Ndv aiv vectors and uses thereof

Abstract

The present invention provides recombinant Newcastle disease virus (NDV) viral vectors comprising a heterologous polynucleotide encoding an avian influenza antigen. The recombinant viral vectors are suitable for use in immunogenic compositions and vaccines, and can provide protection against avian influenza and other pathogens when administered to an avian.

Description

NDV AIV VECTORS AND USES THEREOFSEQUENCE LISTING

[0001] This application contains a Sequence Listing in accordance with 37 C.F.R. 1.821 - 1.825. The Sequence Listing associated with this application is submitted electronically as an XML file, compliant with WIPO Standard ST.26. The XML file, titled “23-0047-US-2.xml,” created on July 24, 2024, and 29,233 byte in size, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to recombinant Newcastle disease virus (NDV) viral vectors comprising a foreign avian influenza H9 haemagglutinin (AIV H9-HA) gene. The recombinant viral vectors are suitable for use in immunogenic compositions and vaccines, and can provide protection against avian influenza and other pathogens.BACKGROUND OF THE INVENTION

[0003] Avian influenza (AIV), sometimes called avian flu, and commonly recognized as bird flu refers to influenza caused by influenza viruses adapted to birds. AIV is a segmented, singlestrand, negative sense RNA virus belonging to the family of Orthomyxoviridae, and is classified as a type A influenza virus. Type A virus is the most frequent cause of animal and human influenza. This type occurs in numerous strains or subtypes that are differentiated mainly on the basis of two surface lipid-enveloped membrane proteins, hemagglutinin (HA) and neuraminidase (NA). HA facilitates entry of the virus into host cells, and NA assists in the release of progeny virus from infected cells (de Jong et al., J Clin Virol. 35(1):2-13, 2006). Influenza type A viruses are divided into subtypes based on their specific HA and NA content. There are 16 different HA subtypes, and 9 different NA subtypes. Many different combinations of HA and NA proteins are possible. Subtypes of influenza A virus are named according to their HA and NA surface proteins. Avian influenza H9N2 virus is considered a low-pathogenic virus that is endemic to poultry populations. Low pathogenicity avian influenza H9N2 has adverse effects on poultry production and poses a significant cross-species transmission and zoonotic threat.

[0004] Newcastle disease virus (NDV) belongs to the Paramyxovirinae family and the Avulavirus genus. NDV replicates in respiratory and gastrointestinal tracts, in the oviduct, and forsome isolates, in the nerve system. The transmission is aerogenic and by oral and fecal routes. NDV causes a highly contagious and fatal disease affecting all species of birds, and can infect some mammalian species. The disease can vary from clinically unapparent to highly virulent forms, depending on the virus strain and the host species. The continuous spectrum of virulence displayed by NDV strains enabled the grouping of them into three different pathotypes: lentogenic, mesogenic, and velogenic (Alexander, D. J., Diseases of Poultry, Iowa State Uni. Press, Ames IA, 541-569, 1997). Lentogenic strains do not usually cause disease in adult chickens and are widely used as live vaccines in poultry industries in the United States and other countries. Viruses of intermediate virulence are termed mesogenic, while viruses that cause high mortality are termed velogenic. The disease has a worldwide distribution and remains a constant major threat to commercial poultry production.

[0005] The NDV genome is a non-segmented negative strand of RNA of approximately 15kb. The genomic RNA contains six genes that encode the following proteins in the order of: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutinin-neuraminidase (HN) and large polymerase protein (L). Two additional proteins, V and W, of unknown function are produced by RNA editing during P gene transcription (Steward et al., 1993, Journal of General Virology 74:2539-2547).

[0006] Several studies have highlighted the potential of Newcastle disease virus (NDV) to be used as a vaccine vector for avian diseases (Krishnamurthy et al., Virology 278, 168-182,2000; Huang et al., J. Gen. Virol. 82, 1729-1736, 2001; Nakaya et al., J. Virol. 75, 11868-11873, 2001; Park et al. PNAS 103, 8203-8208, 2006; Veits et al PNAS 103, 8197-8202, 2006; Ge et al. J. Virol. 81, 150-158, 2007; Romer- Ober dorfer et al. Vaccine 26, 2307-2313, 2008). In particular, Nagy et al., Vaccine 34(23), 2537-2545, 2016, describes recombinant NDV vectors encoding a consensus H9-HA sequence based on Chinese and Middle Eastern AIV isolates, or encoding a chimeric protein comprising an ectodomain of H9-HA from a Hong Kong AIV isolate and a transmembrane domain of a Newcastle disease virus (NDV) fusion (F) protein. However, the alleged results disclosed in Nagy et al. (2016) are limited to serological response tests which are not sufficient to indicate that the disclosed recombinant NDV vectors will be efficacious, and it is also indicated that these alleged results are highly antigen-specific.

[0007] The development of highly effective influenza virus vaccines for poultry would be of benefit to both human and veterinary health. In particular, it would be of benefit to provide acombined NDV and AIV vector vaccine which is effective even in stringent clinical conditions, with a level of efficacy to reach ambitious clinical endpoints such as protection against clinical signs. It would be of benefit to provide such vaccines further to the attempts that may have already been made in the art. However, it is not always straightforward to predict how successful a particular type of AIV HA antigen will be in a NDV vector context. This is particularly the case when that type of AIV HA antigen has not previously been tested in this context in vivo, or is a novel antigen.

[0008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.SUMMARY OF THE INVENTION

[0009] The present invention generally provides recombinant NDV vectors comprising a heterologous polynucleotide encoding an avian influenza (AIV) H9 haemagglutinin antigen (AIV H9-HA, also referred to simply as H9), derived from a Saudi Arabia AIV H9N2 strain or encoding a H9 COBRA antigen. The recombinant viral vectors can be used in immunogenic compositions and vaccines to provide avians with protection against avian influenza and Newcastle disease.

[0010] In a first aspect, the present invention provides a recombinant Newcastle disease virus (NDV) vector, wherein the recombinant NDV vector comprises a heterologous polynucleotide encoding an AIV subtype H9 haemagglutinin (AIV H9-HA) antigen, wherein:(i) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80% identity to SEQ ID NO: 1, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4; or(ii) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80% identity to SEQ ID NO: 2, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3.

[0011] This recombinant NDV vector comprising the AIV H9-HA is capable of inducing a protective immune response against avian influenza virus (AIV) in an avian.

[0012] In a second aspect, the present invention provides one or more nucleic acid molecules comprising the recombinant NDV vector of the invention.

[0013] In a third aspect, the present invention provides a cell comprising the recombinant NDV vector or the nucleic acid molecules of the invention.

[0014] In a fourth aspect, the present invention provides a composition comprising the recombinant NDV vector, the nucleic acid molecules or the cell of the invention.

[0015] In a fifth aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in a method of inducing a protective immune response against AIV in an avian. Alternatively or additionally in a fifth aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in a method of inducing a protective immune response against NDV in an avian

[0016] In a sixth aspect, the present invention provides a method of inducing a protective immune response against AIV in an avian, comprising administering the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian. Alternatively or additionally in a sixth aspect, the present invention provides a method of inducing a protective immune response against NDV in an avian, comprising administering the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian.

[0017] In a seventh aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in the manufacture of a medicament for inducing a protective immune response against AIV in an avian. Alternatively or additionally in a seventh aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in the manufacture of a medicament for inducing a protective immune response against NDV in an avian.

[0018] In an eighth aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in a method of reducing AIV shedding in an avian.

[0019] In a ninth aspect, the present invention provides a method of reducing AIV shedding in an avian, comprising administering the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian.

[0020] In a tenth aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in the manufacture of a medicament for reducing AIV shedding in an avian.

[0021] In an eleventh aspect, the present invention provides a method of manufacturing the recombinant NDV vector of the invention, wherein the method comprises:a) providing one or more nucleic acids encoding a NDV vector; b) providing a polynucleotide encoding an AIV H9-HA antigen; and c) recombinantly combining the nucleic acids encoding a NDV vector and the polynucleotide encoding an AIV H9-HA antigen.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows a pFR128 plasmid map.

