EP3596205A1 - Vaccin recombinant contre le ndv - Google Patents

Vaccin recombinant contre le ndv

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Publication number
EP3596205A1
EP3596205A1 EP18706537.0A EP18706537A EP3596205A1 EP 3596205 A1 EP3596205 A1 EP 3596205A1 EP 18706537 A EP18706537 A EP 18706537A EP 3596205 A1 EP3596205 A1 EP 3596205A1
Authority
EP
European Patent Office
Prior art keywords
ndv
protein
seq
recombinant
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18706537.0A
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German (de)
English (en)
Inventor
Emmanuel Albina
Haijin LIU
Renata Servan De Almeida
Patricia GIL
Olivier Fridolin MAMINIAINA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre de Cooperation Internationalel en Recherche Agronomique pour le Development CIRAD
AxLR SATT du Languedoc Roussillon SAS
Fofifa
Original Assignee
Centre de Cooperation Internationalel en Recherche Agronomique pour le Development CIRAD
AxLR SATT du Languedoc Roussillon SAS
Fofifa
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Application filed by Centre de Cooperation Internationalel en Recherche Agronomique pour le Development CIRAD, AxLR SATT du Languedoc Roussillon SAS, Fofifa filed Critical Centre de Cooperation Internationalel en Recherche Agronomique pour le Development CIRAD
Publication of EP3596205A1 publication Critical patent/EP3596205A1/fr
Pending legal-status Critical Current

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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • C12N7/025—Packaging cell lines, e.g. transcomplementing cell lines, for production of virus
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18151—Methods of production or purification of viral material

Definitions

  • the present invention relates to a recombinant attenuated Newcastle Disease Virus (NDV) comprising nucleotide sequences encoding F and HN proteins of a virulent strain from intermediate genotype XI wherein the nucleic acid sequence encoding F protein contains at least one mutation in the cleavage site leading to asymptomatic infection.
  • NDV Newcastle Disease Virus
  • Newcastle disease is one the most fatal diseases of birds, representing a high threat for the poultry industry owing to its sanitary and economic impacts around the world (Dimitrov et al., 2017).
  • the causative agent is a virulent Newcastle disease virus (NDV), an enveloped virus that belongs to the Avulavirus genus, Paramyxoviridae family.
  • the genome of NDV is a 15 kilobases (Kb) negative-sense single-strand RNA molecule structured as 3'-Leader-NP-P- M-F-HN-L-Trailer-5' with six coding segments surrounded by the leader and trailer viral polymerase promoters (de Leeuw and Peeters, 1999).
  • NDV nucleocapsid
  • P phosphoprotein
  • M matrix
  • F hemagglutinin- neuraminidase
  • L large protein
  • V and W two nonstructural proteins, V and W from P gene editing.
  • N, P and L form the viral polymerase complex replicates and transcribes the viral genome.
  • These three proteins play a crucial role in virus rescue by reverse genetics.
  • the host of NDV is avian, although virus can be infrequently isolated from other animals, such as pig and mink (Zhao et al., 2017).
  • ND vaccines have been used to control disease since 1950s, but many ND outbreaks still happen in poultry under the pressure of vaccination, which have led to consider that current vaccines may not be efficient enough to prevent ND (Dimitrov et al., 2017).
  • the present invention provides a new attenuated vaccine strain, in particular generated by reverse genetics.
  • This recombinant vaccine was based on the worldwide used vaccine strain, LaSota (genotype II) (Genbank accession numbers AY845400.2, AF077761 or JF950510), in which the F and HN genes have been replaced by those of NDV MG-725 strain.
  • MG-725 strain was isolated from poultry in Madagascar, in 2008 and classified as genotype XI (Maminiaina et al., 2010) (Genbank accession number HQ266602.1 ).
  • genotype XI NDV only occurs in Madagascar (Dimitrov et al., 2016).
  • genotype XI owning unique evolution way from an ancestor belonging to genotype IV, which in turn makes genotype XI viruses different from any others in terms of F and HN protein (de Almeida et al., 2013; Maminiaina et al., 2010).
  • This genotype has also an original F1/F2 cleavage site motif with five basic amino acids ( 2 R-R-R-R-R 6 ), which is consistent with character of NDV velogenic strain (Peeters et al., 1999).
  • viruses from genotype XI have some unique mutations on F and HN proteins. Some of these mutations are supposed to play a role in the interaction with NDV neutralizing antibodies, potentially involved in the protection against viral shedding (data not shown).
  • the F protein's cleavage site of MG-725 (between positions 4483 and 4900 of the nucleotide sequence encoding F protein) was mutated towards a lentogenic motif (RRRRRF to GRQGRL).
  • This vaccine candidate was tested and compared to the parental LaSota strain in a vaccine/challenge experiment in chickens involving different velogenic strains of genotype II, VII and XI. It is demonstrated that the recombinant attenuated NDV is able to totally protect chickens and stop viral shedding from homologous or heterologous virulent strains.
  • a first subject matter of the invention is a recombinant attenuated Newcastle Disease Virus (NDV) comprising at least nucleotide sequences encoding F and HN proteins of a virulent strain MG-725 (Genotype XI , GenBank Accession number HQ266602.1 ) or derivative thereof wherein the nucleotide sequence encoding the F protein (SEQ ID NO: 25) comprises at least a mutation in cleavage site represented by the amino acid sequence of formula (I) from positions number 1 12 to 1 17 2 ⁇ - ⁇ 2 - 3- ⁇ 4- 5 - 6 7 wherein X-i to X 5 are independently selected from basic or non-basic amino-acids, the total number of basic amino-acids in the said formula (I) being equal or inferior to 4, preferably equal or inferior to 3, more preferably equal to 2.
