EP3596205A1 - Ndv recombinant vaccine - Google Patents

Abstract

The invention relates to a recombinant attenuated Newcastle Disease Virus (NDV), comprising nucleotide sequences encoding F and HN proteins of a strain of genotype XI wherein the nucleic acid sequence encoding F protein contains at least one mutation in the cleavage site leading to lentogenic genotype.

Description

NDV recombinant vaccine

FIELD OF THE INVENTION

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. BACKGROUND OF THE INVENTION

Newcastle disease (ND) 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). This genome encodes six structural proteins, nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin- neuraminidase (HN), large protein (L) and two nonstructural proteins, V and W from P gene editing (Dortmans et al., 2010). Among these proteins, 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).

So there is still a need of providing new efficient ND recombinant vaccines.

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 ). Unlike worldwide spread of genotype II and VII viruses, genotype XI NDV only occurs in Madagascar (Dimitrov et al., 2016). The special geographic condition probably induces 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 ( ²R-R-R-R-R ⁶), which is consistent with character of NDV velogenic strain (Peeters et al., 1999). Moreover, based on silico analyses of F and HN protein structures, 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).

In order to modify the virulence of the recombinant vaccine of the invention, 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.

SUMMARY OF THE INVENTION

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 ²Χι-Χ₂- 3-Χ4- ₅- 6 ⁷ wherein X-i to X₅ 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.

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.

Another subject matter of the invention is an in vitro method of reverse genetics for preparing a attenuated Newcastle Disease Virus (NDV) according to the invention, comprising at least the steps of:

(i) Co-transfecting eukaryotic host cells, in particular mammal cells, preferably baby hamster kidney cells (BHK-21 ) with a plasmids system comprising a. 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 ²X-|-X₂-X₃- X4-X₅-X₆ ⁷ wherein X-i to X₅ 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. ,

b. three helper plasmids comprising respectively the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),

or alternatively a unique pNPL helper plasmid comprising the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),

or alternatively two 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,

optionally amplification of the recombinant virus into chicken embryos, and recovering the recombinant attenuated NDV.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Recombinant attenuated NDV

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 ²Xi -X₂- ₃-X4- 5- 6 ⁷ wherein X-i to X₅ 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.

The 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. In particular, 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.

The expression "recombinant NDV" according to the invention 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 » according to the invention 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, more particularly a reduced infection capacity, 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.

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. In order for the NDV to infect a cell, it is necessary for the precursor glycoprotein F0 to be cleaved into F1 and F2. 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.

The molecular basis for the different level of pathogenicity (more than 10 different genotypes) is known to be linked to the sequence of cleavage site of the precursor of the fusion protein F. At this position, a pathogenic NDV strain for example has at least one extra pair of basic amino-acids motif ²X-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.

According to the invention, 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".

For the NDV, 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. In particular, 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.

In a particular embodiment, in the cleavage site represented by the amino acid sequence of formula (I) from positions number 1 12 to 1 17 ²Xi-X2-X3-X4-Xs-X6 ⁷ of the F protein wherein Xi to X₅ are independently selected from basic or non-basic amino-acids, 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. In a particular embodiment, non-basic amino acid is one of glycine, glutamine or glutamic acid

In a preferred embodiment, the basic amino acid arginine or lysine is replaced by a non-basic amino acid selected from glycine or glutamine.

With respect to related nucleotide sequence encoding F protein, it 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.

Amino acid Xi at position number 112 ( ²Xi) corresponds to nucleotides 334-336 of the coding sequence (CDS) of the F gene.

In a particular embodiment, the mutated cleavage site is represented by amino acid sequence of formula (I) ²X-|-X2-X3-X4-X5-X6 ⁷ wherein X₂ and/or X₅ are independently arginine (R) or lysine (K), preferably arginine (R), X₆ is a non-basic amino-acid and at least two of Xi , X₃, and X* are independently selected from the group consisting of non-basic amino-acids, preferably glycine, glutamine or glutamic acid. In a particular embodiment, X₂ is a threonine (T) and X₅ is an arginine (R) or lysine (K).

In another particular embodiment, X₂ and X₅ are independently arginine (R) or lysine (K), preferably arginine (R). In a particular embodiment and preferred embodiment, 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". In a particular embodiment, 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). In a particular embodiment, 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). In a particular and preferred embodiment, 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). In particular, 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). In a particular and preferred embodiment, 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.

The said recombinant attenuated NDV is named rLaSota/M-Fmu-HN.

In another particular embodiment, 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).

Reverse genetic system for NDV recombinant

The Examples describe a reverse genetic system that may be used to make recombinant attenuated NDV. Skilled person will appreciate that in view of the teachings of this disclosure, alternative embodiments of systems may be provided and utilized to practice embodiments of this invention and to make the disclosed recombinant attenuated NDV and compositions.