[0023] FIG. 2 shows AIV H9-HA expression (IFI) from vAVW48.

[0024] FIG. 3 shows a pFR211 Plasmid map.

[0025] FIG. 4 shows a diagram for constructing the transcription plasmid pFR211.

[0026] FIG. 5 shows H9-HA expression results from vAVW68 and vAVW48 (positive control).

[0027] FIG. 6 shows evolutions of mean daily clinical scores by groups. Onset of clinical signs in CTL group = 2 dpc, slight and mild respiratory signs. Lower mean daily clinical signs than CTL in groups NDV & NDV COBRA. 1 chicken found dead in G3 with opaque and thick air sacs.

[0028] FIG. 7 shows a schedule of the study.

[0029] FIG. 8 shows results from viral shedding measured by QRT-PCR in SPF chickens. The titers are significantly different between Gia and Gib on D27 and D29 (ANOVA test p=0.008 and Kruskal -Wallis test p<0.001 respectively). The treatment with vAVW48 allowed a quicker decrease of the viral shedding in SPF chickens. These results are in accordance with clinical observations.

[0030] FIG. 9 is a bar graph showing anti-H9 antibody titers four different H9N2 isolates used as antigens in the HI test: A / chicken / Irak / AVl 342 / 2011 (Irak), A / chicken / Saudi Arabia / WNB9510 / 2010 (Arabia), A / av. / Azerbaidjan / l lvirl344-l / 2011 (Azerbaidjan) and A / Ck / Iordan / 436-1 / 2010 (Iordan).

[0031] FIG. 10 is a graph showing dispersions of individual AUC of viral RNA excretion by three groups, namely NDV-H9(COBRA), NDV-H9 and Controls.DETAILED DESCRIPTION

[0032] The present invention generally provides recombinant NDV vectors comprising a heterologous polynucleotide encoding an avian influenza AIV H9-HA antigen. In particular, thepresent invention provides novel recombinant NDV vectors comprising either an AIV H9-HA polynucleotide from a H9N2 Saudi Arabia strain of AIV, or an AIV H9-HA COBRA polynucleotide. The AIV H9-HA polynucleotides and antigens of the present invention can be defined with reference to SEQ ID NOs: 1 and 4 (Saudi Arabia strain H9-HA) or SEQ ID NOs: 2 and 3 (COBRA H9-HA) respectively.

[0033] The recombinant viral vectors of the invention may be used in immunogenic compositions and vaccines to provide avians with protection against avian influenza and Newcastle disease. In particular, the recombinant viral vectors of the present invention may provide a combined NDV and AIV vector vaccine which is surprisingly effective even in stringent clinical conditions, with an unexpected level of efficacy that can achieve ambitious clinical endpoints such as protection against clinical signs. Benefits of the viral vectors of the invention include the capability to induce a protective immune response against AIV as well as NDV, including a reduction in clinical signs, even when administered just once and to avians of six or fewer days of age, with demonstrated efficacy via the oculo-nasal route of administration.

[0034] In a first aspect, the present invention provides a recombinant Newcastle disease virus (NDV) vector, wherein the recombinant NDV vector comprises a heterologous polynucleotide encoding an AIV subtype H9 haemagglutinin (AIV H9-HA) antigen, wherein:(i) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80% identity to SEQ ID NO: 1, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4; or(ii) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80% identity to SEQ ID NO: 2, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3.

[0035] In an embodiment of (i) such NDV vector is capable of inducing a protective immune response against avian influenza virus (AIV) in an avian. In an embodiment of (i), the AIV H9- HA antigen is a Saudi Arabia strain H9-HA antigen. In an embodiment of (i), the polynucleotide encoding the AIV H9-HA antigen is a Saudi Arabia strain H9-HA polynucleotide. In an embodiment of (ii) such NDV vector is capable of inducing a protective immune response against avian influenza virus (AIV) in an avian. In an embodiment of (ii), the AIV H9-HA antigen is a H9-HA COBRA antigen. In an embodiment of (ii), the polynucleotide encoding the AIV H9-HA antigen is a H9-HA COBRA polynucleotide.

[0036] In an embodiment, the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.0% 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% or 100.0% identity to SEQ ID NO: 1 or any fragments thereof. In an embodiment, the AIV H9-HA antigen comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.0% 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% or 100.0% identity to SEQ ID NO: 4 or any fragments thereof.

[0037] In an embodiment, the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.0% 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% or 100.0% identity to SEQ ID NO: 2 or any fragments thereof. In an embodiment, the AIV H9-HA antigen comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.0% 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% or 100.0% identity to SEQ ID NO: 3 or any fragments thereof.

[0038] In an embodiment, herein it will be understood that “fragments thereof’ relate to polypeptide or polynucleotide sequences having an equivalent function to the polypeptide or polynucleotide sequence in respect of which they are defined, i.e. SEQ ID NOs: 1 to 4. In anembodiment, equivalent function means that a polynucleotide fragment encodes an AIV H9-HA antigen. In an embodiment, equivalent function means that a polypeptide fragment comprises an AIV H9-HA antigen. In an embodiment, an AIV H9-HA antigen is defined as a sequence that is capable of inducing an immune response, preferably a protective immune response, against AIV, preferably H9N2 AIV.

[0039] In an embodiment of the recombinant NDV vector of the invention, either:(i) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 95% identity to SEQ ID NO: 1, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 4; or(ii) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 85% identity to SEQ ID NO: 2, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 3.

[0040] In an embodiment of the recombinant NDV vector of the invention, either:(i) the polynucleotide encoding the AIV H9-HA antigen comprises the sequence of SEQ ID NO: 1, and / or the AIV H9-HA antigen comprises the amino acid sequence of SEQ ID NO: 4; or(ii) the polynucleotide encoding the AIV H9-HA antigen comprises the sequence of SEQ ID NO: 2, and / or the AIV H9-HA antigen comprises the amino acid sequence of SEQ ID NO: 3.

[0041] In an embodiment of the recombinant NDV vector of the invention, either:(i) the polynucleotide encoding the AIV H9-HA antigen consists of the sequence of SEQ ID NO: 1, and / or the AIV H9-HA antigen consists of the amino acid sequence of SEQ ID NO: 4; or(ii) the polynucleotide encoding the AIV H9-HA antigen consists of the sequence of SEQ ID NO: 2, and / or the AIV H9-HA antigen consists of the amino acid sequence of SEQ ID NO: 3.

[0042] Thus, it will be understood that in an embodiment the present invention relates to particular H9-HA antigens, which may be defined with reference to SEQ ID NOs 1-4, that are advantageous in a NDV vector context for the reasons outlined herein.

[0043] In some embodiments, the heterologous polynucleotide encoding the avian influenza H9 antigen is from strain A / avian / Saudi Arabia / 910135 / 2006 (H9N2), and encodes a polypeptidedefined by GenBank No: ACY80655.1, which is incorporated by reference herein in its entirety. In embodiments, the H9 avian influenza polynucleotide has been computationally optimized to express a broadly reactive antigen (e.g., a COBRA H9).

[0044] In a preferred embodiment of the recombinant NDV vector of the invention, the genome of the NDV vector comprises a sequence having at least 80% identity to SEQ ID NO: 5, into which the heterologous polynucleotide encoding the AIV H9-HA antigen has been inserted. In an embodiment of this type, the genome of the NDV vector comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 5, into which the heterologous polynucleotide encoding the AIV H9-HA antigen has been inserted. Thus, in an embodiment, it will be understood that the recombinant NDV vector of the invention comprises a NDV vector genome of SEQ ID NO: 5 (or a sequence having % identity to SEQ ID NO: 5 as defined herein), that a heterologous polynucleotide encoding an AIV H9-HA antigen of the invention has been inserted into. In an embodiment, the recombinant NDV vector of the invention comprises a NDV vector genome sequence defined in relation to SEQ ID NO: 5 that is interrupted by the AIV H9-HA polynucleotide of the invention as well as any other recombinant elements included in the AIV H9-HA antigen expression cassette.