  • NDV Newcastle Disease Virus
  • Another subject matter of the invention is a recombinant attenuated NDV comprising at least a nucleotide sequence having at least 90%, preferably at least 91 %, 92%, 93%, 94%, more preferably at least 95%, 96%, 97%, 98%, 99% and even preferably 100% identity with nucleotide sequence encoding a mutated F protein Fmu (SEQ ID NO: 25) and a nucleotide sequence having at least 90%, preferably at least 91 %, 92%, 93%, 94%, more preferably at least 95%, 96%, 97%, 98%, 99% and even preferably 100% identity with nucleotide sequence encoding HN protein (SEQ ID NO: 26).
  • the invention also concerns an immunogenic composition or vaccine comprising a recombinant attenuated NDV according to the invention and at least one ingredient selected from excipients, adjuvants and mixtures thereof.
  • Another subject matter of the invention is the recombinant attenuated NDV of the invention or the immunogenic composition of the invention for use in inducing a protective immune response in a subject.
  • the invention also relates to the recombinant attenuated NDV of the invention or the composition of the invention for use in the manufacture of a vaccine for the prophylaxis and/or treatment of a NDV infection in a subject in need thereof, in particular for birds.
  • the invention also relates to the recombinant attenuated NDV of the invention or the composition of the invention for protecting a bird against Newcastle disease and for reducing viral shedding.
  • NDV Newcastle Disease Virus
  • a pGenome plasmid comprising at least a nucleotide sequence encoding a virus genome of LaSota (GenBank Accession numbers AY845400.2, AF077761 or JF950510) wherein the nucleotide sequences encoding F and HN proteins are replaced by nucleotide sequences encoding F and HN proteins of MG-725 (GenBank Accession number HQ266602.1 ) and wherein the nucleotide sequence encoding the F protein (SEQ ID NO: 25) comprises at least a mutation in cleavage site represented by the amino acid sequence of formula (I) from positions number 112 to 117 2 X-
  • helper plasmids comprising respectively the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),
  • pNPL helper plasmid comprising the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),
  • helper plasmids one of them comprising the sequences encoding two of the three structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L) and the other one comprising the sequence encoding the remaining structural viral protein, culturing host cells under conditions for replication and transcription of the recombinant virus,
  • a first subject matter of the invention is a recombinant attenuated Newcastle Disease Virus (NDV) comprising at least nucleotide sequences encoding F and HN proteins of a virulent strain MG-725 (Genotype XI , GenBank Accession number HQ266602.1 ) or derivative thereof wherein the nucleotide sequence encoding the F protein (SEQ I D NO: 25) comprises at least a mutation in cleavage site represented by the amino acid sequence of formula (I) from positions number 1 12 to 1 17 2 Xi -X 2 - 3 -X4- 5- 6 7 wherein X-i to X 5 are independently selected from basic or non-basic amino-acids, the total number of basic amino-acids in the said formula (I) being equal or inferior to 4, preferably equal or inferior to 3, more preferably equal to 2.
  • NDV Newcastle Disease Virus
  • nucleotide sequences encoding respectively NP, P, M, L, F and HN proteins of the NDV are available in GenBank under Accession numbers and versions.
  • the skilled in the art will be able to select the related sequences to produce recombinant sequences and recombinant or rescued virus according to the invention, either by synthetic method or reverse genetic method.
  • the man skilled in the art will use the nucleotide sequences as identified in the disclosure as 'SEQ ID NO:' and the Table 1 hereunder.
  • recombinant NDV designates a virus obtained by synthetic or reverse genetics, preferably reverse genetics i.e. is one which has been manipulated in vitro, e.g. using recombinant DNA techniques to introduce changes to the viral genome.
  • a « recombinant attenuated NDV » refers to a recombinant ND virus (RNA) or (RNA, DNA or cDNA) clone, which comprises heterologous F and HN proteins, and which has a reduced pathogenic phenotype compared to the wild-type pathogenic NDV (i.e., compared to the infectious and/or virulent NDV), more particularly compared to a wild- type virus of the same genus, species, type or subtype (i.e., compared to an infectious and/or virulent virus of the same genus, species, type or subtype).
  • a reduced pathogenic phenotype encompasses a reduced infection capacity and/or a reduced replication capacity, and/or a reduced and/or restricted tissue tropism, and/or a default or defect in the assembly of the viral particles, more particularly a reduced infection capacity.
  • a reduced pathogenic phenotype encompasses a (viral) infection, which is impeded, 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 thus provides a recombinant attenuated NDV or clone thereof which is able to replicate to an extent that is sufficient for inducing an immune response but that is not sufficient for inducing a disease.
  • MG-725 strain By 'derivative of MG-725 strain (Genotype XI, Genbank accession number HQ266602.1 )' according to the invention, it means a strain (native or recombinant one) having less than 10% of variability on the complete genome sequence of MG-725 strain, based on the mean interpopulational evolutionary distance (inferred from the complete F gene sequences) set as the cutoff to define distinct genotypes (Diego G Diel et al., 2012).
  • the "F protein” belongs to the type I membrane glycoprotein group and forms a trimeric structure (trimer).