In a particular embodiment, 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). In another particular embodiment, 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). In a particular embodiment, the invention uses the "2-plasmids system" disclosed in the publication of Liu et al. (2017).

In another particular embodiment, 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.

So the invention concerns an in vitro method of reverse genetics for preparing a attenuated Newcastle Disease Virus (NDV) according to the invention, comprising at least the steps of:

(i) Co-transfecting eukaryotic host cells, in particular mammal cells, preferably baby hamster kidney cells (BHK-21 ) with a plasmid system comprising a. 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 ²X-|-X₂-X₃- X4-X5-X6 ⁷ wherein Xi to X₅ 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. ,

b. three helper plasmids comprising respectively the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),

(ii) culturing host cells under conditions for replication and transcription of the recombinant virus,

(iii) optionally amplification of the recombinant virus into chicken embryos, and

(iv) recovering the recombinant attenuated NDV. In an alternative embodiment, the in vitro method of reverse genetics for preparing a attenuated Newcastle Disease Virus (NDV) according to the invention, comprising at least the steps of:

(i) Co-transfecting eukaryotic host cells, in particular mammal cells, preferably baby hamster kidney cells (BHK-21 ) with a plasmid system comprising a. 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 ²X-|-X₂-X₃- X4-X₅-X₆ ¹¹⁷ wherein X-i to X₅ 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. ,

b. a unique pNPL helper plasmid comprising the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),

(ii) culturing host cells under conditions for replication and transcription of the recombinant virus,

(iii) optionally amplification of the recombinant virus into chicken embryos, and

(iv) recovering the recombinant attenuated NDV.

In another alternative embodiment, the in vitro method of reverse genetics for preparing an attenuated Newcastle Disease Virus (NDV) according to the invention, comprising at least the steps of:

(i) Co-transfecting eukaryotic host cells, in particular mammal cells, preferably baby hamster kidney cells (BHK-21 ) with a plasmid system comprising:

a. 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 ²X-|-X₂-X₃- X4-X₅-X₆ ¹¹⁷ wherein X-i to X₅ 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. ,

b. 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,

(ii) culturing host cells under conditions for replication and transcription of the recombinant virus,

(iii) optionally amplification of the recombinant virus into chicken embryos, and

(iv) recovering the recombinant attenuated NDV. In a particular embodiment, the in vitro method of rescuing a recombinant attenuated NDV according to the invention comprises at least the steps of:

(i) Co-transfecting baby hamster kidney cells (BHK-21 ) with a plasmid system comprising

a. a pGenome plasmid comprising the nucleotide sequence SEQ ID NO: 5

(pLaSota/M-Fmu-HN) ,

b. three 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,

(ii) culturing host cells under conditions for replication and transcription of the recombinant virus,

(iii) optionally amplification of the recombinant virus into chicken embryos, and

(iv) recovering the recombinant attenuated NDV

In an alternative embodiment, the in vitro method of rescuing a recombinant attenuated NDV according to the invention comprises at least the steps of:

(i) Co-transfecting baby hamster kidney cells (BHK-21 ) with a plasmid system comprising

a. a pGenome plasmid comprising the nucleotide sequence SEQ ID NO: 5

(pLaSota/M-Fmu-HN) ,

b. a unique 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 ),

(ii) culturing host cells under conditions for replication and transcription of the recombinant virus,

(iii) optionally amplification of the recombinant virus into chicken embryos, and

(iv) recovering the recombinant attenuated NDV In another alternative embodiment, the in vitro method of rescuing a recombinant attenuated NDV according to the invention comprises at least the steps of:

(i) Co-transfecting baby hamster kidney cells (BHK-21 ) with a plasmid system comprising a. pGenome plasmid comprising the nucleotide sequence SEQ ID NO: 5 (pLaSota/M-Fmu-HN) ,

b. two helper plasmids, one of them 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,

(iii) optionally amplification of the recombinant virus into chicken embryos, and

(iv) recovering the recombinant attenuated NDV

The co-transfecting technologies for step (i) and culturing conditions for step (ii) are well known from the man skilled in the art.

The term "rescuing a recombinant virus" according to the invention 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.

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. In a particular embodiment, the pNPL plasmid comprises three independent expression cassettes under a promoter, in particular pCMV promoter to express N, P, L. In some embodiments, 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.

In some embodiments, the host cells are eukaryotic cells, in particular mammal cells, preferably baby hamster kidney cells (BHK-21 ). In particular embodiments, the host cells are transfected with an amount of two-plasmid system ranging from 1 μg to 20 μg, in particular 2 to 20 μg, and preferably 3 to 5 μg (total amount of both plasmids).

In some embodiments, the in vitro method of rescuing negative RNA viruses in host cells additionally comprises a step of virus amplification into chicken embryos. In particular, this additional step is managed between culturing step (ii) and recovering step (iii) of the method. In particular for 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.