[0045] In embodiments, the backbone vector is an NDV Villegas-Glisson / University of Georgia (VG / GA) strain (U.S. Patent Nos. 5,118,502 and 8,871,220; ATCC No. VR 2239).

[0046] In a preferred embodiment of the recombinant NDV vector of the invention, the heterologous polynucleotide encoding the AIV H9-HA antigen is inserted between the P and M genes of the NDV vector genome. In a preferred embodiment, the heterologous polynucleotide encoding the AIV H9-HA antigen is inserted between the P and M genes of the NDV vector genome defined with reference to SEQ ID NO: 5.

[0047] In embodiments, the recombinant NDV vectors comprise a promoter. In embodiments, the promoter is selected from a murine cytomegalovirus (mCMV) promoter, a vaccinia virus H6 promoter, a T7 promoter and a NDV genome promoter. In embodiments, the promoter driving antigen expression is a NDV genome promoter. In embodiments, the expression of the AIV H9- HA antigen is regulated by the promoter. In a preferred embodiment, the expression of the AIV H9-HA antigen is regulated by the NDV genome promoter. In a preferred embodiment, the heterologous polynucleotide encoding the AIV H9-HA antigen is inserted between the P and M genes and the expression of the AIV H9-HA antigen is regulated by the NDV genome promoter.In a preferred embodiment, the heterologous polynucleotide encoding the AIV H9-HA antigen is inserted between the P and M genes of the NDV vector genome of SEQ ID NO: 5 (or a sequence having % identity to SEQ ID NO: 5 as defined herein) and the expression of the AIV H9-HA antigen is regulated by the NDV genome promoter.

[0048] In embodiments, the transcription of the entire NDV genome is under the control of the NDV genome promoter. Each gene in the NDV genome is flanked by transcription start and transcription stop sequences. Therefore, the inserted heterologous polynucleotide encoding the AIV antigen is also flanked by virus- specific transcription start and transcription stop sequences. It should thus be understood in this context that it is the genomic RNA of the NDV virus that is under the control of the NDV genome promoter and it does not mean that the heterologous polynucleotide encoding the AIV antigen is flanked by the NDV genome promoter. Alternatively, a recombinant promoter, e.g. a heterologous non-NDV promoter, may be used to control the expression of the polynucleotide encoding the AIV antigen. In some such alternative embodiments, the recombinant promoter is located 5’ of, such as directly 5’ of, the polynucleotide encoding the AIV antigen.

[0049] In embodiments, the recombinant vectors comprise a polyadenylation (poly A) signal. In embodiments, the polyA signal is a simian virus 40 (SV40) polyA tail. In embodiments, expression of the avian influenza H9 antigen is influenced by the polyA signal. In embodiments, the polyA signal is located downstream of the heterologous polynucleotide encoding the avian influenza H9 antigen.

[0050] The recombinant NDV vector of the invention is useful as a vaccine against both NDV and AIV, which can be administered to an avian to induce heterologous protection against NDV and AIV. In an embodiment of the recombinant NDV vector of the invention, the recombinant NDV vector is capable of inducing a protective immune response against NDV. In an embodiment, the recombinant NDV vector is capable of inducing a protective immune response against AIV. In an embodiment, the recombinant NDV vector is capable of inducing a protective immune response against NDV and AIV. In an embodiment, the protective immune response comprises a reduction of clinical signs caused by AIV. In an embodiment, the protective immune response comprises a reduction of clinical signs caused by NDV. In an embodiment, the clinical signs are respiratory signs. In an embodiment, the protective immune response comprises reducing shedding caused by AIV in an avian. In an embodiment, the recombinant NDV vector of the invention is capable ofreducing shedding caused by AIV in an avian. In an embodiment, the recombinant NDV vector of the invention is capable of reducing shedding caused by AIV in an avian following AIV infection or challenge. In an embodiment, the protective immune response comprises reducing clinical signs caused by NDV and / or AIV as well as reducing shedding caused by AIV. In an embodiment, the reduction is relative to the clinical signs of an equivalent unvaccinated avian, such as an avian that has not received an NDV and / or AIV vaccine and has been infected or challenged with NDV and / or AIV.

[0051] In an embodiment of the recombinant NDV vector of the invention, one dose of the recombinant NDV vector is capable of inducing a protective immune response against AIV and / or NDV. In an embodiment, this means that a protective immune response against AIV and / or NDV will be induced in an avian that has received (only) a single administration of the recombinant NDV vector of the invention. In an embodiment, this means that a protective immune response against AIV and / or NDV will be induced in an avian that has received (only) a single administration of any AIV and / or NDV vaccine or antigen, wherein the single administration is of the recombinant NDV vector of the invention. Thus, it will be understood that in an embodiment one dose alone of the recombinant vector is capable of inducing a protective immune response against AIV and / or NDV. In an embodiment, it will be understood that the protective immune response is induced when (only) a single dose of the recombinant NDV vector of the invention has been administered. In an embodiment, “one dose” means that a second or further dose of the recombinant NDV vector of the invention is not necessary for the induction of the protective immune response. In an embodiment, “one dose” means that a second or further dose of any NDV and / or AIV vaccine or antigen is not necessary for the induction of the protective immune response. In an embodiment “one dose” means that the avian has not been primed against the same avian pathogen(s) to which the invention relates. For example, the avian has not been primed with the same vaccine, vector or antigen to which the invention relates, or any other type of antigen (e.g. killed vaccine, subunit vaccine, etc). In an embodiment, the one dose is about 6.0 loglO EID50.

[0052] In an embodiment of the recombinant NDV vector of the invention, the recombinant NDV vector is capable of inducing a protective immune response against AIV and / or NDV when administered to an avian of six or fewer days of age, five or fewer days of age, four or fewer days of age, three or fewer days of age, two or fewer days of age, one or fewer day of age, or one dayof age. In an embodiment, the recombinant NDV vector of the invention will be capable of inducing a protective immune response against AIV and / or NDV even when administered to an avian of one day of age.

[0053] In a second aspect, the present invention provides one or more nucleic acid molecules comprising the recombinant NDV vector of the invention.

[0054] In a third aspect, the present invention provides a cell comprising the recombinant NDV vector or the nucleic acid molecules of the invention.Compositions

[0055] In a fourth aspect, the present invention provides a composition comprising the recombinant NDV vector, the nucleic acid molecules or the cell of the invention.

[0056] The present invention also provides immunogenic compositions and vaccines comprising any of the recombinant vectors described herein. In embodiments, the recombinant viral vectors of the present invention can be used in immunogenic compositions and vaccines to elicit an immune response against, and / or provide avians with protection against avian influenza, and / or Newcastle disease.

[0057] In some embodiments, the immunogenic compositions are effective to elicit an immune response in an avian, when administered to the avian. In some embodiments, the immunogenic compositions are effective to induce an immune response in an avian, when administered to the avian. In some embodiments, the immunogenic compositions are effective to stimulate an immune response in an avian, when administered to the avian. In embodiments, the immunogenic compositions of the present invention are formulated such that they are safe and effective to elicit immunity against avian influenza, and / or Newcastle disease when administered to an avian.

[0058] In some embodiments, the vaccines are effective to elicit a protective immune response in an avian, when administered to the avian. In some embodiments, the vaccines are effective to induce a protective immune response in an avian, when administered to the avian. In some embodiments, the vaccines are effective to stimulate a protective immune response in an avian, when administered to the avian. In embodiments, the vaccines of the present invention are formulated such that they are safe and effective to elicit protective immunity against avian influenza, and / or Newcastle disease when administered to an avian.