  • the F protein is made as a non-active precursor form (F0) and is divided into the disulfide linked subunits F1 and F2 when the precursor F0 molecule passes through Golgi membranes.
  • F0 non-active precursor form
  • F1 and F2 the precursor glycoprotein F0
  • F1 and F2 the precursor glycoprotein F0
  • F1 and F2 the precursor glycoprotein F0
  • This post-translational cleavage is intervened by proteases of a host cell. If the cleavage does not occur, non-infectious virions are generated and the virus replication cannot progress.
  • a pathogenic NDV strain for example has at least one extra pair of basic amino-acids motif 2 X-R-X-R/K-R-F 7 and can be cleaved by a wide range of proteases of the furin family in different host cells.
  • the recombinant attenuated NDV has a genotype coding for a F protein cleavage site which has less than 4 basic amino acids, in particular less than 3 basic amino acids, and preferably only two basic amino acids, for example in amino acids positions number 1 12 to 1 17 for NDV virus. It is also said that the F protein cleavage site is modified or mutated (Fmu). And said genotype is named "genotype with a lentogenic-like F protein cleavage site".
  • the velogenic strains have five basic amino acids, while the lentogenic strains or recombinant attenuated NDV according to the invention have preferably two basic amino acids. This difference makes the F protein of virulent strains more prone to be cleaved by various proteases present in various tissues and the virus is then activated to amplify whereas the F protein of attenuated strains or recombinant attenuated NDV is only cleaved in environments like the digestive and respiratory tracts or in vitro, in cell culture medium containing trypsin.
  • the "HN protein” belongs to the type II membrane glycoprotein and forms a tetramer on the surface of the viral envelope, to penetrate into a cell membrane.
  • a “mutation” as used herein, refers to a change in nucleic acid or polypeptide sequence relative to a reference sequence (which is preferably a naturally-occurring normal or « wild- type » or « reference » sequence), and includes translocations, deletions, insertions, and substitutions/point mutations. In a particular embodiment, the mutation is a substitution/point mutation.
  • a mutation by "substitution” as used with respect to amino acids refers to the replacement of one amino acid residue by any other amino acid residue, excepted the substituted amino acid residue.
  • the sequence encoding a virus genome comprises a mutation within the cleavage site of the F protein, to be lentogenic-like as the LaSota strain.
  • a basic amino acid is replaced by a non-basic amino acid
  • 'Basic amino acid' is one of the arginine, lysine, or histidine, preferably arginine or lysine.
  • 'Non-basic amino acid' is one of the 1 non-basic amino acids.
  • non-basic amino acid is one of glycine, glutamine or glutamic acid
  • the basic amino acid arginine or lysine is replaced by a non-basic amino acid selected from glycine or glutamine.
  • nucleotide sequence encoding F protein means the substitution, in the cleavage site between nucleotide positions 334 and 351 of the coding sequence (CDS) of the F gene, of the arginine (R) codon consisting of agg, egg, aga, or cgc by glycine (G) codon consisting of ggg, ggc, gga, or glutamine (Q) codon consisting of cag or caa, or glutamic acid (E) codon consisting of gaa or gag.
  • CDS coding sequence
  • Amino acid Xi at position number 112 ( 2 Xi) corresponds to nucleotides 334-336 of the coding sequence (CDS) of the F gene.
  • the mutated cleavage site is represented by amino acid sequence of formula (I) 2 X-
  • X 2 is a threonine (T) and X 5 is an arginine (R) or lysine (K).
  • X 2 and X 5 are independently arginine (R) or lysine (K), preferably arginine (R).
  • the mutated cleavage site in the nucleotide sequence encoding F protein is GRQGRL (SEQ ID NO: 27).
  • the invention also concerns a recombinant attenuated NDV comprising at least a nucleotide sequence having at least 90%, preferably at least 91 %, 92%, 93%, 94%, more preferably at least 95%, 96%, 97%, 98%, 99% and even preferably 100% identity with nucleotide sequence encoding a mutated F protein Fmu (SEQ ID NO: 25) and a nucleotide sequence having at least 90%, preferably at least 91 %, 92%, 93%, 94%, more preferably at least 95%, 96%, 97%, 98%, 99% and even preferably 100% identity with nucleotide sequence encoding HN protein (SEQ ID NO: 26).
  • the percent identities referred to in the context of the disclosure of the present invention are determined on the basis of a global alignment of sequences to be compared, i.e., on an alignment of the sequences taken in their entirety over their entire length using any algorithm well-known to a person skilled in the art, such as the algorithm of Needleman and Wunsch (1970).
  • This sequence comparison may be performed using any software well-known to a person skilled in the art, for example the Needle software by using the "Gap open” parameter equal to 10.0, the “Gap extend” parameter equal to 0.5 and a "Blosum 62" matrix.
  • the Needle software is for example available on the website ebi.ac.uk under the name "Align”.
  • the recombinant attenuated NDV according to the invention additionally comprises nucleotide sequences encoding the NP, P, M, and L proteins of a lentogenic strain, in particular LaSota strain (genotype II, GenBank Accession numbers AY845400.2, AF077761 or JF950510).
  • the nucleotide sequences encoding respectively the NP, P, M, and L proteins are of a strain belonging to genotype II or III, in particular genotype II (old genotypes).
  • the nucleotide sequences encoding respectively the NP, P, M, and L proteins are of lentogenic LaSota strain (15,186 base pairs linear RNA, Accession numbers GenBank AY845400.2, AF077761 or JF950510).
