titrated on C6/36 mosquito cells. Figure 1 B: Supernatants from SK-N-SH infected cells collected at 24h and 48h post-infection were titrated on C6/36 mosquito cells.
Figures 2A and 2B. M-I36F virus is attenuated and induces production of antibodies in C57BL/6 mice. Figure 2A: Three-week-old C57BL/6 female mice were injected intraperitoneally with 103 ffu of RP9 virus, or with different doses from 103 to 106 ffu of M-I36F mutant virus. Survival percentages were calculated (****: P < 0.00001 ). Figure 2B: Sera were collected 27 days after inoculation from the mice that survived and were diluted. Dilutions were analyzed by ELISA, using purified and inactivated JEV particles as target. Dilution to 1/100 of serum from mice injected with DPBS was used as negative control (***: P < 0.001 ).
Figures 3A and 3B. The replacement by phenylalanine of the amino acid, which is at position 36 in protein M of a Flavivirus other than JEV, i.e., in the Yellow Fever Virus, does not lead to attenuation [cf. example 2 below]. Figure 3A: schematic representation of a while-type infectious Yellow Fever Virus [YF-Asibi], which has been modified by replacement of the amino acid at position 36 in protein M by the amino acid phenylalanine (L36F) and by replacement of the amino acid at position 95 in protein NS4B by the amino acid Met (I95M) [YF-Asibimut]. Figure 3B: titration results of this mutated YFV (YF-Asibimut) and of the wild-type parent (i.e., non mutated) YFV (YF-Asibi), showing that mutation does not lead to attenuation in YFV.
Figure 4. The replacement in protein M of JEV of the amino acid at position 36 by alanine instead of phenylalanine does not lead to attenuation [cf. example 3 below]. Figure 4 shows the results of the titration of the viral particles produced by the wild-type (infectious) JEV and by the I36A mutated JEV in BHK21 cells and in C6/36 cells. WT = wild-type (i.e. , infectious) JEV; I36A = wild-type JEV modified by I36A mutation in protein M. DETAILED DESCRIPTION OF THE INVENTION
The inventors introduced a mutation into the M protein of a JEV infectious clone, in particular a point mutation into the M protein of a JEV infectious clone and showed that infection of mammalian cells resulted in a reduced number of secreted viral particles relative to the wild-type virus. Interestingly, when mosquito cells were infected, the inventors did not observe any difference between the wild-type and the mutant viruses infectious cycles. In order to elucidate at which step of the viral life cycle the mutant viral particles are impaired in their production in mammalian cells, the inventors designed several reporter systems that uncoupled the entry, replication and assembly of JEV. Using these systems, the inventors showed that the mutation in the M protein strongly impacted the assembly of genuine viral particles in mammalian cells. Moreover, the mutant virus was severely attenuated in vivo in a mouse model of viral encephalitis, when compared to the wild-type virus.
The inventors thus identified an amino acid residue at position 36 in the M protein of JEV that plays a major role in the assembly of JEV particles in mammalian cells. More particularly, the inventors found that the replacement of the amino acid which is at position 36 in protein M of JEV by an amino acid other than isoleucine (I), more particularly by the amino acid phenylalanine (F), leads to attenuation.
Interestingly, this mutation did not impact particle assembly in mosquito cells suggesting different mechanisms/cellular partner(s) for viral particle assembly between mammals and mosquitoes. These results indicated that the M protein of JEV, in particular the ectoM of JEV, contained viral determinants for viral attenuation. The amino acid at position 5 in protein M of JEV may further be mutated, more particularly replaced by an amino acid other than glutamine (Q), more particularly replaced by the amino acid proline (P). Please see e.g., Brault et al. 201 1 (Virology 417: 369-378) and EP 1 809 325 (or US 8,029,802 B2).
The application accordingly relates to a live and attenuated JEV, which is obtainable by mutation of the endogenous M protein of a wild-type and/or infectious and/or virulent JEV, wherein said mutation comprises or consists of the replacement of the amino acid at position 36 in the sequence of said endogenous M protein (i.e., at position 36 is the sequence of the endogenous polyprotein sequence of said wild-type and/or infectious and/or virulent JEV) by an amino acid other than isoleucine (I), more particularly by the amino acid phenylalanine (F), and optionally by the replacement of the amino acid at position 5 in the sequence of said endogenous M protein (i.e., at position 36 is the sequence of the endogenous polyprotein sequence of said wild-type and/or infectious and/or virulent JEV) by an amino acid other than glutamine (Q), more particularly by the amino acid proline (P). The expression "a live and attenuated JEV is intended in accordance with its ordinary meaning in the field. More particularly, the expression "a live and attenuated JEV" designates a JEV that has a reduced pathogenic phenotype compared to a wild-type and/or infectious and/or virulent JEV. More particularly, a live and attenuated JEV does not cause encephalitis (e.g., because of reduced replication capacity and/or because of reduced and/or restricted tissue tropism, and/or because of default or defect in assembly of the JEV viral particles, more particularly by default or defect in assembly of the JEV viral particles), but is able to stimulate an immune response when administered to a host, especially a human, more particularly a protecting immune response protecting against JEV infection (seroneutralization). The term "immune response" includes one or several from antibody production, seroneutralization, induction of cell-mediated immunity, complement activation, development of immunological tolerance, alteration of cytokine production and alteration of chemokine production, more particularly (neutralizing) antibody production or seroneutralization, more particularly seroneutralization.
More particularly, a live and attenuated JEV of the application shows a default or defect in assembly of the JEV viral particles (such as a reduced or inhibited rate of viral particle production), more particularly a default or defect in assembly of the JEV viral particles (such as a reduced or inhibited rate of viral particle production), which is sufficient to not cause encephalitis.
Please see example 1 below, as well as Figures 1A and 1 B. More particularly, a live and attenuated JEV of the application stimulates said immune response (more particularly said anti-JEV (neutralizing) antibody production or JEV seroneutralization, more particularly said JEV seroneutralization).
Please see example 1 below, as well as Figures 2A and 2B.
More particularly, a live and attenuated JEV of the application stimulates said immune response (more particularly said anti-JEV antibody production or JEV seroneutralization, more particularly said JEV seroneutralization), but shows a default in assembly of the JEV viral particles (such as a reduced or inhibited rate of viral particle production), more particularly a default in assembly of the JEV viral particles (such as a reduced or inhibited rate of viral particle production), which is sufficient to not cause encephalitis.
Please see example 1 below as well as Figures 1A, 1 B, 2A and 2B, which notably demonstrate:
- a 100% survival rate of the mice, which have received the live and attenuated JEV of the application (36F mutated M protein), - the production of anti-JEV (neutralizing) antibodies, or JEV seroneutralization, and which further demonstrates that
- the mutation at position 36 of the protein M of JEV is sufficient to achieve said 100% survival rate (and is also sufficient to achieve said anti- JEV (neutralizing) antibodies, or said JEV seroneutralization).
The wild-type and/or infectious and/or virulent JEV to be mutated for attenuation can e.g., be a JEV of genotype 3 (G3), such as the strain RP9 (GENBANK® accession number KF907505).
The nucleotide sequence of the polynucleotide encoding the endogenous M protein of JEV strain RP9 (infectious or virulent JEV), as well as the amino acid sequence of the endogenous M protein of JEV strain RP9 may for example consist of the sequences disclosed as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.
SEQ ID NO: 1 (cDNA sequence of the endogenous protein M of JEV RP9): tccgtgtc ggtccaaaca catggggaga
gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cgatatctca tgaaaactga gaactggatc ataaggaatc ctggctatgc tttcctggcg gcggtacttg gctggatgct tggcagtaac aacggtcaac gcgtggtatt caccatcctc ctgctgctgg ttgctccggc ttacagt
SEQ ID NO: 2 (endogenous protein M of JEV RP9):
SVSVQTHGESSLV KKEA LDSTKATRYLMKTEIWIII^PGYAFLAAVLG LGSITOGQRVVFTILLL LVAPAYS The live and attenuated JEV of the application can e.g., be a JEV of genotype 3 (G3).
In other words, the application generally relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) M protein, wherein the amino acid sequence of said (mutated JEV) M protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 93%, or at least 94%, or at least 96% or at least 97% or at least 98%, more particularly at least 96% or at least 97% or at least 98%, more particularly at least 97% or at least 98% identical to the sequence of SEQ ID NO: 2, and
(b) wherein the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine.
The application more particularly relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) M protein, wherein the amino acid sequence of said (mutated JEV) M protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 98% identical to the sequence of SEQ ID NO: 2, and
(b) wherein the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine. The application also relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) M protein, wherein the amino acid sequence of said (mutated JEV) M protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 93%, or at least 94%, or at least 96% or at least 97%, more particularly at least 96% or at least 97%, more particularly at least 97% identical to the sequence of SEQ ID NO: 2, and
(b) wherein the amino acid at position 5 in said sequence of 75 amino acids is the amino acid proline, and the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine.