[0059] In embodiments, the immunogenic compositions and vaccines comprise a pharmaceutically or veterinarily acceptable carrier, excipient, and / or adjuvant. Examples ofsuitable pharmaceutically or veterinarily acceptable carriers include 0.9% NaCl (e.g., saline) solution, phosphate buffer, poly-(L-glutamate), lactated Ringer’s injection diluent (sodium chloride, sodium lactate, potassium chloride and calcium chloride), and polyvinylpyrrolidone. Examples of pharmaceutically or veterinarily acceptable adjuvants include, (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units, (3) an oil in water emulsion, (4) cation lipids containing a quaternary ammonium salt, e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, and / or (7) saponin.

[0060] In embodiments, the immunogenic compositions and vaccines are formulated for administration to an avian. In embodiments, the immunogenic compositions and vaccines are formulated for administration by one or more of the following routes: aerosol (spray), ocular, nasal, oculonasal, oral, subcutaneous injection, and / or intramuscular injection.

[0061] In embodiments, the immunogenic compositions and vaccines can contain a titer of recombinant vector from 102to 103, 103to 104, 104to 105, 105to 106, 106to 107, 107to 108, 108to 109, 109to 1010, 103to 108, 104to 108, or 105to 107, per dose. The recombinant vector may be titrated based on any virus titration methods including, but not limited to, FFA (Focus Forming Assay) or FFU (Focus Forming Unit), TCIDso (50% Tissue Culture Infective Dose), EIDso (50% Egg Infective Dose), PFU (Plaque Forming Units), and FAIDso (50% Fluorescent Antibody Infectious Dose). In embodiments, the dose volumes can be between 0.01 and 10 ml, between 0.01 and 5 ml, between 0.01 and 1 ml, or 0.01 and 0.5 ml.Methods of use

[0062] In an embodiment, it is understood that the recombinant NDV vector of the present invention surprisingly induces a protective immune response against AIV due to the presence of an AIV H9-HA gene / antigen of the invention as defined herein (as sequences consisting of, comprising or having % identity to SEQ ID NOs 1 to 4). In an embodiment, it is understood that the recombinant NDV vector of the present invention will induce a protective immune response against NDV due to the presence of the NDV vector genome (e.g. the backbone of the recombinant NDV vector of the invention), which is preferably SEQ ID NO: 5 (or a sequence with % identity to SEQ ID NO: 5 as defined herein).

[0063] In a fifth aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in a method of inducinga protective immune response against AIV in an avian. Alternatively or additionally in a fifth aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in a method of inducing a protective immune response against NDV in an avian.

[0064] In a sixth aspect, the present invention provides a method of inducing a protective immune response against AIV in an avian, comprising administering the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian. Alternatively or additionally in a sixth aspect, the present invention provides a method of inducing a protective immune response against NDV in an avian, comprising administering the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian.

[0065] In a seventh aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in the manufacture of a medicament for inducing a protective immune response against AIV in an avian. Alternatively or additionally in a seventh aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in the manufacture of a medicament for inducing a protective immune response against NDV in an avian.

[0066] In an embodiment, inducing a protective immune response comprises reducing clinical signs associated with infection or challenge by AIV. In an embodiment, inducing a protective immune response comprises reducing clinical signs associated with infection or challenge by NDV. In an embodiment, the clinical signs are respiratory clinical signs.

[0067] In an eighth aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in a method of reducing AIV shedding in an avian.

[0068] In a ninth aspect, the present invention provides a method of reducing AIV shedding in an avian, comprising administering the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian.

[0069] In a tenth aspect, the present invention provides the recombinant NDV vector, the nucleic acid molecules, the cell or the composition of the invention, for use in the manufacture of a medicament for reducing AIV shedding in an avian.

[0070] In an embodiment of the medical uses / methods of the present invention, the recombinant NDV vector is administered once. In an embodiment, this means that the recombinantNDV vector is only administered once. In an embodiment, this means that second / further administrations of the recombinant NDV vector are not carried out. In an embodiment, the recombinant NDV vector is administered once and the protective immune response is obtained after such single administration. In another embodiment, this means that the initial administration of the recombinant NDV vector only comprises one dose to induce a protective immune response, but the administration of one or more booster administrations comprising further doses at a substantially later date are not excluded. In an embodiment, the one dose is about 6.0 logl 0 EID50.

[0071] In an embodiment of the medical uses / methods of the present invention, the recombinant NDV vector is administered to an avian at six or fewer days of age, five or fewer days of age four or fewer days of age, three or fewer days of age, two or fewer days of age, or one or fewer days of age, preferably to an avian of one day of age. In an embodiment, the recombinant NDV vector of the invention is administered to an avian of one day of age and induces a protective immune response against AIV. Alternatively or additionally in an embodiment, the recombinant NDV vector of the invention is administered to an avian of one day of age and induces a protective immune response against NDV.

[0072] In a preferred embodiment of the medical uses / methods of the present invention, the recombinant NDV vector is administered by the oculo-nasal route.

[0073] The present invention also provides methods of immunizing, methods for eliciting an immune response and methods for eliciting a protective immune response in an avian using any of the recombinant vectors, immunogenic compositions, and / or vaccines described herein. In embodiments, the avian is a chicken.

[0074] In embodiments, the methods comprise administering to an avianl a recombinant vector according to the present invention.

[0075] In embodiments, the methods comprise administering to an avian an immunogenic composition according to the present invention. In embodiments, the methods are effective to elicit, induce, and / or stimulate an immune response against avian influenza and / or Newcastle disease in an avian.

[0076] In embodiments, the methods comprise administering to an avian a vaccine comprising an effective amount of a recombinant vector according to the present invention. In embodiments, the avian is vaccinated / immunized against avian influenza and / or Newcastle disease. In embodiments, the methods are effective to elicit, induce, and / or stimulate a protective immuneresponse against avian influenza and / or Newcastle disease in an avian, and thereby reduce and / or prevent clinical signs associated with subsequent avian influenza and / or Newcastle disease exposure, infection, challenge and / or disease in the avian, relative to a non-vaccinated control avian of the same species. In embodiments, the protective immune response is effective to provide the avian with protection against subsequent avian influenza virus and / or Newcastle disease virus infection or challenge, and clinical disease and signs associated therewith.

[0077] In embodiments, the recombinant vectors, immunogenic compositions, and / or vaccines may be administered in ovo 1 to 4 days before hatching. In embodiments, the recombinant vectors, immunogenic compositions, and / or vaccines may be administered to a 1 day old, 2 day old, 3 day old, 4 day old, 5 day old, 6 day old, 7 day old, 8 day old, 9 day old, 10 day old, 11 day old, 12 day old, 13 day old, 14 day old, 15 day old, 16 day old, 17 day old, 18 day old, 19 day old, 20 day old, or 21 day old chicken. A variety of administration routes may be used such as aerosol (spray), ocular, nasal, oculonasal, oral, subcutaneous injection, and intramuscular injection. In an embodiment, the administration routes are used in 1 day old chicks.

[0078] In an embodiment, the avians that the recombinant NDV vectors of the invention are administered to are chickens. In an embodiment, the avians are SPF chickens. In an embodiment, the avians are 1 day old SPF chickens.Co-administration

[0079] In certain embodiments, the recombinant NDV vectors of the invention may be coadministered to an avian together with one or more additional avian vaccines.

[0080] In one embodiment, the additional avian vaccine provides protection against another avian pathogen, for example, infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV) or avian influenza subtype H5 (AIV H5).

[0081] In one embodiment, the additional avian vaccine is a HVT-vectored vaccine. In one embodiment, the HVT-vectored vaccine comprises a heterologous polynucleotide encoding an IBDV antigen, such as an IBDV VP2 antigen. In one embodiment, the HVT-vectored vaccine comprises a heterologous polynucleotide encoding an ILTV antigen, such as an ILTV glycoprotein D (gD) antigen. In one embodiment, the HVT-vectored vaccine comprises a heterologous polynucleotide encoding an avian influenza H5 hemagglutinin (AIV H5-HA) antigen.