  • the recombinant attenuated NDV according to the invention comprises:
  • nucleotide sequence encoding NP protein of LaSota strain comprises 1470 bases (SEQ ID NO: 29);
  • nucleotide sequence encoding P protein of LaSota strain comprises 1188 bases(SEQ ID NO: 30);
  • nucleotide sequence encoding M protein of LaSota strain comprises 1095 bases (SEQ ID NO: 31 );
  • nucleotide sequence encoding L protein of LaSota strain comprises 6615 bases (SEQ ID NO: 32).
  • the recombinant attenuated NDV according to the invention comprises a nucleotide sequence having at least 90%, preferably at least 91 %, 92%, 93%, 94%, more preferably at least 95%, 96%, 97%, 98%, 99% and even preferably 100% identity with the nucleotide sequence SEQ ID NO: 28 (LaSota/M-Fmu-HN).
  • This nucleotide sequence comprises a nucleotide sequence encoding the virus genome of Newcastle Disease Virus (NDV) from LaSota wherein the nucleotide sequence encoding F protein is replaced by a sequence encoding Fmu protein (SEQ ID NO: 25) of M-725 and the nucleotide sequence encoding HN protein of LaSota is replaced by a nucleotide sequence encoding HN protein of MG-725.
  • NDV Newcastle Disease Virus
  • the said recombinant attenuated NDV is named rLaSota/M-Fmu-HN.
  • the recombinant attenuated NDV according to the invention comprises at least a sequence encoding the virus genome of Newcastle Disease Virus (NDV) from recombinant MG-725 strain having a mutated protein Fmu (SEQ ID NO: 25).
  • NDV Newcastle Disease Virus
  • the reverse genetic system used in the context of the invention is a "4-plasmids system", comprising a pGenome plasmid comprising at least a nucleotide sequence encoding a virus genome of NDV and three help plasmids comprising respectively the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L).
  • N nucleocapsid protein
  • P phosphoprotein
  • L large protein
  • the reverse genetic system used in the context of the invention is a "2-plasmids system", comprising a pGenome plasmid comprising at least a nucleotide sequence encoding a virus genome of NDV and a unique helper plasmid comprising respectively the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L).
  • N nucleocapsid protein
  • P phosphoprotein
  • L large protein
  • the invention uses the "2-plasmids system” disclosed in the publication of Liu et al. (2017).
  • the reverse genetic system used in the context of the invention is a "3-plasmids system", comprising a pGenome plasmid comprising at least a nucleotide sequence encoding a virus genome of NDV and two helper plasmids, one of them comprising the sequences encoding two out of the three structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L), and the other one comprising the sequence encoding the remaining structural viral protein.
  • N nucleocapsid protein
  • P phosphoprotein
  • L large protein
  • NDV Newcastle Disease Virus
  • a pGenome plasmid comprising at least a nucleotide sequence encoding a virus genome of LaSota (GenBank Accession numbers AY845400.2, AF077761 or JF950510) wherein the nucleotide sequences encoding F and HN proteins are replaced by nucleotide sequences encoding F and HN proteins of MG-725 (GenBank Accession number HQ266602.1 ) and wherein the nucleotide sequence encoding the F protein (SEQ ID NO: 25) comprises at least a mutation in cleavage site represented by the amino acid sequence of formula (I) from positions number 112 to 1 17 2 X-
  • helper plasmids comprising respectively the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),
  • NDV Newcastle Disease Virus
  • a pGenome plasmid comprising at least a nucleotide sequence encoding a virus genome of LaSota (GenBank Accession numbers AY845400.2, AF077761 or JF950510) wherein the nucleotide sequences encoding F and HN proteins are replaced by nucleotide sequences encoding F and HN proteins of MG-725 (GenBank Accession number HQ266602.1 ) and wherein the nucleotide sequence encoding the F protein (SEQ ID NO: 25) comprises at least a mutation in cleavage site represented by the amino acid sequence of formula (I) from positions number 112 to 1 17 2 X-
  • pNPL helper plasmid comprising the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),
  • NDV Newcastle Disease Virus
  • a pGenome plasmid comprising at least a nucleotide sequence encoding a virus genome of LaSota (GenBank Accession numbers AY845400.2, AF077761 or JF950510) wherein the nucleotide sequences encoding F and HN proteins are replaced by nucleotide sequences encoding F and HN proteins of MG-725 (GenBank Accession number HQ266602.1 ) and wherein the nucleotide sequence encoding the F protein (SEQ ID NO: 25) comprises at least a mutation in cleavage site represented by the amino acid sequence of formula (I) from positions number 112 to 117 2 X-
  • helper plasmids one of them comprising the sequences encoding two out of the three structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L), and the other one comprising the sequence encoding the remaining structural viral protein,
  • the in vitro method of rescuing a recombinant attenuated NDV according to the invention comprises at least the steps of:
  • a. a pGenome plasmid comprising the nucleotide sequence SEQ ID NO: 5
  • helper plasmids comprising respectively the sequences encoding the structural viral proteins nucleocapsid protein (NP) (pLaSo-NP SEQ ID NO: 18) , phosphoprotein (pLaSo-P SEQ ID NO: 19) and large protein (pLaSo- L SEQ ID NO: 20) of l_aSota strain,
  • the in vitro method of rescuing a recombinant attenuated NDV according to the invention comprises at least the steps of:
  • a. a pGenome plasmid comprising the nucleotide sequence SEQ ID NO: 5
  • pNPL helper plasmid comprising the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L) (pLaSo-NPL SEQ ID NO: 21 ),
  • the in vitro method of rescuing a recombinant attenuated NDV according to the invention comprises at least the steps of:
  • helper plasmids comprising the sequences encoding two out of the three structural viral proteins nucleocapsid protein (NP) (pLaSo- NP SEQ ID NO: 18) , phosphoprotein (pLaSo-P SEQ ID NO: 19) and large protein (pLaSo-L SEQ ID NO: 20) of LaSota strain, and the other one comprising the sequence encoding the remaining structural viral protein culturing host cells under conditions for replication and transcription of the recombinant virus,
  • NP nucleocapsid protein
  • pLaSo-P SEQ ID NO: 19 phosphoprotein
  • pLaSo-L SEQ ID NO: 20 large protein
  • step (i) The co-transfecting technologies for step (i) and culturing conditions for step (ii) are well known from the man skilled in the art.