Advantageously, said mutated M protein replaces an endogenous M protein, more particularly the endogenous M protein of a wild-type and/or infectious and/or virulent JEV, more particularly the endogenous M protein, the sequence of which is SEQ ID NO: 2.
In other words, the live and attenuated JEV does advantageously not comprise (nor codes for) the endogenous M protein of an infectious and/or virulent JEV, more particularly the M protein of SEQ ID NO: 2. A particular nucleotide sequence of the polynucleotide encoding said mutated JEV M protein as well as a particular amino acid sequence of said mutated JEV M protein are the sequences disclosed as SEQ ID NO: 3 and SEQ ID NO: 4 respectively.
SEQ ID NO: 3 (cDNA sequence coding for a mutated JEV M protein of the application):
tccgtgtc ggtccaaaca catggggaga
gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cgatatctca tgaaaactga gaactggnnn ataaggaatc ctggctatgc tttcctggcg gcggtacttg gctggatgct tggcagtaac aacggtcaac gcgtggtatt caccatcctc ctgctgctgg ttgctccggc ttacagt
wherein nnn = a codon coding for phenylalanine (TTT or TTC).
SEQ ID NO: 4 (mutated JEV M protein of the application):
SVSVQTHGESSLV KKEAWLDSTKATRYLMKTE W^
LVAPAYS
wherein X = F (phenylalanine).
In other words, the live and attenuated JEV of the application can e.g., be a JEV, which comprises or codes for a (mutated JEV ) M protein, wherein said (mutated JEV) protein M comprises or consists of the protein of SEQ ID NO: 4.
Another particular nucleotide sequence of the polynucleotide encoding a mutated JEV M protein of the application as well as another particular amino acid sequence of said mutated M protein are the sequences disclosed as SEQ ID NO: 9 and SEQ ID NO: 10 respectively.
SEQ ID NO: 9 (cDNA sequence coding for a mutated JEV M protein of the application):
tccgtgtc ggtcnnniaca catggggaga
gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cgatatctca tgaaaactga gaactggnnn^ ataaggaatc ctggctatgc tttcctggcg gcggtacttg gctggatgct tggcagtaac aacggtcaac gcgtggtatt caccatcctc ctgctgctgg ttgctccggc ttacagt wherein nnni = a codon coding for proline (CCT or CCC or CCA or CCG) and nnn2 = a codon coding for phenylalanine (TTT or TTC).
SEQ ID NO: 10 (mutated JEV M protein of the application):
SVSV iTHGESSLV KKEA LDSTKATRYLMKTE WX21RNPGYAFLAAVLGWMLGS NGQRWFTILL LLVAPAYS
wherein Xi = P (proline) and X2 = F (phenylalanine).
In other words, the live and attenuated JEV of the application can e.g., be a JEV, which comprises or codes for a (mutated) M protein, wherein said (mutated) protein M comprises or consists of the protein of SEQ ID NO: 10.
In a particular embodiment, the live and attenuated JEV of the application comprises the RNA version of the (cDNA) nucleotide sequence of SEQ ID NO: 7 or of SEQ ID NO: 1 1 (the sequence of SEQ ID NO: 7 or of SEQ ID NO: 1 1 codes for a mutated ectoM of the application; cf. below).
In a particular embodiment, the live and attenuated JEV of the application comprises the RNA version of the (cDNA) nucleotide sequence of SEQ ID NO: 3 or of SEQ ID NO: 9 (the sequence of SEQ ID NO: 3 or of SEQ ID NO: 9 codes for a mutated M of the application; cf. above).
In a particular embodiment, the live and attenuated JEV of the application comprises the RNA version of the (cDNA) nucleotide sequence insert carried by the plasmid stbl2 / pBR322-JEV-RP9 (M-I36F), which has been deposited under the terms of the Budapest Treaty at the Collection Nationale de Culture de Microorganismes (CNCM) under deposit number I- 4902, on November 4, 2014.
The address of CNCM is: Collection Nationale de Culture de
Microrganismes, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris CEDEX 15, France.
Plasmid stbl2/pBR322-JEV-RP9(M-l36F) deposited at the CNCM under I-4902 was obtained from the JEV RP9 strain, mutated by replacement of the codon, which in the protein M codes for the amino acid at position 36 (i.e., isoleucine), by a codon coding for the amino acid phenylalanine (I36F mutation). The expression "RNA version of a (cDNA) nucleotide sequence" means the (RNA) sequence, which results from the replacement of each nucleotide T of said (cDNA) nucleotide sequence by the nucleotide U. Advantageously, the live and attenuated JEV of the application shows a default or detect in the assembly of the viral particles (e.g. , a reduced production rate of (correctly) assembled viral particles).
Advantageously, the live and attenuated JEV of the application shows said default or defect in a mammalian cell, but not in a mosquito cell.
By comparison, an infectious JEV (such as the JEV strain RP9) does not show this defect in a mosquito cell and does neither show it in a mammalian cell.
In other words, the live and attenuated JEV of the application shows a default or detect in the assembly of the viral particles in a mammalian cell (e.g. , a reduced production rate of (correctly) assembled viral particles), compared to an infectious JEV (such as the JEV strain RP9; complete genome of JEV RP9 = GENBANK® accession number KF907505; polyprotein of JEV RP9 = GENBANK® accession number AHK05344.1 ).
Said mammalian cell can e.g. , be a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g. , a cell of the Vero cell line [ATCC® CCL-81™]) or a human cell (e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216™] or of the SK-N-SH cell line [ATCC® HTB-1 1™]). More particularly, said mammalian cell can e.g. , be a human cell (e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216™] and/or of the SK-N-SH cell line [ATCC® HTB-1 1™]).
Said mosquito cell can e.g. , be an Aedes cell, an Aedes albopictus cell or a cell of the C6/36 cell line [ATCC® CRL-1660™].
Advantageously, the live and attenuated JEV of the application shows said default or defect in a human cell (e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216™] and/or of the SK-N-SH cell line [ATCC® HTB-1 1™]), but not in a mosquito cell (e.g. , be an Aedes cell, an Aedes frugipreda cell, an Aedes albopictus, or a cell of the C6/36 cell line [ATCC® CRL-1660™]). Advantageously, the live and attenuated JEV of the application shows said default or defect in a cell of the HEK293T cell line [ATCC® CRL-3216™] and/or of the SK-N-SH cell line [ATCC® HTB-1 1™]), but not in a cell of the C6/36 cell line [ATCC® CRL-1660™].
Please see example 1 below, which notably demonstrates a 100% survival rate of mice, which have received the live and attenuated JEV of the application, and which further demonstrates that the mutation at position 36 of the protein M of JEV is sufficient to achieve said 100% survival rate. Advantageously, the live and attenuated JEV of the application induces JEV neutralizing antibodies, more particularly JEV sero- neutralization, more particularly in a mammalian host (such as a rodent, a monkey or a human). Please see example 1 below and Figures 2A and 2B. The application also relates to the (mutated JEV) M protein of the live and attenuated JEV of the application.
The application thus relates to a (mutated JEV M) protein, wherein the amino acid sequence of said (mutated JEV M) protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 93%, or at least 94%, or at least
96% or at least 97% or at least 98%, more particularly at least 96% or at least 97% or at least 98%, more particularly at least 97% or at least 98% identical to the sequence of SEQ ID NO: 2, and
(b) wherein the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine. An example of such a (mutated JEV M) protein is the protein of SEQ ID NO: 4 or of SEQ ID NO: 10 (cf. above).
In a particular embodiment, the application relates to a (mutated JEV
M) protein, wherein the amino acid sequence of said (mutated JEV M) protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 97% or at least 98%, more particularly at least 98% identical to the sequence of SEQ ID NO: 2, and (b) wherein the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine. An example of such a (mutated JEV M) protein is the protein of SEQ ID NO: 4 or of SEQ ID NO: 10, more particularly of SEQ ID NO: 4 (cf. above).
In a particular embodiment, the application relates to a (mutated JEV
M) protein, wherein the amino acid sequence of said (mutated JEV M) protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 93%, or at least 94%, or at least 96% or at least 97%, more particularly at least 96% or at least 97%, more particularly at least 97% identical to the sequence of SEQ ID NO: 2, and
(b) wherein the amino acid at position 5 in said sequence of 75 amino acids is the amino acid proline, and the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine. An example of such a (mutated JEV M) protein is the protein of SEQ ID NO: 10 (cf. above).
The application also relates to a nucleic acid, more particularly a cDNA, DNA or RNA nucleic acid, coding for said (mutated JEV M protein) of the application, as well as to expression vectors comprising this nucleic acid inserted in an expression cassette, and cells, more particularly recombinant cells, comprising or transformed or transfected or infected by such an expression vector.
Examples of nucleic acid coding for said (mutated JEV M protein) of the application include the nucleic acid of SEQ ID NO: 3 or of SEQ ID NO: 9.