[0082] In one embodiment, the co-administered HVT-vectored vaccine is one sold under the trade name Vaxxitek®. In one embodiment, the co-administered HVT-vectored vaccine comprisesa heterologous polynucleotide encoding an IBDV VP2 antigen and a heterologous polynucleotide encoding an ILTV gD antigen, for example as described in WO2018112501 Al, the contents of which are incorporated herein in its entirety. In one embodiment, the co-administered HVT- vectored vaccine comprises a heterologous polynucleotide encoding an IBDV VP2 antigen and a heterologous polynucleotide encoding an AIV H5-HA antigen, for example as described in WO2021257706A1, the contents of which are incorporated herein in its entirety.

[0083] In one embodiment, the co-administered vaccines may be given concurrently or simultaneously, for example in the same or separate doses. In another embodiment, the coadministered vaccines may be given at different timings according to a given schedule or regime.Methods of manufacture

[0084] In an eleventh aspect, the present invention provides a method of manufacturing the recombinant NDV vector of the invention, wherein the method comprises: a) providing one or more nucleic acids encoding a NDV vector; b) providing a polynucleotide encoding an AIV H9-HA antigen; and c) recombinantly combining the nucleic acids encoding a NDV vector and the polynucleotide encoding an AIV H9-HA antigen.

[0085] In an embodiment, the polynucleotide encoding an AIV H9-HA antigen is a polynucleotide of the invention as defined herein as any of SEQ ID NOs: 1 to 4 (or a sequence with % identity to any of SEQ ID NOs: 1 to 4 as defined herein). In an embodiment, the one or more nucleic acids encoding a NDV vector encode the NDV vector of SEQ ID NO: 5 (or a sequence with % identity to SEQ ID NO: 5 as defined herein).

[0086] In an embodiment, the method of manufacture further comprises step d) generating recombinant NDV particles from the recombinant nucleic acid obtained in step c). In an embodiment, the NDV particles are infectious NDV particles. In an embodiment, step d) of the method of manufacture involves a reverse genetics system for NDV. In an embodiment, step d) of the method of manufacture involves transcribing the recombinant nucleic acid obtained in step c). In an embodiment, the recombinant nucleic acid is comprised in a transcription plasmid. In an embodiment, the recombinant nucleic acid is transcribable / transcribed from a T7 promoter (i.e. a promoter recognised by T7 RNA polymerase). In an embodiment, step d) of the method of manufacture comprises expressing one or more nucleic acids encoding the NDV N, P and L proteins. For the avoidance of doubt, in an embodiment, the method steps a), b), c) (and d)) aretypically in this order (step a) followed by step b) followed by step c) followed by step d) when present).

[0087] The present invention also provides methods of making recombinant NDV vectors comprising an avian influenza H9 polynucleotide that encodes a hemagglutinin antigen.

[0088] In embodiments, the methods comprise inserting an avian influenza H9 polynucleotide into a NDV backbone vector. In embodiments, the H9 polynucleotide is from strain A / avian / Saudi Arabia / 910135 / 2006 (H9N2). In embodiments, the H9 avian influenza polynucleotide has been computationally optimized to express a broadly reactive antigen (e.g., a COBRA H9).

[0089] In embodiments, the methods further comprise inserting a promoter into the backbone vector. In embodiments, the heterologous polynucleotide encoding the avian influenza H9 antigen is operably linked to a promoter, and expression of the H9 antigen is regulated by the promoter.

[0090] In embodiments, the methods further comprise inserting a polyadenylation (poly A) signal into the backbone vector. In embodiments, the polyA signal is a simian virus 40 (SV40) polyA tail. In embodiments, the polyA signal is inserted downstream of the H9 polynucleotide.General definitions

[0091] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a”, “an”, and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0092] It is noted that this disclosure may contain the terms “comprising,” “consisting essentially of,” and “consisting of,” and variants thereof, and these terms have the meaning attributed to them in U.S. patent law. It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting essentially of’ and / or “consisting of’ are also provided.

[0093] As used herein, the term "about" means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50 means in the range of 45-55. Numerical ranges recited hereinby endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5).

[0094] As used herein, an “adjuvant” is a substance that is able to favor or amplify the cascade of immunological events, ultimately leading to a better immunological response (e.g., the integrated bodily response to an antigen). An adjuvant is in general not required for the immunological response to occur, but favors or amplifies this response.

[0095] As used herein, the terms “antigen” or “immunogen” mean a substance that induces a specific immune response in a host animal (e.g., an immune response of the humoral and / or cellular type directed against the antigen). The antigen may be a whole organism, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, and the like.

[0096] As used herein, the term “avian” includes, for example, chicken, chick, broiler, capon, duck, goose, turkey, grouse, quail, swan, squab, pigeon, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary. The term “avian” also includes an individual avian in all stages of development, including embryonic and fetal stages. In a preferred embodiment, an avian is a chicken.

[0097] As used herein, the term “carrier” refers to a solvent or diluent in which a recombinant vector is formulated and / or administered. Pharmaceutically and veterinarily acceptable carriers can be sterile liquids such as water and / or oils. Examples of suitable oil-based carriers can include petroleum oils, animal oils, vegetable oils, and oils of synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Examples of suitable aqueous carriers can include water and aqueous solutions (e.g. aqueous saline solution, aqueous dextrose solution, glycerol solution, etc.).

[0098] As used herein, the term “gene” is used broadly to refer to any segment of polynucleotide associated with a biological function.

[0099] As used herein, the term “genome” refers to the heritable genetic information of a host organism. The genome contemplated in the present invention can refer to the DNA or RNA of a virus or pathogenic organism. The RNA may be a positive strand or a negative strand RNA.

[0100] As used herein, the term “heterologous” means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a differentsource, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.

[0101] As used herein, the terms “identity” and “sequence identity” refer to a relationship between two or more sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides / amino acids are identical. The total number of such position identities is then divided by the total number of nucleotides / amino acids in the shorter of the two sequences to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G, Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are also codified in publicly available computer programs which determine sequence identity between given sequences.

[0102] As used herein, the term “immunogenic composition” refers to a composition that comprises at least one antigen which elicits an immunological response in a host to which the immunogenic composition is administered.

[0103] As used herein, a “protective immune response” comprises an “immunological response” to a composition or vaccine which is the development in the host of a cellular and / or antibody-mediated immune response to a composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically toan antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display a protective or therapeutic immunological response such that resistance to new infection will be enhanced and / or the clinical severity of subsequent disease reduced. Such protection can be demonstrated by a reduction in clinical disease signs relative to those normally displayed by an infected host, a lack of clinical disease signs relative to those normally displayed by an infected host, a quicker recovery time relative to that normally displayed by an infected host, and / or a lowered pathogen count relative to that normally found in an infected host.

[0104] As used herein, the terms “nucleic acid” and “polynucleotide” refer to RNA, DNA, and derivatives thereof.

[0105] As used herein, the terms “pharmaceutically acceptable” and “veterinarily acceptable” are used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical or veterinary product. When it is used, for example, to describe an excipient in a pharmaceutical or veterinary vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient.

[0106] As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of consecutive amino acid residues.

[0107] As used herein, the term “protection” does not require complete protection from any indication of infection. For example, protection can mean that, after challenge, clinical signs of the underlying infection are at least reduced, and / or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the clinical signs are reduced and / or eliminated. It is understood that reduced, as used in this context, means relative to the state of the infection, including the molecular state of the infection, not just the physiological state of the infection.

[0108] As used herein, the term “recombinant” in the context of a polynucleotide or protein means a polynucleotide or protein with a semisynthetic or synthetic origin which either does not occur in nature or is linked to another a polynucleotide or protein in an arrangement not found in nature. A recombinant polynucleotide or protein can be made by modifying, altering, or engineering a polynucleotide or protein from its native form or structure to a non-native form or structure. The modification, alteration or engineering of a polynucleotide or protein may include, for example, the deletion of one or more nucleotides or amino acids, the substitution of one or more nucleotides or amino acids, and / or the insertion of one or more nucleotides or amino acids.