So in a particular embodiment, 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.

The co-transfecting technologies for step (i) and culturing conditions for step (ii) are well known from the man skilled in the art.

In a non-limitative embodiment, the method of rescuing NDV virus may comprise the following steps:

1 ) preparation of the plasmids constructions:

. extraction RNA from NDV strain ;

. cDNA generation based on viral RNA;

. 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;

. pGenome construction: 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 μg pCMV-NDV (pGenome) and 1.5 μg pNPL (pNPL plasmid) are transfected by Lipofectamin into BHK-21 cells;

3) optionally amplification of the rescued RNA virus:

3 days after transfection, the transfected cells with 200μΙ_ 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;

4) recovering the rescued virus: the allantoic fluids are harvested. Then, rescuing virus is confirmed with hemagglutination assay (HA) and qRT-PCR.

Immunogenic composition or vaccine

Another subject matter of the invention 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. In a particular embodiment, the composition comprises the attenuated recombinant NDV rLaSota/M-Fmu-HN of the invention.

The term "composition" encompasses pharmaceutical composition, antiviral composition, immunogenic composition and vaccine, more particularly antiviral composition, immunogenic composition and vaccine. The 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. In certain embodiments, 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. In some embodiments, 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.

In certain embodiments, 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. In some embodiments, 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). In some embodiments, a subcutaneous or intravenous route is used.

In a particular embodiment, an immunogenic composition provided herein is administered to a subject by ocular or intranasal route.

Uses of recombinant virus or vaccine

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. By "reducing viral shedding" according to the invention, it 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.

In some embodiment, 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.

In a particular embodiment, 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.

The term "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. As a skilled person will appreciate 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.

In some embodiments, 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. 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. For avian paramyxoviruses and in particular NDV, the subject in need thereof is typically a bird, preferably domestic birds, more preferably, poultry, duck, goose or pigeon.

DESCRIPTION OF THE FIGURES

Figure 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. Then, 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. Immediately after, the HN gene from LaSota was replaced by the one of MG-725 strain using Fsel and Pad enzymes.

Figure 2. Differences of challenge NDV strains. (A) With the F gene of 41 NDV strains representing different genotypes, the phylogenetic tree was constructed by using the Neighbor-Joining method with 1000 bootstraps based on the Kimura 2-parameter model in MEGA6. A discrete Gamma distributed (Gamma parameter = 0.685) was applied for modeling rate among sites. The tree was drawn according to branch lengths measured by scale and representing the number of substitutions per site. The vaccine and challenge strains were indicated by bold dot and triangle, respectively. (B and C) Different amino acids of F and HN protein between three challenge strains were marked in blue and red, respectively (black on Fig.2), by using NDV F (PDB: 3 MAW) and HN (PDB: 3T1 E) protein structures in PyMOL.

Figure 3. Protection conferred by the rLaSota and rLaSota/M-Fmu-HN vaccines against rMG- 725 (Genotype XI), GB Texas (genotype II, Accession number GU978777) and NDV/EG/CK/104/12 (Genotype VII) challenges by intramuscular route. (A) Two of three sacrificed chickens vaccinated with rLaSota strain showed genotype XI strain RNA in trachea, but no viral RNA could be detected in tissues from bird vaccinated with the rLaSota/M-Fmu- HN strain. Two of three sacrificed chickens vaccinated with the rLaSota had genotype II RNA in the tracheal and nasal. After challenge with the genotype VII, none of the vaccinated showed virus RNA in their tissues. In contrast, all tissues of unvaccinated birds had viral RNA and the spleen followed by the respiratory tract had the highest loads. (B and C) 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.

The invention will be now illustrated by the following non-limitative examples.

EXAMPLES

Materials and Methods

Cells and virus

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% C0₂. 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. Miller from Southeast Poultry Research Laboratory, Unite States. 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 μηι 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

ID (plasmids, complete cleavage

NO: nucleotide genome or sites

sequences) gene

1 pMG-725 MG-725 Plasmid with complete Velogenic- genome of MG-725 like^a

2 pMG-725/Fmu MG-725 Plasmid with genome of Lentogenic- MG-725 wherein cleavage like site of the F protein in MG- 725 was modified to that

of LaSota

3 pLaSota LaSota Plasmid with the complete Lentogenic- genome of LaSota like

4 pLaSota/M- LaSota Plasmid with LaSota Lentogenic- Fmu genome, wherein the F like

gene of LaSota was

replaced by that of MG- 725/Fmu

5 pLaSota/M- LaSota Plasmid with LaSota Lentogenic- Fmu-HN genome, wherein the F like

gene of LaSota was

replaced by that of MG- 725/Fmu and HN gene of

LaSota was replaced by

that of MG-725

6 M-F-change-F Primer for amplification of

Fmu gene from pMG- 715/Fmu plasmid

7 M-F-change-R Primer for amplification of

Fmu gene from pMG- 715/Fmu plasmid

8 M-HN-change- Primer for amplification of

F HN gene from pMG- 715/Fmu plasmid

M-HN-change- Primer for amplification of R HN gene from pMG- 715/Fmu plasmid

M-HN-change- Primer for amplification of L-F non-coding region between F and HN of LaSota strain