  • rescue a recombinant virus encompasses any process well known from the man skilled in the art allowing the generation of an infectious viral clone from a cDNA of the virus genome.
  • a "host cell modification”, as used herein, refers to any genetic modification of the cell allowing permanent or transient expression of deleted and/or mutated gene(s) to complement the said deleted and/or mutated gene(s) in the rescued virus genome. Modifications include insertional cell genome mutagenesis based on transposons or viruses and cell genome editing by specific nucleases (e.g. TALEN or CRIPR/Cas9) and homologous recombination.
  • specific nucleases e.g. TALEN or CRIPR/Cas9
  • the helper plasmid pNPL used in the alternative method of the invention comprises at least the sequences of the structural viral proteins of NDV, consisting of nucleocapsid protein (N), phosphoprotein (P) and large protein (L), under control of a promoter, in particular a pCMV promoter.
  • the pNPL plasmid comprises three independent expression cassettes under a promoter, in particular pCMV promoter to express N, P, L.
  • the weight ratio between the first plasmid pGenome and the helper plasmid pNPL ranges from 9: 1 to 1 :9, and is preferably 1 :1.
  • the host cells are eukaryotic cells, in particular mammal cells, preferably baby hamster kidney cells (BHK-21 ).
  • the host cells are transfected with an amount of two-plasmid system ranging from 1 ⁇ g to 20 ⁇ g, in particular 2 to 20 ⁇ g, and preferably 3 to 5 ⁇ g (total amount of both plasmids).
  • the in vitro method of rescuing negative RNA viruses in host cells additionally comprises a step of virus amplification into chicken embryos.
  • this additional step is managed between culturing step (ii) and recovering step (iii) of the method.
  • NDV the host cells in which the infectious virus clone is generated and their supernatants are collected and injected into 10 day old SPF chicken embryos for virus amplification.
  • the in vitro method of rescuing negative RNA virus according to the invention additionally comprises a step of amplification of RNA virus into chicken embryos between steps (ii) and step (iii). And in particular, the host cells transformed (transfected) with the two-plasmid system and their supernatants are collected and injected into 10 day old SPF chicken embryos for virus amplification.
  • step (i) The co-transfecting technologies for step (i) and culturing conditions for step (ii) are well known from the man skilled in the art.
  • the method of rescuing NDV virus may comprise the following steps:
  • pNPL plasmid construction amplification of N, P, L genes of NDV from cDNA by PCR and cloning into a plasmid, between a CMV promoter and polyA sequences; then N, P and L genes with CMV promoter and polyA are amplified from pN, pP, and pL by PCR and then cloned into pCMV plasmid to generate pNPL plasmid;
  • CMV promoter and polyA replace T7 promoter and terminator of a pKS plasmid and two ribozymes are inserted between CMV promoter and polyA to be the pCMV plasmid; then with PCR and restriction, the full genome of virus is assembled on pCMV plasmid, between both ribozymes, to get the pCMV-NDV (pGenome); 2) co-transfection of the host cell with the said plasmids pGenome and pNPL and culture under conditions for replication and transcription of the virus:
  • BHK-21 cells are seeded on the 6-well plate and cultured at 37°C, 5% C02 for overnight; then 1.5 ⁇ g pCMV-NDV (pGenome) and 1.5 ⁇ g pNPL (pNPL plasmid) are transfected by Lipofectamin into BHK-21 cells;
  • the transfected cells with 200 ⁇ _ supernatants are collected and injected into allantoic cavity of 10-days old chicken embryo. This chicken embryo is incubated at 37°C for 3 days and then put at 4°C for overnight;
  • compositions or vaccine comprising a recombinant attenuated NDV according to the invention and at least one ingredient selected from excipients, adjuvants and mixtures thereof.
  • the composition comprises the attenuated recombinant NDV rLaSota/M-Fmu-HN of the invention.
  • composition encompasses pharmaceutical composition, antiviral composition, immunogenic composition and vaccine, more particularly antiviral composition, immunogenic composition and vaccine.
  • composition of the application comprises at least one attenuated recombinant NDV of the invention and at least one ingredient selected from excipients, adjuvants and mixtures thereof.
  • the invention also includes immunogenic compositions comprising attenuated recombinant NDV as described herein.
  • the immunogenic compositions can be formulated according to standard procedures in the art.
  • the immunogenic compositions are administered in combination with an adjuvant.