[0109] As used herein, a “vaccine” is an immunogenic composition that is suitable for administration to an avian which, upon administration to the avian, induces an immune response strong enough to aid in the protection from a clinical disease arising from an infection with a wildtype pathogenic micro-organism (e.g., strong enough for aiding in the curing of, ameliorating of, protection against, and / or prevention of a clinical disease and / or clinical signs associated therewith).

[0110] The invention will now be further described by way of the following non-limiting examples.EXAMPLESEXAMPLE 1: Construction of vAVW48 expressing AIV H9

[0111] This Example details the construction and characterization of a Newcastle Disease Virus (NDV) vector expressing avian influenza virus H9 gene from an H9N2 Saudi Arabia strain (vAVW48).A. Objective

[0112] A plasmid harboring the whole genome of NDV VG / GA Avinew strain (GenBank KC906188) was generated that has been modified to allow insertion of the avian influenza virus H9 gene which comes from an H9N2 Saudi Arabia strain.B. NDV genome and insert genes characteristics

[0113] Vector: NDV transcription plasmid pIV29 (NDV AVINEW vaccine strain).

[0114] Insert: H9 gene from Influenza A virus (A / avian / Saudi Arabia / 910135 / 2006(H9N2)) strain (GenBank: ACY80655.1). The H9 gene has a sequence according to SEQ ID NO: 1 and encodes a polypeptide sequence according to SEQ ID NO: 4.

[0115] The H9 Saudi Arabia gene was synthesized and sub cloned in the pIV37 by GeneArt (no. 1166890) by Asci and Pmel restriction sites.

[0116] C. Construction of transcription plasmid

[0117] Plasmid name: pFR 128.

[0118] Promoter: T7.

[0119] Polyadenylation signal: none.

[0120] Resistance gene: Ampicillin. 1

[0121] Insertion sites in pIV29 plasmid: Pacl / Fsel.

[0122] Host cells: Escherichia coli KI 2 strain NEB 10-beta (NEB, France).

[0123] Plasmid Map: A map of plasmid pFR 128 is shown in FIG. 1.1). Generation of a NDV Avinew vector expressing an avian influenza virus H9 gene from an H9N2 Saudi Arabia strain using reverse genetics methodology (vAVW48)

[0124] The NDV is a negative RNA virus and the generation of genetically modified NDV virus can be accomplished with a reverse genetics system. The transcription of a full length genomic viral RNA and the simultaneous expression of NP, P and L proteins permit the assembly of RNP and the transcription of positive RNA into negative RNA genome. It initiates the normal replication cycle of NDV virus and permit the generation of infectious particles. To generate modified NDV particles, the following reagents and conditions were used:

[0125] Transcription plasmid pFR128: The plasmid map is shown in FIG. 1. The amount transfected was 2 pg.

[0126] Expression NP plasmid pIV32: The amount transfected was 1 pg.

[0127] Expression P plasmid pIV33: The amount transfected was 0.5 pg.

[0128] Expression L plasmid pIV34: The amount transfected was 0.5 pg.

[0129] T7 RNA polymerase plasmid pNS151: The amount transfected was 1 pg.

[0130] Cells and transfection: (a) CHO cells, 0.5 x 106cells / well (6-well plate); (b) Medium used : MEM glutamax + 5% SVF (GIBCO); (c) Confluency: subconfluent; (d) Transfection Medium: MEM (Gibco); (e) Transfecting agent: Lipofectamine LTX plus reagent (Invitrogen); (f) Incubation medium: Opti MEM medium (Gibco) without SVF and supplemented with TPCK trypsin (Worthington) at 0.5pg / ml and 1 pg / m (lot : X5H8120) note: this medium is used to replace the transfection mix ; (g) Incubation time: 96h; (h) Harvest: clarified supernatant.

[0131] Egg passages: 10-days embryonated SPF eggs. Egg passage 2 had a titer of 8.7 loglO EID50 / ml.E. Characterization

[0132] Expression of the inserted gene: (a) Cells: CHO KI cells; (b) MOI: 1 and 10 for IFI technique. 10 and 100 for Western Blot technique; (c) Incubation time: 48h; (d) Antibody: chicken sera H9N2 000919; (e) Ascites anti-NDV HN antibody SOU4D6D (only for IFI); (f) Techniques: IFI, Western Blot.

[0133] Sequence of the inserted gene: (a) RNA extraction kit: QiAmpViral RNA QIAGEN ref52904; (b) RT-PCR kit: One step RT-PCR QIAGEN ref 210210; (c) Primers: oFR09 and oFRIO; (d) Results: H9 gene sequence is validated.

[0134] Expression results can be seen in FIG. 2.EXAMPLE 2: Construction of vAVW68 expressing AIV H9

[0135] This Example details the construction and characterization of a Newcastle Disease Virus (NDV) vector expressing avian influenza virus H9 COBRA gene (vAVW68).A. Objective

[0136] A plasmid harboring the modified Avinew NDV genome was generated in which was inserted the avian influenza virus H9 COBRA gene.B. NDV genome and insert genes characteristics

[0137] Vector: NDV transcription plasmid pIV29 (a NDV AVINEW vaccine strain)

[0138] Insert: synthetic, H9 COBRA gene (Computationally Optimized Broadly Reactive Antigen) The H9 COBRA gene was synthesized by Eurofin MWG (no. 11104239917) and sub cloned in the pIV37 by Asci and Pmel restriction sites. The H9 COBRA gene has the nucleotide sequence according to SEQ ID NO: 2 and encodes a protein having the amino acid sequence according to SEQ ID NO: 3.C. Construction of transcription plasmid

[0139] Plasmid name: pFR211.

[0140] Promoter: T7.

[0141] Polyadenylation signal: none.

[0142] Resistance gene: Ampicillin.

[0143] Insertion sites in pIV29 plasmid: Pacl / Fsel.

[0144] Host cells: Escherichia coli KI 2 strain NEB 10-beta (NEB, France).

[0145] Plasmid Map: A map of plasmid pFR211 is shown in FIG. 3. FIG. 4 shows a diagram for constructing the transcription plasmid pFR211.D. Generation of a NDV Avinew vector expressing an avian influenza virus H9 COBRA GENE using reverse genetics methodology (vAVW68)

[0146] The NDV is a negative RNA virus and the generation of genetically modified NDV virus can be accomplished with a reverse genetics system. The transcription of a full lengthgenomic viral RNA and the simultaneous expression of NP, P and L proteins permit the assembly of RNP and the transcription of positive RNA into negative RNA genome. It initiates the normal replication cycle of NDV virus and permit the generation of infectious particles. To generate modified NDV particles, the following reagents and conditions were used:

[0147] Transcription plasmid pFR211 : The plasmid map is shown in FIG. 3. The amount transfected was 5.6 pg.

[0148] Expression NP plasmid pIV32: The amount transfected was 2.8 pg.

[0149] Expression P plasmid pIV33: The amount transfected was 1.4 pg.

[0150] Expression L plasmid pIV34: The amount transfected was 1.4 pg.

[0151] T7 RNA polymerase plasmid pNS151: The amount transfected was 2.8 pg.

[0152] Cells and transfection: (a) CHO cells, 1.0 x 106cells / well (6- well plate); (b) Medium used : MEM glutamax + 5% SVF (GIBCO); (c) Confluency: subconfluent; (d) Transfection Medium: MEM (Gibco); (e) Transfecting agent: Lipofectamine LTX plus reagent (Invitrogen); (f) Incubation medium: Opti MEM medium (Gibco) without SVF and supplemented with TPCK trypsin (Worthington) at 0.5 pg / ml and 1 pg / m. Note: this medium is used to replace the transfection mix ; (g) Incubation time: 72h; (h) Harvest: clarified supernatant.