M-HN-change- Primer for amplification of L-R non-coding region between F and HN of LaSota strain

LaSo-NP-F Primer for amplification of

NP gene of LaSota strain

LaSo-NP-R Primer for amplification of

NP gene of LaSota strain

LaSo-P-F Primer for amplification of

P gene of LaSota strain

LaSo-P-R Primer for amplification of

P gene of LaSota strain

LaSo-L-F Primer for amplification of

L gene of LaSota strain

LaSo-L-R Primer for amplification of

L gene of LaSota strain pLaSo-NP LaSota Plasmid with NP gene of

LaSota strain

pLaSo-P LaSota Plasmid with P gene of

LaSota strain

pLaSo-L LaSota Plasmid with L gene of

LaSota strain

pLaSo-NPL LaSota Plasmid with NP, P and L genes of LaSota

F259 Primer for detection of F gene

3 F488 Primer for detection of F

gene

F MG-725 Nucleotide sequence of F Velogenic- gene of MG-725 like^a 5 (Accession number

Genbank HQ266602)

5 Fmu MG-725 Nucleotide sequence of F Lentogenic- gene of MG-725 mutated like in cleavage site to 10 reproduce LaSota

lentogenic like genotype

HN MG-725 Nucleotide sequence of

HN gene of MG-725

(Accession number 15 Genbank HQ266602)

7 GRQGRL F cleavage site amino acid

sequence

8 rLaSota-M- Recombinant nucleotide

Fmu-HN sequence, wherein the F 20 gene of LaSota was

replaced by that of MG- 725/Fmu and HN gene of

LaSota was replaced by

that of MG-725 259 NP NP gene of LaSota

0 P P gene of La Sota

1 M M gene of LaSota

L L gene of LaSota

F protein cleavage sites are ²RRRRRF¹¹⁷.

F protein cleavage sites are ²GRQGRL¹¹⁷. Construction of rLaSota/M-Fmu-HN strain

For the cloning strategy, the Fmu gene (SEQ ID NO: 25, cleavage site of MG-725 strain's F gene was mutated to the LaSota) was first amplified from pMG-725/Fmu plasmid (SEQ ID NO: 2) by M-F-change-F (5'-TCCCCGCGGGCAAGATGGGCTCTAAATCTTCTAC-'3= SEQ ID NO: 6) and M-F-change-R (5 -

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 HN gene (SEQ ID NO: 26) of MG-725 strain was amplified from pMG-715/Fmu plasmid (SEQ ID NO: 3) using M-HN-change-F (5 - ACCGACAACAGTCCTCAATCATGGACCATGTAGTTAGCAG-'3= SEQ ID NO: 8) and M- HN-change-R (5'-CCTTAATTAATCAAGTCCTGCCATCCTTGAGAATCTCCACT-'3= SEQ ID NO: 9) primers. The non-coding region between F and HN genes of LaSota strain was generated by M-HN-change-L-F (5'-GCACATCTGCTCTCATTACCT-'3= SEQ I D NO: 10) and M-HN-change-L-R (5'-GATTGAGGACTGTTGTCGGT-'3= SEQ ID NO: 1 1 ) primers. 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). Fragments bearing NP, P and L gene of LaSota strain were generated from wild LaSota strain by RT-PCR using LaSo-NP-F (5'- CCGCTCGAGATGTCTTCCGTATTTGATGAGTA-'3= SEQ ID NO: 12) and LaSo-NP-R(5'- ATTTGCGGCCGCTCAATACCCCCAGTCG-'3= SEQ ID NO: 13), LaSo-P-F (5 - CCGCTCGAGATGGCCACCTTTACAGATGC-'3= SEQ ID NO: 14) and LaSo-P-R(5'- ATTTGCGGCCGCTTAGCCATTTAGAGCAAG-'3= SEQ ID NO: 15), and LaSo-L-F (5 - GGACTAGTATGGCGAGCTCCGGTCCTG-'3= SEQ ID NO: 16) and LaSo-L-R (5 - ATTTGCGGCCGCTTAAGAGTCACAGTTACTG-'3= SEQ ID NO: 17) primers, respectively. The 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. According to another embodiment, 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. Then, 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 ).

Recovery of rLaSota/M-Fmu-HN strain.