Examples of such (recombinant) cells include:
- a bacterial cell, such as an E. coli cell,
- a yeast cell, such as a S. cerevisiae cell,
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660™]),
- a mammal cell, especially a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g., a cell of the Vero cell line [ATCC® CCL-81 I M]) or a human cell (e.g., a cell of the HEK293T cell line [ATCC® CRL-3216™] or of the SK-N-SH cell line [ATCC® HTB-1 1™]),
more particularly
- a bacterial cell, such as an E. coli cell, or
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660™]),
more particularly
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660™]).
The cell can be in isolated form. The cell of the application can be contained in a culture medium, more particularly a non-naturally occurring culture medium, e.g., an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
The application also relates to a polypeptide, which is a fragment of the (mutated JEV M) protein of the application, more particularly a fragment of said mutated JEV M protein, which comprises or consists of the ectodomain of said (mutated JEV) M protein [mutated ectoM].
The nucleotide sequence of the polynucleotide encoding the endogenous ectoM of the M protein of JEV strain RP9 (infectious or virulent JEV) as well as the amino acid sequence of the endogenous ectoM of the M protein of JEV strain RP9 are the sequences disclosed as SEQ ID NO: 5 and SEQ ID NO: 6 respectively. SEQ ID NO: 5 (cDNA coding for the ectodomain of the M protein of the JEV wild-type strain RP9):
tccgtgtc ggtccaaaca catggggaga
gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cgatatctca tgaaaactga gaactggatc ataaggaatc ct
SEQ ID NO: 6 (ectodomain of the M protein of JEV RP9):
SVSVQTHGESSLV KKEA LDSTKATRYL KTE IIRNP
A particular nucleotide sequence of the polynucleotide encoding a mutated ectoM of the application as well as a particular amino acid sequence of said mutated ectoM are the sequences disclosed as SEQ ID NO: 7 and SEQ ID NO: 8 respectively.
SEQ ID NO: 7 (cDNA coding for a mutated M ectodomain of the application): tccgtgtc ggtccaaaca catggggaga
gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cgatatctca tgaaaactga gaactggnnn ataaggaatc ct
wherein nnn = a codon coding for phenylalanine (TTT or TTC).
SEQ ID NO: 8 (mutated M ectodomain):
SVSVQTHGESSLVNKKEAWLDSTKATRYL KTENWXIRNP
wherein X = F (phenylalanine).
Another particular nucleotide sequence of the polynucleotide encoding a mutated ectoM of the application as well as another particular amino acid sequence of said mutated ectoM are the sequences disclosed as SEQ ID NO: 1 1 and SEQ ID NO: 12 respectively.
SEQ ID NO: 1 1 (cDNA coding for a mutated M ectodomain of the application):
tccgtgtc ggtcnnniaca catggggaga
gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cgatatctca tgaaaactga gaactggnnn^ ataaggaatc ct
wherein nnni = a codon coding for proline (CCT or CCC or CCA or CCG) and nnn2 = a codon coding for phenylalanine (TTT or TTC).
SEQ ID NO: 12 (mutated M ectodomain):
SVSVXiTHGESSLVNKKEAWLDSTKATRYL KTENW¾IRNP
wherein X2 =P (proline) and X2 = F (phenylalanine). The application thus relates to a polypeptide or to a mutated ectodomain (ectoM) of a JEV M protein, wherein the amino acid sequence of said polypeptide or mutated ectoM of a JEV M protein comprises or consists of a sequence, which:
(a) consists of 40 amino acids and is at least 92%, or at least 95%, more particularly at least 95% or at least 97%, more particularly at least 97% identical to the sequence of SEQ ID NO: 6, and
(b) wherein the amino acid at position 36 in said sequence of 40 amino acids is the amino acid phenylalanine. An example of such a polypeptide of the application is the polypeptide of SEQ ID NO: 8 or of SEQ ID NO: 12.
The application more particularly relates to a polypeptide or to a mutated ectodomain (ectoM) of a JEV M protein, wherein the amino acid sequence of said polypeptide or mutated ectoM of a JEV M protein comprises or consists of a sequence, which:
(a) consists of 40 amino acids and is at least 95% or at least 97%, more particularly at least 97% identical to the sequence of SEQ ID NO: 6, and (b) wherein the amino acid at position 36 in said sequence of 40 amino acids is the amino acid phenylalanine. An example of such a polypeptide of the application is the polypeptide of SEQ ID NO: 8 or of SEQ ID NO: 12, more particularly of SEQ ID NO: 8.
The application more particularly relates to a polypeptide or to a mutated ectodomain (ectoM) of a JEV M protein, wherein the amino acid sequence of said polypeptide or mutated ectoM of a JEV M protein comprises or consists of a sequence, which:
(a) consists of 40 amino acids and is at least 95% identical to the sequence of SEQ ID NO: 6, and
(b) wherein the amino acid at position 5 in said sequence of 40 amino acids is the amino acid proline, and the amino acid at position 36 in said sequence of 40 amino acids is the amino acid phenylalanine. An example of such a polypeptide of the application is the polypeptide of SEQ ID NO: 12.
The application also relates to a nucleic acid, more particularly a cDNA, DNA or RNA nucleic acid, coding for said polypeptide or mutated ectoM of the application, as well as to expression vectors comprising this nucleic acid inserted in an expression cassette, and cells, more particularly recombinant cells, comprising or transformed or transfected or infected by such an expression vector.
Examples of nucleic acid coding for said mutated ectoM of the application include the nucleic acid of SEQ ID NO: 7 or of SEQ ID NO: 1 1 .
Examples of such (recombinant) cells include:
- a bacterial cell, such as an E. coli cell,
- a yeast cell, such as a S. cerevisiae cell,
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660™]),
- a mammal cell, especially a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g., a cell of the Vero cell line [ATCC® CCL-81™]) or a human cell (e.g., a cell of the HEK293T cell line [ATCC® CRL-3216™] or of the SK-N-SH cell line [ATCC® HTB-1 1™]),
more particularly
- a bacterial cell, such as an E. coli cell, or
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660™]),
more particularly
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660™]). The cell can be in isolated form. The cell of the application can be contained in a culture medium, more particularly a non-naturally occurring culture medium, e.g., an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
The application generally relates to the JEV mutated M protein or JEV mutated ectoM of the application, as a protein or polypeptide inducing a default or defect in JEV viral assembly, more particularly as a protein or polypeptide inducing a default or defect in JEV viral assembly that achieves JEV attenuation.
The application also relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) protein M, wherein the amino acid sequence of said (mutated JEV) protein M comprises the polypeptide or mutated ectoM of the application.
The application also relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) protein M ectodomain, wherein the sequence of said (mutated JEV) protein M ectodomain comprises or consists of the polypeptide or mutated ectoM of the application.
In a particular embodiment, the live and attenuated JEV of the application comprises the RNA version of the (cDNA) nucleotide sequence of SEQ ID NO: 7 or 1 1 , more particularly the RNA version of the (cDNA) nucleotide sequence of SEQ ID NO: 3 or 9.
Advantageously, said polypeptide or mutated ectoM of the application replaces an endogenous ectoM, more particularly the endogenous ectoM of a wild-type and/or infectious and/or virulent JEV, more particularly the ectoM of SEQ ID NO: 6. In other words, the live and attenuated JEV of the application does advantageously not comprise (nor codes for) the endogenous ectoM of an infectious and/or virulent JEV, more particularly the ectoM of SEQ ID NO: 6. In the live and attenuated JEV of the application, the JEV structural proteins other than protein M, such the JEV protein E and the JEV protein C, more particularly the JEV protein E, can be the JEV structural proteins of an infectious JEV (such as JEV strain RP9).
In the live and attenuated JEV of the application, the JEV nonstructural proteins, such as the JEV proteins NS1 , NS2A, NS2B, NS3, NS4A, NSA4 and NS5, can be the JEV non-structural proteins of an infectious JEV (such as JEV strain RP9).
Indeed, the mutation of JEV M protein as described in the application, more particularly the replacement by phenylalanine of the amino acid, which is at position 36 within JEV M protein, is sufficient to obtain a default or defect in JEV viral particles assembly that achieves JEV attenuation.
Please see example 1 below as well as Figures 1A, 1 B, 2A and 2B, which notably demonstrate:
- a 100% survival rate of the mice, which have received the live and attenuated JEV of the application (36F mutated M protein),
- the production of anti-JEV (neutralizing) antibodies, or JEV seroneutralization, and which further demonstrates that
- the mutation at position 36 of the protein M of JEV is sufficient to achieve said 100% survival rate (and is also sufficient to achieve said anti- JEV (neutralizing) antibodies, or said JEV seroneutralization).
Therefore, the JEV proteins other than protein M, more particularly the JEV protein E, can be non-mutated compared to a wild-type and/or infectious and/or virulent JEV.