  • the adjuvant for administration in combination with a composition described herein may be administered before, concomitantly with, or after administration of said composition.
  • the term "adjuvant" refers to a compound that when administered in conjunction with or as part of a composition described herein enhances and/or boosts the immune response to the attenuated recombinant NDV present in the immunogenic composition.
  • the adjuvants that can be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants, and immunostimulatory adjuvants.
  • the immunogenic compositions comprise the attenuated recombinant NDV alone or, preferably, together with a pharmaceutically acceptable carrier.
  • Suspensions or dispersions of the attenuated recombinant NDV, especially isotonic aqueous suspensions or dispersions, can be used.
  • the pharmaceutical compositions may be sterilized and/or may comprise excipients, e.g., preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dispersing and suspending processes.
  • an immunogenic composition provided herein is administered to a subject by, including but not limited to, oral, ocular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, percutaneous, intranasal and inhalation routes, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle).
  • a subcutaneous or intravenous route is used.
  • an immunogenic composition provided herein is administered to a subject by ocular or intranasal route.
  • Another subject matter of the invention is the recombinant attenuated NDV of the invention or the composition of the invention for use in inducing a protective immune response in a subject.
  • the invention also concerns the recombinant attenuated NDV of the invention or the composition of the invention for use in the manufacture of a vaccine for the prophylaxis and/or treatment of a NDV infection in a subject in need thereof, in particular for birds.
  • the invention also relates to the recombinant attenuated NDV of the invention or the composition of the invention for protecting a bird against Newcastle disease and for reducing viral shedding.
  • reducing viral shedding means a reduction of the virus excreted by infected animals through natural routes, including oro-tracheal, ocular, fecal and feather route. Excretion can be quantified either by direct-contact exposure experiments between infected animals and susceptible animals or by means of oro-tracheal, ocular, fecal or feather sampling on infected animals and then virus titration or viral genome quantification.
  • the said attenuated recombinant NDV or the composition comprising it according to the invention are used in a method for preventing and/or treating a NDV infection and/or of a disease or disorder induced by Newcastle Disease Virus (NDV) in a subject in need thereof.
  • NDV Newcastle Disease Virus
  • the said attenuated recombinant NDV or the composition comprising it according to the invention are used in a method for inducing a protective immune response in a subject in need thereof.
  • immunogenic response is intended in accordance with its ordinary meaning in the field, and includes one or several from antibody production, induction of cell mediated immunity, complement activation, development of immunological tolerance, alteration of cytokine production and alteration of chemokine production, more particularly antibody production.
  • Antibody production encompasses neutralizing antibody production, such as seroneutralization.
  • a protective immune response is one that reduces the risk that a subject will become infected with a NDV and/or reduces the severity of an infection with a NDV. Accordingly, protective immune responses include responses of varying degrees of protection.
  • Such methods may comprise administering an effective amount of attenuated recombinant NDV of the invention, such as in the form of an immunogenic composition comprising an attenuated recombinant NDV of the invention, to the subject in need thereof.
  • the method is a method of inducing a therapeutic immune response against the Newcastle Disease Virus (NDV), in a subject infected or susceptible to be infected with said virus.
  • NDV Newcastle Disease Virus
  • Such methods may comprise administering an effective amount of attenuated recombinant NDV of the invention, such as in the form of an immunogenic composition comprising an attenuated recombinant NDV of the invention, to a subject infected with said virus.
  • NDV Newcastle Disease Virus
  • the subject in need thereof is typically a bird, preferably domestic birds, more preferably, poultry, duck, goose or pigeon.
  • FIG. 1 Construction of pLaSota/M-Fmu-HN plasmid.
  • the pLaSota/M-Fmu-HN was generated from pLaSota plasmid which contained the full-genome of LaSota.
  • Three unique restriction enzymes sites (Sacll, Fsel and Pad) were introduced into the LaSota genome for further manipulations of F and HN genes.
  • the cleavage site of the F protein of the MG-725 strain was first modified from "RRRRRF" to "GRQGRL" by overlap PCR as described.
  • the modified nucleotides or amino acids are underlined and the modified F gene was thereafter named Fmu.
  • the F gene of LaSota was replaced by the one of MG-725 strains with the modified cleavage site Fmu, using Sacll and Fsel enzymes.
  • the HN gene from LaSota was replaced by the one of MG-725 strain using Fsel and Pad enzymes.
  • FIG. 1 Differences of challenge NDV strains.
  • Vaccinated chickens never showed clinical signs and mortality. All unvaccinated chickens suffered from disease and died within 3 to 5 days after challenge. It seems that the challenge with the genotype II was more serious in terms of clinical signs and survival curve.
  • Baby hamster kidney cells (BHK-21 ) were cultured in Eagle's minimum essential medium (Gibco) with 10% fetal bovine serum (PAN-Biotech) at 37°C, 5% C0 2 .
  • Chemically competent cells, E.coli 10-beta were purchased from New England Biolabs (NEB).
  • the rMG-725 strain was produced by reverse genetics based on the full genome of the NDV/chicken/Madagascar/2008 strain (MG-725, Genbank accession number HQ266602.1 )) a virulent strain isolate from Madagascar and belonging to genotype XI (Liu et al., 2017).
  • the virulent NDV/EG/CK/104/12 strain belonging to genotype VII was kindly provided by Patti J.