Recombinant rLaSota/M-Fmu-HN strain was generated by reverse genetics as previously described. Briefly, 4* 10⁵ BHK-21 cells were grown overnight in 6-well plates. Supernatants were discarded and cells were washed twice with Opti-MEM (Gibco). Then, 5 μg of pLaSota/M-Fmu-HN (SEQ ID NO: 5), 2 μg of pLaSo-NP (SEQ ID NO: 18), 2 μg of pLaSo-P (SEQ ID NO: 19) and 1 μg of pLaSo-L (SEQ ID NO: 20) were cotransfected with 20 μΙ_ Lipofectamine 2000 (Invitrogen).

In another embodiment, the three helper plasmids are replaced by a unique helper plasmid pLaSo-NPL (SEQ ID NO: 21 ).

After 6 h, the transfection mixture was replaced by MEM medium with 10% FBS. Three days after transfection, the cells with 200 μΙ_ 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.

In order to replace F and HN genes in the LaSota strain, 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). With 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.

Example 2: Growth ability and pathogenicity index tests

The growth characteristics of rLaSota and rLaSota/M-Fmu-HN strain were checked on embryonated chicken eggs. One hundred 50% egg infective doses (EID₅₀) 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 EID₅₀ method. To test pathogenicity of strains, the mean death time index (MDT) in 9-day-old embryonated SPF chicken eggs and the intracerebral pathogenicity index (ICPI) in 1 -day-old SPF chickens were used. To determine MDT, 10-fold serial dilutions of the infective allantoic liquid were prepared in sterile phosphate-buffered saline (PBS). One hundred microliters of 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. Briefly, Fresh infective allantoic fluids with HA titres > 2⁴ were diluted 1/10 in sterile isotonic saline without antibiotics and used as inoculum. Fifty μΙ 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. Dead individuals were scored as 2 at each of the remaining daily observations after death.). The ICPI is the mean score of 10 chicks over 8 days. The growth ability of rescued strains was verified on eggs. The F and HN genes replacement did not affect the viral growth. The titers of rLaSota and rLaSota/M-Fmu-HN, after three days of amplification, were up to 3.16 <10⁹ and 3.98x10⁹ EID₅₀/ml, respectively (Table 2). TABLE 2: Properties of rescued strains.

a: Hemagglutination; : 50% egg infectious doses;

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).

In addition, the 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. Based on the World Organization for Animal Health (OIE) guidelines, a NDV strain with MDT value higher than 90 h and ICPI value close to 0.00 is considered as a lentogenic strain, while a strain with MDT value less than 60 h and ICPI approaching 2.00 is considered as a velogenic strain. Consequently, rLaSota and rLaSota/M- Fmu-HN strains were confirmed as avirulent strains while rMG-725 was virulent. These results suggested that the replacement of the F and HN genes of LaSota by the Fmu and HN genes of MG-725 strain did not increase the viral virulence of the vaccine strain.

Example 3: Immunization and challenge

Two groups of 39 two-week old specific pathogen free (SPF) chickens were vaccinated by rLaSota and rLaSota/M-Fmu-HN strains, respectively. Each chicken was vaccinated with 10⁶ EI D50 (50% egg infective dose) of the corresponding vaccine strain through the intranasal and eye routes. Vaccine strains were diluted in PBS. Chickens from another group received 100 μί sterile PBS without virus through the same route and were used as sham-vaccine control group. Blood were collected from all chickens three weeks after immunization, to study the serum NDV antibody response. Then, the two vaccinated groups were randomly split into three subgroups (n=13 per subgroup) and challenges. Virulent challenges occurred three weeks post vaccination and each chicken received 10⁵ ELD₅₀ (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. 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.

Three virulent strains, GB Texas (Accession number GU978777), EG/CK/104/12 and rMG- 725, were selected for in vivo testing. Based on phylogenetic tree of F gene, GB Texas and rLaSota strains were from genotype II and EG/CK/104/12 was from genotype VII, but rMG- 725 and rLaSota/M-Fmu-HN belonged to genotype XI (Fig. 2). In terms of isolated and identified time of genotypes, these strains represented old, popular and newly emerged genotypes (Dimitrov et al., 2016). Different amino acids of F and HN protein of those strains were marked based on proteins' structure. Those differences not only located at the head areas, but also were in the stalk areas of F and HN protein (Fig. 2A and B). Furthermore, F protein similarity between those velogenic strains and vaccine rLaSota strain were 96.8%, 88.1 % and 87.4%, respectively, while these values were 86.5%, 86.6% and 99.1 % for vaccine rLaSota/M-Fmu-HN strain (Table 3). The HN protein between those velogenic and vaccinated strains showed same similarity. These results show that these challenge and vaccine strains were quite different and can be used to test whether the genotype mismatch is responsible for failure of ND vaccine. TABLE 3: Amino acid similarity of F and HN protein between challenge and vaccine strains.