Hence, the application also relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) polyprotein, wherein the amino acid sequence of said (mutated JEV) polyprotein comprises the (mutated JEV) protein M of the application, more particularly the polypeptide or mutated ectoM of the application. The application thus relates to a live and attenuated JEV, which comprises or codes for a (mutated JEV) polyprotein, wherein the amino acid sequence of said (mutated JEV) polyprotein comprises or consists of the protein of SEQ ID NO: 13 (JEV RP9 polyprotein, wherein protein M is I36F mutated), or the protein of SEQ ID NO: 14 (JEV RP9 polyprotein, wherein protein M is Q5P and I36F mutated).
The sequence of SEQ ID NO: 13 is:
1 mtkkpggpgk nrainmlkrg lprvfplvgv krwmslldg rgpvrfvlal itffkftala
61 ptkallgrwk aveksvamkh ltsfkrelgt lidavnkrgr kqnkrggneg simwlaslav
121 viacagamkl snfqgkllmt inntdiadvi viptskgenr cwvraidvgy mcedtityec
181 pkltmgndpe dvdcwcdnqe vyvqygrctr trhskrsrrs vsvqthgess lvnkkeawld
241 stkatrylmk tenwfirnpg yaflaavlg mlgsnngqrv vftillllva paysfnclgm
301 gnrdfiegas gat vdlvle gdscltiman dkptldvrmi nieasqlaev rsycyhasvt
361 distvarcpt tgeahnekra dssyvckqgf tdrg gngcg lfgkgsidtc akfsctskai
421 grtiqpenik yevgifvhgt ttsenhgnys aqygasqaak ftvtpnapsi tlklgdygev
481 tldceprsgl nteafyvmtv gsksflvhre wfhdlalpwt spsstawrnr ellmefeeah
541 atkqswalg sqegglhqal agaiweyss svkltsghlk crlkmdklal kgttygmcte
601 kfsfaknpad tghgtwiel sysgsdgpck ipivsvasln dmtpvgrlvt vnpfvatssa
661 nskvlvemep pfgdsyiwg rgdkqinhhw hkagstlgka fsttlkgaqr laalgdtawd
721 fgsiggvfns igkavhqyfg gafrtlfggm switqglmga lllwmgvnar drsialafla
781 tggvlvflat nvhadtgcai ditrkemrcg sgifvhndve awvdrykylp etprslakiv
841 hkahkegvcg vrsvtrlehq mweavrdeln vllkenavdl svwnkpvgr yrsapkrlsm
901 tqekfemg k awgksilfap elanstfwd gpetkecpde hrawnsmqie dfgfgitstr 961 vwlkireest decdgaiigt avkghvavhs dlsywiesry ndtwklerav fgevksctwp
1021 ethtlwgdgv eeseliipht vagpkskhnr regyktqnqg pwdengivld fdycpgtkvt
1081 itedcgkrgp svrtttdsgk litdwccrsc slpplrfrte ngcwygmeir pvrhdettlv
1141 rsqydafnge mvdpfqlgll vmflatqevl rkrwtarlti pavlgallvl mlggitytdl
1201 arywlvaaa faeansggdv lhlaliavfk iqpaflvmnm lstrwtnqen wlvlgaaff
1261 qlasvdlqig vhgilnaaai awmivraitf pttssvtmpv lalltpgmra lyldtyriil
1321 lvigicsllq erkktmakkk gavllglalt stgwfsptti aaglmvcnpn kkrgwpatef
1381 lsavglmfai vgglaeldie smsipfmlag lmavsywsg katdmwlera adiswemdaa
1441 itgssrrldv kldedgdfhl iddpgvpwkv wvlrmscigl aaltpwaivp aafgy ltlk
1501 ttkrggvfwd tpspkpcskg dtttgvyrim argilgtyqa gvgvmyenvf htlwhttrga
1561 aimsgegklt py gsvkedr iayggpwrfd rkwngtddvq viwepgkaa vniqtkpgvf
1621 rtpfgevgav sldyprgtsg spildsngdi iglygngvel gdgsyvsaiv qgdrqeepvp
1681 eaytpnmlrk rqmtvldlhp gsgktrkilp qiikdaiqqr lrtavlaptr waaemaeal
1741 rglpvryqts avqrehqgne ivdvmchatl thrlmspnrv pnynlfvmde ahftdpasia
1801 argyiatkve lgeaaaifmt atppgttdpf pdsnapihdl qdeipdraws sgyewiteya
1861 gktvwfvasv kmgneiamcl qragkkviql nrksydteyp kckngdwdfv ittdisemga
1921 nfgasrvidc rksvkptile egegrvilgn pspitsasaa qrrgrvgrnp nqygdeyhyg
1981 gatseddsnl ahwteakiml dnihmpnglv aqlygperek aftmdgeyrl rgeekknfle
2041 llrtadlpvw laykvasngi qytdrkwcfd gprtnailed nteveivtrm gerkilkprw
2101 Idarvyadhq alkwfkdfaa gkrsavsfie vlgrmpehfm gktrealdtm ylvataekgg
2161 kahrmaleel pdaletitli vaitvmtggf fllmmqrkgi gkmglgalvl tlatfflwaa 2221 evpgtkiagt llialllmw lipepekqrs qtdnqlavfl icvltwgw aaneygmlek
2281 tkadlksmfg gktqasgltg lpsmaldlrp atawalyggs twltpllkh litseyvtts
2341 lasinsqags lfvlprgvpf tdldltvglv flgcwgqitl ttfltamvla tlhygymlpg
2401 wqaealraaq rrtaagimkn awdgmvatd vpelerttpl mqkkvgqyll igvsvaaflv
2461 npnvttvrea gvlvtaatlt lwdngasavw nsttatglch vmrgsylagg siawtlikna
2521 dkpslkrgrp ggrtlgeq k eklnamsree ffkyrreaii evdrtearra rrennivggh
2581 pvsrgsaklr wlvekgfvsp igkvidlgcg rggwsyyaat Ikkvqevrgy tkggagheep
2641 mlmqsygwnl vslksgvdvf ykpsepsdtl fcdigessps peveeqrtlr vlemtsdwlh
2701 rgprefcikv lcpympkvie kmevlqrrfg gglvrlplsr nsnhemywvs gaagnwhav
2761 nmtsqyllgr mdrtvwrgpk yeedvnlgsg travgkgevh snqekikkri qklkeefatt
2821 whkdpehpyr twtyhgsyev katgsasslv ngwklmskp wdaianvttm amtdttpfgq
2881 qrvfkekvdt kapeppagak evlnettnwl waylsrekrp rlctkeefik kvnsnaalga
2941 vfaeqnqwst areavddprf wemvdeeren hlrgechtci ynmmgkrekk pgefgkakgs
3001 raiwfmwlga rylefealgf Inedhwlsre nsgggvegsg vqklgyilrd iagkqggkmy
3061 addtagwdtr itrtdlenea kvlelldgeh rmlaraiiel tyrhkwkvm rpaaegktvm
3121 dvisredqrg sgqyvtyaln tftniavqlv rlmeaegvig pqhleqlprk nkiavrtwlf
3181 engeervtrm aisgddcwk plddrfatal hflnamskvr kdiqewkpsh gwhdwqqypf
3241 csnhfqeivm kdgrsiwpc rgqdeligra rispgagwnv kdtaclakay aqmwlllyfh
3301 rrdlrlmana icsavpvdwv ptgrtswsih skgewmtted mlq wnrvwi eenewmmdkt
3361 pitswtdvpy vgkrediwcg sligtrsrat waeniyaain qyravigken yvdymtslrr
3421 yedvliqedr vi The sequence of SEQ ID NO: 14 is:
1 mtkkpggpgk nrainmlkrg lprvfplvgv krwmslldg rgpvrfvlal itffkftala
61 ptkallgrwk aveksvamkh ltsfkrelgt lidavnkrgr kqnkrggneg simwlaslav
121 viacagamkl snfqgkllmt inntdiadvi viptskgenr cwvraidvgy mcedtityec
181 pkltmgndpe dvdcwcdnqe vyvqygrctr trhskrsrrs vsvpthgess lvnkkeawld
241 stkatrylmk tenwfirnpg yaflaavlg mlgsnngqrv vftillllva paysfnclgm
301 gnrdfiegas gatwvdlvle gdscltiman dkptldvrmi nieasqlaev rsycyhasvt
361 distvarcpt tgeahnekra dssyvckqgf tdrgwgngcg lfgkgsidtc akfsctskai
421 grtiqpenik yevgifvhgt ttsenhgnys aqygasqaak ftvtpnapsi tlklgdygev
481 tldceprsgl nteafyvmtv gsksflvhre wfhdlalpwt spsstawrnr ellmefeeah
541 atkqswalg sqegglhqal agaiweyss svkltsghlk crlkmdklal kgttygmcte
601 kfsfaknpad tghgtwiel sysgsdgpck ipivsvasln dmtpvgrlvt vnpfvatssa
661 nskvlvemep pfgdsyiwg rgdkqinhhw hkagstlgka fsttlkgaqr laalgdtawd
721 fgsiggvfns igkavhqyfg gafrtlfggm switqglmga lllwmgvnar drsialafla
781 tggvlvflat nvhadtgcai ditrkemrcg sgifvhndve awvdrykylp etprslakiv
841 hkahkegvcg vrsvtrlehq mweavrdeln vllkenavdl svwnkpvgr yrsapkrlsm
901 tqekfemgwk awgksilfap elanstfwd gpetkecpde hrawnsmqie dfgfgitstr
961 vwlkireest decdgaiigt avkghvavhs dlsywiesry ndtwklerav fgevksctwp
1021 ethtlwgdgv eeseliipht vagpkskhnr regyktqnqg pwdengivld fdycpgtkvt
1081 itedcgkrgp svrtttdsgk litdwccrsc slpplrfrte ngcwygmeir pvrhdettlv
1141 rsqydafnge mvdpfqlgll vmflatqevl rkrwtarlti pavlgallvl mlggitytdl 1201 arywlvaaa faeansggdv lhlaliavfk iqpaflvmnm lstrwtnqen wlvlgaaff
1261 qlasvdlqig vhgilnaaai awmivraitf pttssvtmpv lalltpgmra lyldtyriil