[0153] Egg passages: 10-days embryonated SPF eggs. Egg passage 2 had a titer of 8.44 loglO EID50 / ml.E. Characterization

[0154] Expression of the inserted gene: (a) Cells: CHO KI cells; (b) MOI: 0.1 and 1; (c) Incubation time: 72h; (d) Antibody: chicken sera H9N2 000919; (e) Ascites anti-NDV HN antibody SOU4D6D; (f) Techniques: IFI.

[0155] Expression results can be seen in FIG. 5.EXAMPLE 3: AIV challenge study

[0156] This Example details the development and validation of an Avian Influenza H9N2 (Al H9N2) challenge model with Saudi Arabia 2010 strain administered by nebulization in three weeks-old SPF chickens and evaluation of the protective effect of vAVW48.A. Objectives, design of the study, vaccines and vaccination

[0157] The objective of the study was to develop an H9N2 challenge model and evaluate the protective effect of a NDV vector candidate (vAW48) in SPF chicks. The design of the experiment is shown in table 1 and in FIG. 7.

[0158] One-day-old chicks were used in the study.Table 1 : definition of groups.Status of Groups Number of DO D22 animals animals on DO Treatment ChallengeGia 20 Strain vAVW48 AI H9N2SPF Gib 10 / AI H9N2Glc 10 / Placebo

[0159] The vAVW48 is a NDV vector expressing HA of A / avian / Saudi Arabia / 910135 / 2006. It was diluted in mineral water and administered by oculo-nasal route at a dose of 6.0 loglO EID50 / bird.

[0160] Birds were observed daily for clinical signs between DO and D22.B. Serology

[0161] Blood samples were taken from all birds of Gia and Glc at DI 9. HI titers were evaluated against avian influenza H9N2 with A / chicken / Saudi Arabia / WNB9510 / 2010 (Arabia) and A / chicken / Irak / AVI 342 / 2011 (Irak) antigens. Anti-NDV HI titers were also evaluated using La Sota strain as antigen.

[0162] Results are shown in Table 2. The vAVW48 candidate induced H9N2 antibodies against both tested antigens as well as anti-NDV HI titers.Table 2: Mean (± standard deviation) H9N2 and NDV HI titers (in loglO) induced by vAVW48 in SPF broiler chicks at D19. Negative threshold: titer < 4 HI unit (0.6 loglO).C. H9N2 challenge and clinical protection

[0163] At D22, all chicks were challenged with the A / chicken / Saudi Arabia / WNB9510 / 2010 H9N2 strain, also referred to herein as Saudi Arabia 2010. The diluent for the challenge strain was physiological buffered saline (PBS) pH: 7.1. This H9N2 challenge strain was thawed and diluted in PBS to obtain a challenge suspension with a titer of 9.0 loglO DIO50 / ml (being 8.54 loglO DI050 / 0.35 ml). All birds were challenged with 0.35 ml of the challenge solution by nebulization. In details, 10 animals from a same group were gathered in a nebulization box (plastic box, size [L50 / 139.5 / h33 cm]) and nebulized for about 20 minutes with a 347-CN003. nebulizer.

[0164] Animals were kept in closed contact in the nebulization box for about 3 to 5 minutes and then, they were moved back to their isolators.

[0165] Chickens of all groups were daily monitored from D22 (before challenge) until the end of the study (which was D32). For these monitoring, particular attentions were paid to respiratory and ocular symptoms. All other observed clinical signs were recorded as well. Respiratory and ocular symptoms were scored as detailed in Table 3.Table 3: Scoring of respiratory and ocular signs after H9N2 challenge.Observation NotedNothing to report 0Respiratory Slight: slight rales, sneeze 1 signs Moderate: severe rales, coughing 2Severe: gasp, respiratory distress 3Nothing to report 0Slight: conjunctivitis with eyelid oedema and excessive tearing 1Ocular Moderate: conjunctivitis with eyelid oedema and excessive tearing + periorbital2 signs swellingSevere: conjunctivitis with eyelid oedema and excessive tearing + periorbital 3 swelling + pasty yellow exudates

[0166] A summary of clinical signs and results is provided in Table 4 below.

[0167] Respiratory clinical signs were observed in 100% of unvaccinated challenged birds.The vAVW48 vaccine decreased frequency and average severity of clinical signs.Table 4: summary of clinical signs and mortality after challenge.D. H9N2 shedding

[0168] On D25, D27, and D29 (3, 5, and 7 days post challenge), an oro-pharyngeal swabbing was carried out on 10 chickens per group. The swabs were shipped to a laboratory for detection of the oro-pharyngeal H9N2 excretion by qRT-PCR.

[0169] FIG. 8 shows viral shedding results for chickens. QRT-PCR titers were significantly different between Gia and Gib on D27 and D29 (ANOVA test p=0.008 and Kruskal- Wallis test p<0.001 respectively). The treatment with vAVW48 allowed a quicker decrease of the viral shedding. These results are in accordance with clinical observations.E. Conclusion

[0170] The Al H9N2 challenge model was fully validated in SPF layer-type chickens, with effect on specific morbidity.

[0171] vAVW48 recombinant administered at a dose of 6.0 loglO EID50 / bird by the oculonasal route at one day of age reduced frequency and average severity of clinical signs after challenge, protected against the effect of the challenge on the growth in SPF birds and also allowed a quicker decrease of the shedding of the challenge virus.EXAMPLE 4: AIV challenge study with AIV H9 COBRA

[0172] This Example details the efficacy assessment of two recombinant NDV candidate vaccines expressing the H9 of Avian Influenza H9N2, administered to one-day-old SPF chickens against an Avian Influenza H9N2 (Saudi Arabia 2010) virulent challenge carried out 21 days after vaccination.A. Objectives, design of the study, vaccines and vaccination

[0173] The objective of this study was to assess the efficacy of two recombinant NDV candidate vaccines expressing the H9 of Avian Influenza H9N2, when administered to SPF chicksat one day of age, against a H9N2 virulent challenge (strain Saudi Arabia 2010) carried out 21 days post- vaccination. To reach this objective, the following parameters were monitored after challenge: (i) respiratory and ocular symptoms (clinical monitoring); and (ii) viral excretion.

[0174] Vector vaccine candidates used in the study: the NDV vector vAVW48 (NDV-H9) was expressing the HA gene of the A / avian / Saudi Arabia / 910135 / 2006 strain and the NDV vector vAVW68 (NDV-H9(COBRA)) was expressing a consensus (COBRA) HA gene. The vaccine candidates are described in Example 1 and Example 2. The NDV vector vaccines were diluted in mineral water.

[0175] At DO, 12 one-day-old SPF (white leghorn) chicks per group were vaccinated with the dose shown in Table 5. The NDV vector candidates were administered by the oculo-nasal route.

[0176] Chicks were observed every day in the course of daily care and maintenance from DO to D21.Table 5: Group constitution and treatment.B. Serology

[0177] Blood samples were taken from all birds at D20 for serology. Sera were analyzed for titration of anti-H9 antibodies (antigen IRAK), using a hemagglutination-inhibition (HI) technique, according to standard operating procedure (based on e.g. Kaufmann, L etal., JoVE 130: e55833 (2017)). Four different H9N2 isolates were used as antigen in the HI test: A / chicken / Irak / AV1342 / 2011 (Irak), A / chicken / Saudi Arabia / WNB9510 / 2010 (Arabia), A / av. / Azerbaidjan / l lvirl344-l / 2011 (Azerbaidjan) and A / Ck / Jordan / 436- 1 / 2010 (Jordan).

[0178] Results are shown at FIG. 9 (Mean HI titers in loglO induced by vAVW68 (NDV- H9(COBRA)) and vAVW048 (NDV-H9) against H9N2 Irak, Arabia, Azerbaidjan and Jordan antigens). HI titers against Arabia and Azerbaijani antigens were slightly higher than those against Irak or Jordan antigens. There were no differences between HI titers induced by NDV-H9 and those induced by NDV-H9(COBRA).