The similarity was deduced with Clustal W method alignment in DNAstar software

Example 4: Serological analysis.

Serum antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) and Hemagglutination Inhibition assay (HI) assays. For ELISA 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. To test antibodies by HI, five viruses were utilized. Briefly, 2-fold serial dilutions of the serum (25 μί) were mixed with 4 HA units of rLaSota, rLaSota/M-Fmu-HN, GB Texas (Accession number GU978777), EG/CK/104/12 or rMG-725 (25 μί). After 1 h, 25 μί of 1 % chicken red blood cells (RBC) were added. The mixture was then incubated at room temperature for 30 min and hemagglutination was recorded.

To evaluate the antibody response in immunized chickens, serums were collected 21 days after vaccination and analyzed by HI and ELISA tests. HI and ELISA titers in the animals vaccinated with both strains were higher than 6 log₂ and 6000, respectively (Table 4). Interestingly, HI titers were always higher when the vaccine and HI test strains used were of the same genotype. In contrast, there was no difference between the two vaccine groups when the HI test strain was of genotype VII and HI titers against this genotype were lower than with the others (Table 4). These results indicated both vaccines could induce chicken to generate strong immunity response against NDV from different genotypes. TABLE 4: Evaluation of the antibody response after vaccination. Hl ^a (Log2)

rLaSota GB Texas rLaSota/ rMG-725 NDV/EG/ ELISA

M-Fmu-HN CK/104/12

rLaSota 8±0 7.5 ± 0.55 6.67 ± 0.52 6.83 6.5 ±0.55 8728±2280

± 0.75

rLaSota/ 7±0 7 ± 0 7.33 ± 0.52 7.67 6.5 ±0.55 9154±790 M-Fmu-HN ±0.52

Unvaccinat 0 0 0 0 0 40.78 ed ±21.80 a: Hemagglutination inhibition; : Average value ± Standard deviation.

Example 5: Virological analysis

Two methods, qRT-PCR and egg incubation, were used to check virus shedding from swabs or virus replication in tissues. Briefly, total RNA using Biomek robot was extracted from swabs and tissues by NucleoSpin RNA virus core kit (MACHEREY-NAGEL). Viral RNA was quantified by qRT-PCR based on the F gene detection with primers F259 (5'- ACAYTGACYACTTTGCTCA-'3= SEQ ID NO: 22) and F488 (5'- TGCACAGCYTCATTGGTTGC-'3= SEQ ID NO: 23) according to Brilliant Π Ultra-Fast SYBR Green QRT-PCR Master mix kit (Agilent). In addition, 200 μΙ of the cloacal and tracheal swabs and tissue homogenization were inoculated into SPF embryonated eggs. Allantoic fluids from dead chicken embryo or five days later after incubation, showing hemagglutination, were considered as virus positives.

Both vaccines protect chickens from challenge and block viral shedding from different virulent NDV strains.

Chickens immunized with rLaSota/M-Fmu-HN or rLaSota strain were challenged with 10⁵ ELD₅₀ of GB-Texas (genotype II), EG/CK/104/12 (genotype VII) and rMG-725 (genotype XI) strain via the intramuscular route, 21 days after vaccination.

Chickens were observed daily for 10 days. Clinical signs and death were scored. After challenge, none of the immunized chickens showed any clinical signs, but unvaccinated chickens became sick from the third day after the challenge and all died by 5 days post- challenge (Fig.3B and C). To check viral shedding, oral and cloacal swabs were tested for viral RNA and infectivity at 3- day, 5-day, 7-days and 10-day after challenge. The viral RNA could not be detected in any swabs from chickens vaccinated with rLaSota/M-Fmu-HN or rLaSota. In addition, no virus was isolated from swabs collected from immunized chickens. In contrast, almost all swabs from unvaccinated and challenged chickens contain viral RNA and infectious viral particles at 3-day after the challenge (Table 5).

TABLE 5: Viral shedding in oral and cloacal swabs collected from chickens after challenges with genotype XI, genotype II and genotype VII strains

Swabs

Vaccine Challenge 3 d 5 d 7 d 10 d Strain strain Oral Cloac Oral Cloa Oral CloaOral Cloaal -cal cal cal rMG-725 0/13 0/13 0/10 0/10 0/10 0/10 0/10 0/10 a

(Genotype XI)

rLaSota/M- GB Texas 0/13 0/13 0/10 0/10 0/10 0/10 0/10 0/10 Fmu-HN (Genotype II)

(Genotype NDV/EG/CK/104/1 0/13 0/13 0/10 0/10 0/10 0/10 0/10 0/10 XI) 2

(Genotype VII)

rMG-725 0/13 0/13 0/10 0/10 0/10 0/10 0/10 0/10 (Genotype XI)