1321 lvigicsllq erkktmakkk gavllglalt stgwfsptti aaglmvcnpn kkrgwpatef
1381 lsavglmfai vgglaeldie smsipfmlag lmavsywsg katdmwlera adiswemdaa
1441 itgssrrldv kldedgdfhl iddpgvpwkv wvlrmscigl aaltpwaivp aafgy ltlk
1501 ttkrggvfwd tpspkpcskg dtttgvyrim argilgtyqa gvgvmyenvf htlwhttrga
1561 aimsgegklt py gsvkedr iayggpwrfd rkwngtddvq viwepgkaa vniqtkpgvf
1621 rtpfgevgav sldyprgtsg spildsngdi iglygngvel gdgsyvsaiv qgdrqeepvp
1681 eaytpnmlrk rqmtvldlhp gsgktrkilp qiikdaiqqr lrtavlaptr waaemaeal
1741 rglpvryqts avqrehqgne ivdvmchatl thrlmspnrv pnynlfvmde ahftdpasia
1801 argyiatkve lgeaaaifmt atppgttdpf pdsnapihdl qdeipdraws sgyewiteya
1861 gktvwfvasv kmgneiamcl qragkkviql nrksydteyp kckngdwdfv ittdisemga
1921 nfgasrvidc rksvkptile egegrvilgn pspitsasaa qrrgrvgrnp nqygdeyhyg
1981 gatseddsnl ahwteakiml dnihmpnglv aqlygperek aftmdgeyrl rgeekknfle
2041 llrtadlpvw laykvasngi qytdrkwcfd gprtnailed nteveivtrm gerkilkprw
2101 Idarvyadhq alkwfkdfaa gkrsavsfie vlgrmpehfm gktrealdtm ylvataekgg
2161 kahrmaleel pdaletitli vaitvmtggf fllmmqrkgi gkmglgalvl tlatfflwaa
2221 evpgtkiagt llialllmw lipepekqrs qtdnqlavfl icvltwgw aaneygmlek
2281 tkadlksmfg gktqasgltg lpsmaldlrp atawalyggs twltpllkh litseyvtts
2341 lasinsqags lfvlprgvpf tdldltvglv flgcwgqitl ttfltamvla tlhygymlpg
2401 wqaealraaq rrtaagimkn awdgmvatd vpelerttpl mqkkvgqyll igvsvaaflv 2461 npnvttvrea gvlvtaatlt lwdngasavw nsttatglch vmrgsylagg siawtlikna
2521 dkpslkrgrp ggrtlgeq k eklnamsree ffkyrreaii evdrtearra rrennivggh
2581 pvsrgsaklr wlvekgfvsp igkvidlgcg rgg syyaat lkkvqevrgy tkggagheep
2641 mlmqsyg nl vslksgvdvf ykpsepsdtl fcdigessps peveeqrtlr vlemtsdwlh
2701 rgprefcikv lcpympkvie kmevlqrrfg gglvrlplsr nsnhemywvs gaagnwhav
2761 nmtsqyllgr mdrtvwrgpk yeedvnlgsg travgkgevh snqekikkri qklkeefatt
2821 whkdpehpyr twtyhgsyev katgsasslv ngwklmskp wdaianvttm amtdttpfgq
2881 qrvfkekvdt kapeppagak evlnettnwl waylsrekrp rlctkeefik kvnsnaalga
2941 vfaeqnq st areavddprf wemvdeeren hlrgechtci ynmmgkrekk pgefgkakgs
3001 raiwfmwlga rylefealgf lnedhwlsre nsgggvegsg vqklgyilrd iagkqggkmy
3061 addtag dtr itrtdlenea kvlelldgeh rmlaraiiel tyrhkwkvm rpaaegktvm
3121 dvisredqrg sgqwtyaln tftniavqlv rlmeaegvig pqhleqlprk nkiavrtwlf
3181 engeervtrm aisgddcwk plddrfatal hflnamskvr kdiqewkpsh g hdwqqvpf
3241 csnhfqeivm kdgrsiwpc rgqdeligra rispgag nv kdtaclakay aqmwlllyfh
3301 rrdlrlmana icsavpvdwv ptgrtswsih skgewmtted mlqvwnrvwi eenewmmdkt
3361 pitswtdvpy vgkrediwcg sligtrsrat waeniyaain qyravigken yvdymtslrr
3421 yedvliqedr vi Although the mutation of JEV M protein, more particularly the replacement by phenylalanine of the amino acid, which is at position 36 within JEV M protein, is sufficient to achieve a default or defect is JEV viral particle assembly that achieves attenuation, the person of ordinary skill in the art may choose to mutate JEV proteins other than JEV protein M, for example: - to increase the genetic stability of the live and attenuated JEV of the application, and/or
- to reduce mutation reversibility (or to reduce the risk or probability of mutation reversibility), and/or
- to reduce the reversibility of the (attenuation) mutation(s) introduced into JEV M or ectoM (or to reduce the risk or probability of such a reversibility), and/or
- to reduce the reversibility of the 36F mutation introduced into JEV M (or to reduce the risk or probability of such a reversibility), and/or
- to reduce or further reduce JEV viral replication, and/or
- to reduce and/or restrict JEV tissue tropism, and/or
- to reduce or further reduce JEV binding to the host target cells, more particularly:
- to reduce mutation reversibility (or to reduce the risk or probability of mutation reversibility), and/or
- to reduce the reversibility of the (attenuation) mutation(s) introduced into JEV M or ectoM (or to reduce the risk or probability of such a reversibility), and/or
- to reduce the reversibility of the 36F mutation introduced into JEV M (or to reduce the risk or probability of such a reversibility),
more particularly:
- to reduce the reversibility of the (attenuation) mutation(s) introduced into JEV M or ectoM (or to reduce the risk or probability of such a reversibility), and/or
- to reduce the reversibility of the 36F mutation introduced into JEV M
(or to reduce the risk or probability of such a reversibility),
more particularly to reduce the reversibility of the 36F mutation introduced into JEV M (or to reduce the risk or probability of such a reversibility). The person of ordinary skill in the art may thus choose to mutate one or several of proteins E, C, NS1 , NS2A, NS2B, NS3, NS4A, NSA4 and NS5, more particularly to mutate JEV protein E and/or JEV protein C, more particularly to mutate JEV protein E.
Said mutated protein(s) then have a sequence, which differ from the one (the respective ones) of JEV strain RP9, more generally from the one (the respective ones) of an infectious JEV.
Said mutation may comprise or consist of one or several (amino acid) point mutations.
The application also relates to the viral particles or virions of said live attenuated JEV of the application.
The application also relates to a RNA nucleic acid, which is the RNA genomic nucleic acid of the live and attenuated JEV of the application. More particularly, the application relates to the coding sequence (CDS) of said genomic RNA.
The application also relates to a DNA nucleic acid, more particularly to a cDNA nucleic acid, the sequence of which is the retro-transcript or cDNA sequence of the RNA genomic nucleic acid of the application, e.g., according to the universal genetic code. More particularly, the application relates to the coding sequence (CDS) of said DNA or cDNA nucleic acid.
The application also relates to an expression vector, more particularly to a recombinant expression vector, which comprises a nucleic acid (operably) inserted in an expression cassette (for expression of said nucleic acid), wherein said inserted nucleic acid is a nucleic acid of the application, more particularly the RNA genomic nucleic acid of the application or the cDNA or retro-transcript thereof, more particularly the RNA CDS or the cDNA CDS of the application.
The (recombinant) expression vector can e.g., be a prokaryotic expression vector or an eukaryotic expression vector.