  • the GB Texas strain kindly provided by Benedicte Lambrecht from CODA-CERVA, Belgium, is a virulent strain of genotype II (Accession number GU978777).
  • the rLaSota strain was rescued based on the full genome of the LaSota strain (Genbank accession numbers AY845400.2, AF077761 or JF950510). All of these viruses were grown in 10 day-old specific pathogen free (SPF) chicken embryos (Couvoir de Cerveloup, France). After three days of infection or egg death, allantoic liquid was harvested, filtered through 0.22 ⁇ and stored at -80°C.
  • Example 1 Construction and Recovery of rLaSota/M-Fmu-HN strain. TABLE 1. Information on all plasmids and nucleotides sequences used in the present invention. The last four were constructed into the pCI-neo plasmid (Promega) while the rest were built into the pBluescript II SK(+/-) plasmid (Stratagene) SEQ Reference Backbone of Properties F protein
  • F protein cleavage sites are 2 RRRRRF 117 .
  • F protein cleavage sites are 2 GRQGRL 117 . Construction of rLaSota/M-Fmu-HN strain
  • ACCGGCCGGCCTCATCTGTGTTCATATTCTTGTGGTGGCTC-'3 SEQ ID NO: 7) primers. Then, the F gene (SEQ ID NO: 24) of pLaSota plasmid (SEQ ID NO: 3) was replaced by Fmu gene (SEQ ID NO: 25) using Sacl l and Fsel restriction enzymes to obtain pLaSota/M-Fmu plasmid (SEQ ID NO: 4).
  • the two fragments were then assembled by over-lap PCR and inserted in the place of the corresponding region in pLaSota/M-Fmu by Fsel and Pad enzymes to finally get the pLaSota/M-Fmu-HN plasmid (SEQ ID NO: 5).
  • LaSo-P-R(5'- ATTTGCGGCCGCTTAGCCATTTAGAGCAAG-'3 SEQ ID NO: 15)
  • NP and P genes were inserted into pCI-neo plasmid (Promega) between Xholl and Notl sites to obtain pLaSo-NP (SEQ I D NO: 18) and pLaSo-P (SEQ I D NO: 19), while the L gene was cloned in this vector by Spel and Notl enzymes to generate pLaSo-L (SEQ ID NO: 20). All these plasmids were purified by EndoFree plasmid Maxi kit (QIAGEN), aliquoted, stored at -20 °C and sequenced.
  • the T7 RNA polymerase promoter and terminator of pKS plasmid were replaced by the CMV promoter and polyA from pCI-neo.
  • the two ribozymes were then inserted between the CMV promoter and polyA sequences.
  • a fragment with multiple cloning sites was introduced between the two ribozymes to obtain a pCMV vector.
  • the N, P and L genes of the LaSota strain flanked by the CMV promoter and polyA tail were amplified from pN, pP and pL and cloned into the same pCMV vector to generate pNPL plasmid (SEQ ID NO: 21 ).
  • Recombinant rLaSota/M-Fmu-HN strain was generated by reverse genetics as previously described. Briefly, 4* 10 5 BHK-21 cells were grown overnight in 6-well plates. Supernatants were discarded and cells were washed twice with Opti-MEM (Gibco). Then, 5 ⁇ g of pLaSota/M-Fmu-HN (SEQ ID NO: 5), 2 ⁇ g of pLaSo-NP (SEQ ID NO: 18), 2 ⁇ g of pLaSo-P (SEQ ID NO: 19) and 1 ⁇ g of pLaSo-L (SEQ ID NO: 20) were cotransfected with 20 ⁇ _ Lipofectamine 2000 (Invitrogen).
  • the three helper plasmids are replaced by a unique helper plasmid pLaSo-NPL (SEQ ID NO: 21 ).
  • the transfection mixture was replaced by MEM medium with 10% FBS.
  • the cells with 200 ⁇ _ supernatants were collected and injected into 10-day- old SFP embryonated chicken eggs to amplify the rescued virus.
  • the allantoic liquid was harvested at 3 days post-infection and tested for the virus presence by the hemagglutination test (HA).
  • Viral RNA were extracted from HA positive samples and digested with TURBO DNase enzyme (Ambion) to prevent DNA contamination followed by confirmation with qRT- PCR based on F gene. Finally, the recovered virus was passaged once again in SPF embryonated eggs, aliquoted, stored at -80 °C and sequenced.
  • the virus genome was modified to introduce three restriction enzyme sites, Sacl l, Fsel and Pad , before F, between F and HN and behind HN genes, respectively (Fig.1 ). All these modifications were done in non-coding region of F and HN gene.
  • the cleavage site of MG-725 strain's F gene was mutated to that of LaSota by overlap PCR and named as Fmu gene (SEQ I D NO: 25).
  • the MG-725 Fmu gene (SEQ ID NO: 25) and HN gene (SEQ ID NO: 26) were replaced in pLaSota plasmid (SEQ ID NO: 3) to get pLaSota/M-Fmu-HN (SEQ ID NO: 5).
  • the helper plasmids containing respectively the NP, P, and L genes from LaSota strain, or a unique helper plasmid containing all NP, P and L genes the rLaSota and rLaSota/M-Fmu-HN strains were rescued on BHK-21 cells according to a method developed in our laboratory (Liu et al., 2017).
  • the rescued strain was amplified once in 10-days old SPF chicken embryos and confirmed by sequencing.
  • rLaSota and rLaSota/M-Fmu-HN strain were checked on embryonated chicken eggs.