GB Texas 0/13 0/13 0/10 0/10 0/10 0/10 0/10 0/10 rLaSota (Genotype II)

(Genotype II) NDV/EG/CK/104/1 0/13 0/13 0/10 0/10 0/10 0/10 0/10 0/10

2

(Genotype VII)

rMG-725 8 12/1 2/2 2/2 c

(Genotype XI) /13 3

GB Texas 13/1 12/1

(Genotype II) 3 3 Unvaccinate NDV/EG/CK/104/1 13/1 12/1 - - - - - - d 2 3 3

(Genotype VII) ^a: Positive number/total number;

: Viral shedding was detected by qRT-PCR and egg inoculation;

c: No survival chicken.

To check early replication of challenge strains in tissues, three chickens of each group were sacrificed at 3-day post-challenge, and 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).

These results suggested that both vaccines could protect chickens from ND, blocking viral shedding and inhibiting viral replication of virulent NDV strains from different genotypes.

TABLE 6: Viral isolation in tissues of vaccinated or unvaccinated chickens after challenge.

Vaccine Challenge Tissues

strain Strain

Brain Lung Trachea Nasal Spleen Intestine rMG-725 0/3 ³ 0/3 0/3 0/3 0/3 0/3 (Genotype XI)

rLaSota/M- Fmu-HN GB Texas 0/3 0/3 0/3 0/3 0/3 0/3 (Genotype XI) (Genotype II) NDV/EG/CK/104/12 0/3 0/3 0/3 0/3 0/3 0/3 (Genotype VII) rMG-725 0/3 0/3 0/3 0/3 0/3 0/3 (Genotype XI)

rLaSota

(Genotype II) GB Texas 0/3 0/3 0/3 0/3 0/3 0/3

(Genotype II)

NDV/EG/CK/104/12 0/3 0/3 0/3 0/3 0/3 0/3 (Genotype VII) rMG-725 3 /3 3/3 3/3 3/3 3/3 3/3 (Genotype XI)

Unvaccinated GB Texas 3/3 3/3 3/3 3/3 3/3 3/3

(Genotype II)

NDV/EG/CK/104/12 3/3 3/3 3/3 3/3 3/3 3/3 (Genotype VII)

a: Positive number/total number;

: Challenge strains were isolated by egg inoculation.

In terms of antigenic F and HN protein of NDV, one genotype II (rLaSota) and one XI (rLaSota/M-Fmu-HN) attenuated vaccines were generated by reverse genetic. Both vaccines could induce in SPF chickens a high antibody response against NDV regardless of genotypes. Furthermore, these two vaccines exhibited same efficiency based on protecting chickens from ND, blocking viral replication and shedding from genotype II, VII and XI NDV strains. Thus, our results support the assumption that in immunocompetent chickens and correct vaccination, genotype mismatch is not a reason for vaccination failure.

Current ND live vaccines were generated from NDV strains isolated 70 years ago (Dimitrov et al., 2017). These vaccines can still prevent chickens from disease, but hardly block viral shedding when chicken is infected by recent isolates as shown by some recent publications (Liu et al., 2015). Even if it is widely accepted that NDV exists as only one single serotype, mutations often happened in F and HN generating some antigenic diversity (Dimitrov et al., 2016). This diversity can explain why antibodies from immunized chickens react better with the homologous HI test virus. Here, we also demonstrated that the HI titers of sera obtained after vaccination with rLaSota or rLaSota/M-Fmu-HN were lower against heterologous test strains. These observations could support why current live vaccines cannot fully block shedding from new genotypes strains, such as genotype VII, on the expressed condition that differences detected in the antibody titers were high enough to modulate the potency on the virus control. In contradiction with this, the two vaccines were able to totally protect chickens and stop viral shedding from homologous or heterologous virulent strains.

In this study, 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.

In summary, 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.

REFERENCES

- de Almeida, R.S., Hammoumi, S., Gil, P., Briand, F.X., Molia, S., Gaidet, N., Cappelle, J., Chevalier, V, Balanca, G., Traore, A., Grillet, C, Maminiaina, O.F , Guendouz, S., Dakouo,

de Leeuw, O., Peeters, B., 1999. Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the subfamily Paramyxovirinae. The Journal of general virology 80 ( Pt 1 ), 131-136.

Dimitrov, K.M., Afonso, C.L., Yu, Q., Miller, P.J., 2017. Newcastle disease vaccines-A solved problem or a continuous challenge? Veterinary microbiology 206, 126-136.

- Dimitrov, K.M., Ramey, A.M., Qiu, X. , Bahl, J., Afonso, C.L., 2016. Temporal, geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus). Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 39, 22-34.

Dortmans, J.C., Rottier, P.J., Koch, G., Peeters, B.P., 2010. The viral replication complex is associated with the virulence of Newcastle disease virus. Journal of virology 84, 10113-10120.