Examples of (recombinant) prokaryotic expression vector include (recombinant) expression vectors for expression in E. coli. Examples of (recombinant) eukaryotic expression vectors include:
- (recombinant) expression vectors for expression in a yeast cell, such as a S. cerevisiae cell,
- (recombinant) expression vectors for expression in an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the
S2 cell line), a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g. , a cell of the C6/36 cell line [ATCC® CRL-1660™]),
- (recombinant) expression vectors for expression for expression in a mammal cell, especially a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g. , a cell of the Vero cell line [ATCC® CCL-81™]) or a human cell (e.g. , a cell of the HEK293T cell line [ATCC® CRL-3216™] or of the SK-N-SH cell line [ATCC® HTB-1 1™]).
Examples of said (recombinant) prokaryotic expression vectors include (recombinant) plasmids for expression in E. coli, such as the pBR322 plasmid.
Examples of said (recombinant) eukaryotic expression vectors include plasmids, more particularly plasmids for expression in a yeast cell (such the 2-microns plasmid), Baculovirus vectors (e.g. , for expression in an insect cell), and Simian Virus 40 (SV40) vectors (e.g. , for expression in a mammal cell, such as a monkey cell).
A (recombinant) vector generally comprises a cloning site, more particularly a multiple cloning site. The (cloned) nucleic acid can be inserted in said cloning site.
A (recombinant) expression vector comprises an expression cassette wherein said nucleic acid can be inserted.
Said expression cassette generally comprises a promoter and a transcriptional terminator. Suitable promoters include a CMV inducible promoter (such as the CMV inducible TET-ON® promoter, available from CLONTECH LABORATORIES, 1290 Terra Bella Ave., Mountain View, CA 94043 U.S.A.), the promoter of the lac operon, the T7 promoter, the AOX1 promoter, the PGK promoter, the pPolh promoter, the CaMV 35S promoter, the CMV promoter, the CAG promoter, the SV40 promoter, more particularly a CMV inducible promoter (such as the CMV inducible TET-ON® promoter).
The application also relates to a cell, more particularly a host and/or recombinant cell. The cell of the application comprises the live and attenuated JEV of the application, or the mutated M protein of the application, or the mutated ectoM of the application, or the RNA nucleic acid of the application, or the DNA or cDNA nucleic acid of the application, or the expression vector of the application.
The cell of the application can e.g., be a cell, which has been infected, transfected or transformed by the live and attenuated JEV of the application, or the mutated M protein of the application, or the mutated ectoM of the application, or the RNA nucleic acid of the application, or the DNA or cDNA nucleic acid of the application, or the expression vector of the application.
The cell of the application can be infected, transfected or transformed by methods well known to the person skilled in the art, e.g., by chemical transfection (calcium phospate, lipofectamine), lipid-based techniques (liposome), electroporation, photoporation. Said infection, transfection or transformation can be transient or permanent.
Examples of a cell of the application include:
- a bacterial cell, such as an E. coli cell,
- a yeast cell, such as a S. cerevisiae cell,
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g., a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g., a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g., a cell of the C6/36 cell line [ATCC® CRL- 1660™]),
- a mammal cell, especially a rodent cell (such as a mouse cell), a monkey cell, a Cercopithecinae cell, a Cercopithecus aethiops cell (e.g., a cell of the Vero cell line [ATCC® CCL-81™]) or a human cell (e.g., a cell of the HEK293T cell line [ATCC® CRL-3216™] or of the SK-N-SH cell line [ATCC® HTB-1 1™]), more particularly
- a bacterial cell, such as an E. coli cell, or
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g. , a cell of the C6/36 cell line [ATCC® CRL- 1660™]),
more particularly
- an insect cell, such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera cell, a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g. , a cell of the C6/36 cell line [ATCC® CRL- 1660™]).
The cell of the application can be in isolated form. The cell of the application can be contained in a culture medium, more particularly a non- naturally occurring culture medium, e.g. , an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
A particular embodiment of the application is a nucleic acid clone, more particularly a RNA or cDNA clone, more particularly a cDNA clone of the live and attenuated JEV of the application.
A nucleic acid clone of the application can e.g. , be under the form of a (recombinant) cell, more particularly a (recombinant) insect cell [such as a Drosophila cell, a Drosophila melanogaster cell (e.g. , a cell of the S2 cell line), a Spodoptera frugiperda cell (e.g. , a cell of the Sf9 cell line), a mosquito cell, an Aedes cell, an Aedes albopictus cell (e.g. , a cell of the C6/36 cell line [ATCC® CRL-1660™])], wherein said (recombinant) cell or (recombinant) insect cell comprises a live and attenuated JEV of the application, or a (RNA or cDNA) nucleic acid or a vector of the application. An example of cDNA clone of the application is the (recombinant) cell deposited at the Collection Nationale de Culture de Microorganismes (CNCM) under 1-4902, or a (recombinant) cell (more particularly a (recombinant) insect cell) containing the plasmid of said cell, which has been deposited at the Collection Nationale de Culture de Microorganismes (CNCM) under 1-4902.
An example of cDNA clone of the application is a (recombinant) cell (more particularly a (recombinant) insect cell), which comprises or has been infected, transfected or transformed by a (recombinant) expression vector carrying a nucleic acid of the application as an insert for expression thereof.
An example of cDNA clone of the application is a (recombinant) cell (more particularly a (recombinant) insect cell), which comprises a (mutated JEV M) protein of the application or a (mutated JEV M protein ectomain of the application.
A clone or cDNA clone of the application does advantageously not comprise (nor codes for) the M protein of an infectious and/or virulent JEV (such as the JEV M protein of SEQ ID NO: 2).
A clone or cDNA clone of the application does advantageously not comprise (nor codes for) the M protein ectodomain of an infectious and/or virulent JEV (such as the JEV M ectodomain of SEQ ID NO: 6).
Advantageous, the clone or cDNA clone of the application shows a viral particle assembly default or defect in a mammalian cell but not in a mosquito cell, as described above or below illustrated.
Advantageous, the clone or cDNA clone of the application induces the production of JEV neutralizing antibodies, more particularly JEV sero- neutralization, more particularly in a mammalian host (such as a rodent, a monkey or a human), as described above or below illustrated.
Advantageous, the clone or cDNA clone of the application is a live clone or cDNA clone, but does not cause encephalitis (i.e., is an attenuated JEV clone or cDNA). The application also relates to a culture medium comprising the cell or nucleic acid clone of the application, more particularly to a culture medium comprising the cDNA clone of the application. Said culture medium can e.g. , be a non-naturally occurring culture medium, e.g., an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
The application also relates to a non-infectious and non-replicative particle, which comprises the mutated M protein of the live and attenuated JEV of the application.
In a particular embodiment, the application relates to a non-infectious and non-replicative particle, which comprises the mutated ectoM of the M protein of the live and attenuated JEV of the application.
A non-infectious and non-replicative particle of the application does not comprise the (full-length) JEV genomic RNA, nor the cDNA retro- transcript thereof.
A non-infectious and non-replicative particle of the application does advantageously not comprise the protein M or the protein M ectodomain of an infectious and/or virulent JEV.
Examples of non-infectious and non-replicative particles notably include a Virus-Like Particle (VLP).
Said non-infectious and non-replicative particle can be produced by a cell of the application, e.g., by a cell of the application, which has been infected, transfected or transformed by an expression vector of the application, for example an insect cell (e.g., a Drosophila cell such as a S2 cell) infected or transfected by a Baculovirus vector, or a bacterial cell (e.g., a E. coli cell) transfected by a plasmid. The application also relates to a composition, more particularly a pharmaceutical composition, more particularly an immunogenic composition, more particularly a vaccine, comprising the live and attenuated JEV of the application, or the expression vector of the application, or the cell of the application, or the clone or cDNA clone of the application (or at least one of these elements).
The live and attenuated JEV of the application, the expression vector of the application, the cell of the application, or the clone or cDNA clone of the application can be used as active ingredient for immunization, in particular for prophylactic immunization against a JEV infection in a mammalian host, especially in a human or an animal host.
The live and attenuated JEV of the application, the expression vector of the application, the cell of the application, or the clone or cDNA clone of the application can e.g., be used as active ingredient for prophylactic vaccination against JEV.
Advantageously, said composition of the application is suitable for administration into a host, in particular in a mammalian host, especially in a human or an animal host.
Said composition of the application may further comprise a pharmaceutically suitable excipient or carrier and/or vehicle, when used for systemic or local administration. A pharmaceutically suitable excipient or carrier and/or vehicle refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A "pharmaceutically acceptable carrier" is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation; suitable carriers include, but are not limited to, phosphate buffered saline solutions, distilled water, emulsions such as an oil/water emulsions, various types of wetting agents sterile solutions and the like, dextrose, glycerol, saline, ethanol, and combinations thereof.