  • EID 50 egg infective doses
  • One hundred 50% egg infective doses (EID 50 ) of both strains were injected into allantoic cavity of 10-day-old SPF eggs and incubated at 37 °C.
  • Three days after injection allantoic liquids were harvested and viruses titrated by the EID 50 method.
  • MDT mean death time index
  • ICPI intracerebral pathogenicity index
  • MDT 10-fold serial dilutions of the infective allantoic liquid were prepared in sterile phosphate-buffered saline (PBS).
  • PBS sterile phosphate-buffered saline
  • 10 "6 to 10 ⁇ 12 diluted liquids were injected into allantoic cavity of eggs, five eggs per each dilution.
  • the eggs were observed daily, in the morning and afternoon, for six days and the times (in hour) of egg deaths were recorded.
  • MDT is defined as the mean time to achieve 100% of egg death at the highest dilution of the allantoic fluid.
  • the ICPI was tested by the standard procedure.
  • Fresh infective allantoic fluids with HA titres > 2 4 were diluted 1/10 in sterile isotonic saline without antibiotics and used as inoculum. Fifty ⁇ of the diluted virus was injected intracerebrally into each one-day-old SPF chick, 10 chicks per strain, using a 30-gauze needle attached to a 1 ml syringe. The inoculum was injected into the left rear quadrant of the cranium. The birds were examined daily for 8 days. At each observation, the birds were scored: 0 if normal, 1 if sick, and 2 if dead (Birds that were alive but unable to eat or drink were killed humanely and scored as dead at the next observation.
  • c Mean death time pathogenicity index
  • d Intracerebral pathogenicity index.
  • the virulence of rLaSota, rLaSota/M-Fmu-HN and rMG-725 was then confirmed by MDT in chicken embryos. The MDT values of these rescued strains were 96 h, 1 13 h and 49 h, respectively (Table 2).
  • ICPI pathogenicity of these viruses was also checked by ICPI in 1-day old chicks.
  • the ICPI values were 0.00 for rLaSota and rLaSota/M-Fmu-HN strains and 1.82 for rMG-725 similar to the wild type MG-725 strains.
  • OIE World Organization for Animal Health
  • Virulent challenges occurred three weeks post vaccination and each chicken received 10 5 ELD 50 (50% egg lethal dose) of either virulent genotype II strain (GB Texas, Accession number GU978777), virulent genotype VII strain (EG/CK/104/12), or virulent genotype XI (rMG-725) though the intramuscular route.
  • ELD 50 50% egg lethal dose
  • Three chickens of each subgroup were sacrificed 3 days post-challenge and tissues, including brain, lung, trachea, nasal turbinate, spleen, and small intestine, were collected to measure challenge virus replication in the organs. The tissue samples were homogenized in cell culture medium (1 g/10 ml) and clarified by centrifugation.
  • the oral and cloacal swabs were collected from all surviving chickens 3, 5, 7 and 10 days post-challenge for the evaluation of challenge viral shedding.
  • Chickens clinical signs and mortality were recorded daily for 10 days. At each observation, the birds were scored: 0 if normal, 1 if sick, and 2 if dead. Birds that were alive but unable to eat or drink were killed humanely and scored as dead at the next observation. Dead individuals were scored as 2 at each of the remaining daily observations after death. All surviving birds were euthanized at 10 days post-infection. All experimental challenges were performed according to the European Directive 2010/63/UE on the protection of animals used for scientific purposes and approved by the Ethical committee of Animal Experimentation (CEEA) of the Institution (IRTA) in Barcelona.
  • CEEA Ethical committee of Animal Experimentation
  • Serum antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) and Hemagglutination Inhibition assay (HI) assays.
  • ELISA enzyme-linked immunosorbent assay
  • HI Hemagglutination Inhibition assay
  • the commercial kit ID Screen Newcastle Disease Indirect Conventional Vaccines kit from ID-Vet was used on serial 2-fold dilutions of the serum to determine the antibody titers.
  • HI Hemagglutination Inhibition assay
  • RNA virus core kit (MACHEREY-NAGEL).
  • tissue samples from nervous system (brain), respiratory system (lung, trachea and nasal turbinate), lymphoid system (spleen) and digestive tract (small intestine) were tested by qRT-PCR and viral isolation. All unvaccinated birds had high titers of virus in their tissues.
  • the viral RNA load was the highest in spleen followed by the respiratory tract (Fig.3A). Even if viral RNA was detected in a couple of tissues from chickens vaccinated with rLaSota/M-Fmu-HN or rLaSota, no virus could be isolated (Fig. 3A and Table 6).
  • genotype II vaccine totally protected chickens from ND and stopped viral shedding caused by genotype XI strains as well as caused by genotype II or VII NDV. Therefore, these results strictly support that genotype mismatches alone cannot assist NDV to escape vaccine.
  • both of ND genotype II and XI vaccines showed equal efficiency in terms of protecting SPF chickens in good health condition and blocking viral shedding from different genotype viruses' challenge, which supports that genotype mismatches are not a self- sufficient reason of vaccination failure.

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Abstract

L'invention concerne un Virus de la maladie de Newcastle (NDV) atténué recombinant, comprenant des séquences nucléotidiques codant pour les protéines F et HN d'une souche de génotype XI, la séquence d'acide nucléique codant pour la protéine F contenant au moins une mutation dans le site de clivage conduisant à un génotype lentogène.
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