Liu, H., Albina, E., Gil, P., Minet, C, de Almeida, R.S., 2017. Two-plasmid system to increase the rescue efficiency of paramyxoviruses by reverse genetics: The example of rescuing Newcastle Disease Virus. Virology 509, 42-51.

Liu, M.M., Cheng, J.L., Yu, X.H., Qin, Z.M., Tian, F.L. , Zhang, G.Z., 2015. Generation by reverse genetics of an effective attenuated Newcastle disease virus vaccine based on a prevalent highly virulent Chinese strain. Biotechnology letters 37, 1287-1296. Maminiaina, O.F., Gil, P., Briand, F.X., Albina, E., Keita, D., Andriamanivo, H. R., Chevalier, V, Lancelot, R., Martinez, D., Rakotondravao, R., Rajaonarison, J. J., Koko, M., Andriantsimahavandy, A.A, Jestin, V , Servan de Almeida, R., 2010. Newcastle disease virus in Madagascar: identification of an original genotype possibly deriving from a died out ancestor of genotype IV. PloS one 5, e13987.

Peeters, B.P, de Leeuw, O.S., Koch, G., Gielkens, A.L., 1999. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. Journal of virology 73, 5001-5009.

Zhao, P., Sun, L, Sun, X., Li, S., Zhang, W., Pulscher, L.A., Chai, H., Xing, M., 2017. Newcastle disease virus from domestic mink, China, 2014. Veterinary microbiology 198, 104-107.

Claims

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 112 to 117 ²X X2-X3-X4-X5-X6 ⁷ wherein to X₅ 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.

The recombinant attenuated NDV according to claim 1 , wherein the mutated cleavage site is represented by amino acid sequence of formula (I) "^^-Xs-X^Xs-Xe ⁷ wherein X₂ and/or X₅ are independently arginine (R) or lysine (K), preferably arginine (R), X₆ is a non-basic amino-acid and at least two of X-i , X₃, and X4 are independently selected from the group consisting of non-basic amino-acids, preferably glycine or glutamine.

The recombinant attenuated NDV according to claim 1 or 2, wherein the mutated cleavage site in the nucleotide sequence encoding F protein is GRQGRL (SEQ ID NO:27).

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% and even preferably 100% identity with nucleotide sequence encoding HN protein (SEQ ID NO: 26).

The recombinant attenuated NDV according to anyone of claims 1 to 4, comprising additionally 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 recombinant attenuated NDV according to anyone of claims 1 to 5, comprising 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).

. An immunogenic composition or vaccine comprising a recombinant attenuated NDV according to any one of claims 1 to 6 and at least one ingredient selected from excipients, adjuvants and mixtures thereof.

. The recombinant attenuated NDV of any one of claims 1 to 6 or the composition of claim 7 for use in inducing a protective immune response in a subject in need thereof. . The recombinant attenuated NDV of any one of claims 1 to 6 or the composition of claim 7 for use in the manufacture of a vaccine for the prophylaxis and/or treatment of a NDV infection in a subject in need thereof.

0. The recombinant attenuated NDV of any one of claims 1 to 6 or the composition of claim 7 for protecting a bird against Newcastle disease and for reducing viral shedding.

1 . An in vitro method of reverse genetics for preparing a attenuated Newcastle Disease Virus (NDV) according to anyone of claims 1 to 6, comprising at least the steps of:

(i) Co-transfecting eukaryotic host cells, in particular mammal cells, preferably baby hamster kidney cells (BHK-21 ) with a plasmids system comprising

a. 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 ²X X2- X₃-X₄-X₅-X6 ⁷ wherein X-i to X₅ 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. , b. three helper plasmids comprising respectively the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),

or alternatively a unique pNPL helper plasmid comprising the sequences encoding the structural viral proteins nucleocapsid protein (N), phosphoprotein (P) and large protein (L),

or alternatively two 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,

(ii) culturing host cells under conditions for replication and transcription of the recombinant virus,

(iii) optionally amplification of the recombinant virus into chicken embryos, and

(iv) recovering the recombinant attenuated NDV.

12. An in vitro method according to claim 11 , comprising at least the steps of:

(i) Co-transfecting baby hamster kidney cells (BHK-21 ) with a plasmids system comprising

a. a pGenome plasmid comprising the nucleotide sequence SEQ ID NO:

5 (pLaSota/M-Fmu-HN) ,

b. three 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 LaSota strain, or alternatively a unique 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 ),

or alternatively two helper plasmids, one of them comprising the sequences encoding two 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,

(ii) culturing host cells under conditions for replication and transcription of the recombinant virus,

optionally amplification of the recombinant virus into chicken embryos, and

recovering the recombinant attenuated NDV.