Said composition of the application may further comprise an immunogenic adjuvant, such as Freund type adjuvants, generally used in the form of an emulsion with an aqueous phase or can comprise water-insoluble inorganic salts, such as aluminium hydroxide, zinc sulphate, colloidal iron hydroxide, calcium phosphate or calcium chloride. Said composition of the application, the live and attenuated JEV of the application, the expression vector of the application, the cell of the application, or the clone or cDNA clone of the application, in a dose sufficient to elicit an immune antibody response, more particularly an immune antibody response against at least one JEV polypeptide expressed by the live and attenuated JEV of the application, the expression vector of the application, the cell of the application, or the clone or cDNA clone of the application. In a particular embodiment, said immune antibody response is a protective humoral response. The protective humoral response results mainly in maturated antibodies, having a high affinity for their antigen, such as IgG. In a particular embodiment, the protective humoral response induces the production of neutralizing antibodies.
It is considered that the composition of the application (in particular the live and attenuated JEV of the application) has a protective capacity against JEV infection when after challenge of immunized host with JEV, it enables the delay and/or the attenuation of the symptoms usually elicited after infection with said JEV against which protection is sought by the administration of the composition of the application, or when especially the JEV infection is delayed.
According to a particular embodiment, said composition of the application is formulated for an administration through parental route such as subcutaneous (s.c), intradermal (i.d.), intramuscular (i.m.), intraperitoneal (i.p.) or intravenous (i.v.) injection, more particularly intradermal (i.d.) injection.
According to another particular embodiment, said composition of the application is administered in one or multiple administration dose(s), in particular in a prime-boost administration regime. The term " rime-boost regimen" generally encompasses a first administration step eliciting an immune response and one or several later administration step(s) boosting the immune reaction. Accordingly, an efficient prime-boost system can be used for iterative administration, enabling successively priming and boosting the immune response in a host, especially after injections in a host in need thereof. The term "iterative" means that the active principle is administered twice or more to the host. The priming and boosting immunization can be administered to the host at different or identical doses, and injections can be administered at intervals of several weeks, in particular at intervals of four weeks or more.
The quantity to be administered (dosage) depends on the subject to be treated, including the condition of the patient, the state of the individual's immune system, the route of administration and the size of the host. Suitable dosages can be adjusted by the person of average skill in the art.
The application also relates to a method to treat, prevent or protect, more particularly to prevent or protect against a JEV infection in a mammalian host, especially in a human or a non-human animal host, comprising administering said live and attenuated JEV of the application, or said expression vector of the application, or said cell of the application, or said clone or cDNA clone of the application or said composition of the application to said mammalian host.
As used herein, the expression "to protect against JEV infection" refers to a method by which a Japanese encephalitis virus infection is obstructed or delayed, especially when the symptoms accompanying or following the infection are attenuated, delayed or alleviated or when the infecting virus is cleared from the host.
The application also relates to a method to produce a live and attenuated JEV, which comprises producing said live and attenuated JEV of the application, or said expression vector of the application, or said cell of the application, or said clone or cDNA clone of the application or said composition of the application.
The application also relates to a method to produce an immunogenic composition or vaccine against JEV infection, which comprises producing said live and attenuated JEV of the application, e.g., as a clone or cDNA clone in a culture medium, optionally collecting the viral particles or virions produced by said live and attenuated JEV, and formulating said cultured JEV (or said collected viral particles) in a composition suitable for administration to an animal, more particularly to a human. Said culture medium can e.g., be a non-naturally occurring culture medium, e.g., an in vitro cell culture medium, for example a culture medium comprising the Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) or comprising the Leibovitz's 15 (L15, INVITROGEN) culture medium.
The application also relates to a method of (in vitro) attenuation of an infectious and/or virulent JEV, which comprises or consists of mutating the protein M of said infectious and/or virulent JEV, wherein said mutation comprises or consists of the replacement of the amino acid which is at position 36 within the (75 amino acid long) sequence of said protein M by the the amino acid phenylalanine.
The application also relates to a method of (in vitro) attenuation of an infectious and/or virulent JEV, which comprises or consists of mutating the protein M of said infectious and/or virulent JEV, wherein said mutation comprises or consists of the replacement of the amino acid which is at position 5 within the (75 amino acid long) sequence of said protein M by the the amino acid proline, and the replacement of the amino acid which is at position 36 within the (75 amino acid long) sequence of said protein M by the the amino acid phenylalanine.
Advantageously, the attenuated JEV thus produced still is a live virus. Advantageous, the (live and) attenuated JEV thus produced shows a viral particle assembly default or defect in a mammalian cell but not in a mosquito cell, as described above or below illustrated.
Advantageous, the (live and) attenuated JEV thus produced induces the production of JEV neutralizing antibodies, more particularly JEV sero- neutralization, more particularly in a mammalian host (such as a rodent, a monkey or a human), as described above or below illustrated. The application generally relates to the mutation of JEV M protein or fo JEV ectoM, more particularly to the replacement by phenylalanine of the amino acid, which is at position 36 within JEV M protein, as a JEV M or ectoM mutation, which induces a default or defect in JEV viral assembly, more particularly as a JEV M or ectoM mutation inducing a default or defect in JEV viral assembly that achieves JEV attenuation.
The term "comprising", which is synonymous with "including" or "containing", is open-ended, and does not exclude additional, unrecited element(s), ingredient(s) or method step(s), whereas the term "consisting of" is a closed term, which excludes any additional element, step, or ingredient which is not explicitly recited.
The term "essentially consisting of" is a partially open term, which does not exclude additional, unrecited element(s), step(s), or ingredient(s), as long as these additional element(s), step(s) or ingredient(s) do not materially affect the basic and novel properties of the application.
The term "comprising" (or "comprise(s)") hence includes the term "consisting of" ("consist(s) of"), as well as the term "essentially consisting of ("essentially consist(s) of"). Accordingly, the term "comprising" (or "comprise(s)") is, in the present application, meant as more particularly encompassing the term "consisting of" ("consist(s) of"), and the term "essentially consisting of" ("essentially consist(s) of").
In an attempt to help the reader of the present application, the description has been separated in various paragraphs or sections. These separations should not be considered as disconnecting the substance of a paragraph or section from the substance of another paragraph or section. To the contrary, the present description encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated.
Each of the relevant disclosures of all references cited herein is specifically incorporated by reference. The following examples are offered by way of illustration, and not by way of limitation. EXAMPLES
EXAMPLE 1 :
MATERIALS AND METHODS
Cells
Human neuroblastoma cells SK-N-SH (ATCC® HTB-1 1™) and human kidney cells HEK293T (ATCC® CRL-3216™) were cultured at 37°C in Dulbecco's Modified Eagle Medium (DMEM, INVITROGEN) containing 10% of fetal bovine serum (FBS).
Aedes albopictus cells C6/36 [ATCC® CRL-1660™] were cultured at 28°C in Leibovitz's 15 (L15, INVITROGEN) medium containing 10% of FBS and 1 % of penicillin and streptomycin. Plasmids
Plasmid encoding infectious clone RP9 (pBR322-JEV-RP9) was made by Liang et al. (Taiwan, Liang et al. Vaccine, 2009, 27(21): 2746-2754) in 2009 (complete genome of JEV RP9 = GENBANK® accession number KF907505; polyprotein of JEV RP9 = GENBANK® accession number AHK05344.1 ). Transfection of this plasmid into cells leads to production of JEV virions.
Site-directed mutagenesis
Site-directed mutagenesis was conducted on plasmid pBR322-JEV- RP9 by PCR using PHUSION polymerase (Thermo Scientific) and the following primers: FW: 5'-
CATGAAAACTGAGAACTGGTTCATAAGGAATCCTGGCTA-3' (SEQ ID NO: 15), RV: 5'-TAGCCAGGATTCCTTATGAACCAGTTCTCAGTTTTCATG-3' (SEQ ID NO: 16).
PCR products were digested with Dpnl enzyme (NEW ENGLAND
BIOLABS) and used to transform competent bacteria STBL2 (Life Technologies). Bacteria were cultured in rich medium Terrific Broth containing 100 mM of carbenicillin at 30°C during 48h.
Transfections
Transfections into HEK293T cells and C6/36 cells were performed using Lipofectamine2000 reagent (LIFE TECHNOLOGIES), according to the supplier's instructions.
Viruses
Wild type (VVT) and mutant M-I36F viruses were produced by transfection of infectious clone in C6/36 cells using Lipofectamine2000 reagent (LIFE TECHNOLOGIES), according to the supplier's instructions. Supernatants were collected 7 days post-transfection. Infections
Virus infections were performed in 24-well-culture plaques. 105 SK-N- SH cells or 5.105 C6/36 cells were seeded. 24 hours later, they were infected with 200 L of medium containing a given number of viral particles, depending on the MOI. One hour after inoculation, inoculum was replaced by medium containing 2% of FBS.
Titration in ffu/mL
Titrations were conducted on C6/36 cells. 5.105 cells were seeded in 24-well-culture plaques and were infected with viral suspension and serial dilutions by 10 in L15 medium 24h after seeding.
Cells were fixed with paraformaldehyde (PFA) 4% in PBS 54 hours after infection. Cell membranes were permeabilized with PBS containing 0.1 % of Triton X-100. Foci were revealed using murine monoclonal antibody 4G2 and anti-mouse antibody coupled with Horse Radish Peroxidase (HRP) that reacts with Vector VIP Substrate Kit for Peroxidase's reagent (VECTOR LABORATORIES). Mouse experiments
Three-week-old female C57BL/6 mice were obtained from JANVIER LABS (France). They were inoculated intraperitoneally with 103 ffu of virus RP9 or doses from 103 to 106 ffu of virus M-I36F. Mice were monitored daily and mortality was evaluated. 27 days after inoculation, sera were collected from surviving mice and pooled by group.
Statistical analysis Data were analyzed with Prism 5 Software (GRAPHPAD software).
Titers and absorbance (DO) were evaluated for statistically significant differences by t-tests. Survival proportions were evaluated for statistically significant differences by log-rank (MANTEL-COX) test. RESULTS
Mutant M-I36F life cycle is not impaired in mosquito cells, but is impaired in mammalian cells.
Liang et al. have synthesized an infectious molecular clone of JEV genotype 3-strain RP9 {Chen et al., Virology, 1996, 217(1): 220-229 and Lin et al., Virus Res. 1996, 44(1): 45-56) in 2009. They demonstrated that transfection of this molecular clone into BHK21 cells induced production of JEV virions. The inventors used this molecular clone to synthesize a mutant molecular clone containing mutation M-I36F. Mammalian HEK293T cells when transfected with the wt JEV produced JEV virions. However, when transfected with M-I36F mutant infectious clone, less virions were produced (2 log decrease, data not shown).
JEV is an arbovirus that infects both mosquitoes and mammals. Due to the limited amount of infectious particles produced in HEK293T cells, the inventors chose to produce our viruses in Aedes albopictus C6/36 cells by transfection of the two infectious clones into these cells. Viruses were produced in similar quantities (data not shown). To investigate the effect of mutation M-I36F on the virus life cycle in both cell types, Aedes albopictus cells C6/36 and human neuroblastoma cells SK-N-SH were infected at an MOI of 5 (Figures 1A and 1 B). The cells were harvested 24h and 48h post-infection and the supernatants were collected. The supernatants were titrated on C6/36 cells. The inventors determined that the production of infectious viral particles was similar for both viruses in C6/36 cells at 24 and 48h (Figure 1A). However, in mammalian SK-N-SH cells, less infectious viral particles were produced for mutant M-I36F than for WT virus at 24 and 48h (Figure 1 B). The inventors thus demonstrated that the mutant virus M-I36F life cycle is not impaired in C6/36 mosquito cells, while it is impaired in human SK-N-SH cells.
Mutant virus M-I36F is attenuated and induces production of antibodies targeting JEV in mice.
A murine model using C57BL/6 mice for infection by JEV has been described in 201 1 by Larena et al (Larena et al. J Virol. 2011, 85(11): 5446- 5455).
To investigate the effect of mutation M-I36F on JEV pathogenesis in mice, three-week-old female C57BL/6 mice were injected intraperitoneal^ with 103 ffu of WT virus, or from 103 to 106 ffu of M-I36F virus (Figure 2A). Mice injected with WT virus started to die from neurological disease 9 days after inoculation, whereas mice injected with all doses of M-I36F virus survived the inoculation (Figure 2A). These data indicated that mutant M- I36F is attenuated in C57BL/6 mice.
Mice sera were collected 27 days after inoculation and ELISA was conducted on these sera to determine if mice had produced antibodies against JEV. Purified and inactivated JEV particles were used as target for the ELISA test (Figure 2B). Mice injected with WT and M-I36F virus produced antibodies targeting JEV particles (Figure 2B). The produced antibodies were neutralizing (data not shown). However, it seems that mice injected with M- I36F virus produced fewer antibodies than mice injected with RP9 virus, regardless of the dose injected. EXAMPLE 2: 36F mutation in protein M of a Flavivirus other than JEV
An infectious clone of virulent Asibi strain of Yellow Fever Virus (YFV) was produced in the laboratoy. Among the mutations present on the 17D vaccinal strain of YFV, we tested the effect of L36F, located in the M structural protein, together with I95M located in the NS4B non-structural protein, when introduced in the virulent Asibi infectious clone of YFV (Figure 3A). The virus produced after 48h in HepG2 cells from both parental Asibi strain and double mutant of YFV was titrated in BHK21 cells (Figure 3B).
The results show that mutation does not attenuate YFV.
EXAMPLE 3: I36 mutation in protein M of JEV other than I36F Parental and mutant I36A JEV plasm id constructs were transfected in 293T cells, following the methods described in example 1 above. The produced viral particles were titrated in BHK21 and C6/36 cells (Figure 4).
The results show that the replacement of the amino acid, which is at position 36 in protein M of JEV (i.e., the replacement of isoleucine), by alanine, instead of phenylalanine, does not lead to JEV attenuation.

Claims

CLAIMS 1 . A live and attenuated Japanese Encephalitis Virus (JEV), which comprises or codes for a mutated JEV M protein, wherein the amino acid sequence of said mutated M protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 97% or at least 98% identical to the sequence of SEQ ID NO: 2, and
(b) wherein the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine.
2. The live and attenuated JEV of claim 1 , wherein the amino acid sequence of said mutated M protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 98% identical to the sequence of SEQ ID NO: 2, and
(b) wherein the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine.
3. The live and attenuated JEV of claim 2, wherein the amino acid sequence of said mutated M protein comprises or consists of the sequence of SEQ ID NO: 4.
4. The live and attenuated JEV of claim 1 , wherein the amino acid sequence of said mutated M protein comprises or consists of a sequence, which:
(a) consists of 75 amino acids and is at least 97% identical to the sequence of SEQ ID NO: 2, and
(b) wherein the amino acid at position 5 in said sequence of 75 amino acids is proline and the amino acid at position 36 in said sequence of 75 amino acids is the amino acid phenylalanine.
5. The live and attenuated JEV of claim 4, wherein the amino acid sequence of said mutated M protein comprises or consists of the sequence of SEQ ID NO: 10.
6. The live and attenuated JEV of any one of claims 1 -5, which does not comprise and does not code for the protein of SEQ ID NO: 2.
7. The live and attenuated JEV of any one of claims 1 -6, which shows a defect in the assembly of its viral particles in a human cell of the HEK293T cell line [ATCC® CRL-3216™] and/or of the SK-N-SH cell line [ATCC® HTB- 1 1™]), but not in a mosquito cell of the C6/36 cell line [ATCC® CRL-1660™].
8. The live and attenuated JEV of any one of claims 1 -7, which induces JEV neutralizing antibodies.
9. A RNA nucleic acid, which is the RNA genomic nucleic acid of the live and attenuated JEV of any one of claims 1 -8.
10. A cDNA nucleic acid, the sequence of which is the retro-transcript of the RNA genomic nucleic acid of claim 9.
1 1 . A recombinant expression vector, which comprises a nucleic acid inserted in an expression cassette, wherein said inserted nucleic acid is the RNA nucleic acid of claim 9 or the cDNA nucleic acid of claim 10.
12. The recombinant expression vector of claim 1 1 , which is a plasmid, a Baculovirus vector or a SV40 vector.
13. A recombinant cell, which comprises the live and attenuated JEV of any one of claims 1 -8, or the RNA nucleic acid of claim 9, or the cDNA nucleic acid of claim 10, or the recombinant expression vector of claim 1 1 or 12.
14. The recombinant cell of claim 13, which is an insect cell, a Drosophila cell or an Aedes cell.
15. A cDNA clone of the live and attenuated JEV of any one of claims 1 -8.
16. The cDNA clone of claim 15, which is the cell deposited at the Collection Nationale de Culture de Microorganismes (CNCM) under I-4902, or a cell containing the plasmid of the cell, which has been deposited at the Collection Nationale de Culture de Microorganismes (CNCM) under I-4902.
17. The cDNA clone of claim 15 or 16, which shows a defect in the assembly of its viral particles in a human cell of the HEK293T cell line [ATCC® CRL- 3216™] and/or of the SK-N-SH cell line [ATCC® HTB-1 1™]), but not in a mosquito cell of the C6/36 cell line [ATCC® CRL-1660™].
18. The cDNA clone of any one of claims 15-17, which induces JEV neutralizing antibodies.
19. An immunogenic composition, which comprises at least one of:
- the live and attenuated JEV of any one of claims 1 -8 or viral particles thereof,
- the recombinant cell of claim 13 or 14, and
- the cDNA clone of any one of claims 15-18.
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CN117110269B (en) * 2023-10-20 2024-01-26 浙江迪福润丝生物科技有限公司 Screening method of JEV protease inhibitor and inhibition effect evaluation method

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