[0179] These results indicate that the tested vaccine candidates induced significant H9N2 HI titers 3 weeks post-vaccination.C. H9N2 challenge and clinical protection

[0180] At D21, all chicks were challenged with the A / chicken / Saudi Arabia / WNB9510 / 2010 H9N2 strain, also referred to herein as Saudi Arabia 2010. The diluent for the challenge strain was physiological buffered saline (PBS) pH: 7.1. This H9N2 challenge strain was thawed and diluted in PBS to obtain a challenge suspension with a titer of 9.0 loglO DIO50 / ml (being 8.54 loglO DI050 / 0.35 ml). All birds were challenged with 0.35 ml of the challenge solution by nebulization. In details, animals from the same group were gathered in a nebulization box and nebulized for about 20 minutes. Animals were kept in closed contact in the nebulization box for about 3 to 5 minutes and then they were moved back to their isolators. The clinical monitoring after challenge was the same as in the previous example.

[0181] Chickens of all groups were daily monitored from D21 (before challenge) until the end of the study (which was D31). For these monitoring, particular attentions were paid to respiratory and ocular symptoms. All other observed clinical signs were recorded as well. Respiratory and ocular symptoms were scored as detailed in Table 3 in the previous example.

[0182] Results of the clinical monitoring after challenge were determined. In addition, for each animal, a daily clinical score was calculated by adding the scores associated to respiratory and ocular symptoms for each date. For each animal, a global clinical score was calculated by adding all the daily clinical scores from D22 to D31.

[0183] Evolutions of mean daily clinical scores by groups are presented in FIG. 6. Onset of clinical signs in CTL group = 2 dpc, slight and mild respiratory signs. Lower mean daily clinical signs than CTL in groups NDV & NDV COBRA.1). H9N2 shedding

[0184] On D22, D23, D24, D26, D28, and D31 (1, 2, 3, 5, 7, and 10 days post challenge), an oro-pharyngeal swabbing was carried out on all chicks of each group. The swabs were shipped to a laboratory for detection of the oro-pharyngeal H9N2 excretion by qRT-PCR.

[0185] Detection of H9N2 in oro-pharyngeal swabs was performed using a qRT-PCR technique, according to standard operating procedure. When viral excretion was detected in a group, the area under curve (AUC) for the oro-pharyngeal H9N2 excretion from D22 to D31 was determined for each animal according to the trapezium method.

[0186] Virus- excreting groups were compared to the control group on the criterion “AUC” using a Student’s T test (assuming the homogeneity of the variances (Fisher-Snedecor’s test), because the normality of the distributions (Shapiro- Wilks’ test) were assumed.

[0187] For the analysis of viral excretions (AUC calculation), the animals found dead during the challenge period were attributed the last-observed viral excretion value for the next analyzed time points if lacking.

[0188] Results are shown in FIG. 10 (Dispersions of individual AUC of viral RNA excretion by groups - Box plots represent the mini, the lower quartile, the median, the upper quartile and the maxi).

[0189] AUC (FIG. 10) of viral RNA excretion from D22 to D28 in groups G1 and G2 were significantly lower than that of the control group:E. Conclusion

[0190] Administration of the NDV-H9 and NDV-H9 (COBRA) candidates to one-day-old SPF chicks, followed by a challenge with a virulent H9N2 (Saudi Arabia 2010) 21 days post vaccination, resulted in (1) a seroconversion (HI titers) to 4 H9N2 antigens, (2) a significant decrease of respiratory clinical signs, and (3) a significant decrease of the viral RNA excretion during D22 - D28.SEQUENCE LISTINGSEQ ID NO: 1 (H9 gene of H9N2 Saudi Arabia strain) (vAVW48) cgagcgacaaccctgtcctgcttcctctgccccactaaatgatcgcgcagctgcaatcaattcagctatattaaggattaagaaaaaatacgg gtagaatcggagtgccccgattgtgccaagggcgcgccgccaccatggaacccatcagcctgatgatcatcctgctgctggtgacaacca gcaacgccgacaagatctgcatcggccaccagagcaccaacagcaccgagacagtggacaccctgaccgagacaaacgtgcccgtga cccacgccaaagagctgctgcacaccgagcacaacggcatgctgtgcgccaccaacctgggccaccccctgatcctgaacacatgcacc atcgagggcctgatctacggcaaccccagctgcgatctgctgctgggcggacgcgagtggtcctacatcgtggaaagacccagcgccgt gaacggcacatgctaccccggcaacgtggaaaacctggaagaactgagaaccctgttcagcagcagcagctcctaccagagaatccaga tcttccccgacaccatctggaacgtgacctacaccggcaccagcaagagctgcagcgacagcttctacagaaacatgagatggctgaccc agaagaacggcctgtaccccgtgcaggacgcccagttcaccaacaacagaggcaaggacatcctgttcgtgtggggcatccaccacccc cccaccgacaccgcccagaccaacctgtacaccagaaccgacaccaccaccagcgtgaccaccgagaacctggacagaaccttcaagc ccctgatcggccccagacccctggtgaacggcctgatcggcagaatcaactactattggagcgtgctgaagcccggccagaccctgaga gtgcgcagcaacggcaacctgatcgccccttggtttggccacgtgctgagcggcgagagccacggcagaatcctgaaaaccgacctgaa cagcggcaactgcgtggtgcagtgccagaccgagaagggcggcctgaacagcaccctgcccttccacaacatcagcaaatacgccttcg gcacatgccccaagtacatcggcgtgaagtccctgaagctggccatcggcctgaggaacgtgcccgccagaagcagcagaggcctgttc ggcgctatcgccggcttcatcgagggcggctggccaggactggtggccgggtggtacggcttccagcacagcaacgaccagggcgtgg gcatggccgccgacagagacagcacccagaaagccgtggacaagatcaccagcaaagtgaacaacatcgtggacaaaatgaacaagc agtacgagatcatcgaccacgagttcagcgaggtggaaaccagactgaacatgatcaacaacaagatcgacgaccagatccaggacgtg tgggcctacaacgccgagctgctggtgctgctggaaaaccagaaaaccctggacgagcacgacgccaacgtgaacaatctgtacaacaa agtgaagagggccctgggcagcaacgccatggaagatggcaagggctgcttcgagctgtaccacaagtgcgacgaccagtgcatggaa accatccgcaacggcacctacaaccgcggcaagtacaaagaggaaagcagactggaaaggcagaaaatcgagggcgtgaagctggaa agcgagggcacatacaagatcctgaccatctacagcaccgtggccagcagcctggtgctggctatgggactggccgccttcctgttctggg ccatgagcaacggcagctgcagatgcaacatctgcatctgatgaacgtgtttaaactcaccaccgcaacccgcagcagatccctgtccacc cagcaccacacggtatctgcaccaagctcctctctgcaaatccaaggtccaacaccctSEQ ID NO: 2 (H9 COBRA nucleotide sequence) (vAVW68) atggagacaatcagcctgatgacgatactgttggttgtgacaacaagcaatgccgacaagatttgcattgggcaccagtccactaactctaca gagacagtcgatactttgaccgagacaaatgtgcccgtgacccatgccaaggaactgcttcacactgagcataacgggatgctgtgtgcaa ctaatctcggacatccgctcatactcgacacttgtaccatcgagggtcttatctatggcaatccatcctgcgatctgctgctcggaggcagagagtggagttatatcgtggaacgtccttcagctgttaatgggacctgttaccccggcaacgtggagaacctggaagagctgagaacactgttta gctcaagcagcagttaccagagaatccagatcttccccgacacaatttggaatgtgacgtacactgggacatccaaatcatgctcagatagc ttctacaggaatatgcgttggttgacccagaagaacgggctgtatcctgtccaagacgcacagtatacgaacaaccgcggtaaggatattct cttcgtgtggggaatccaccatccgccaaccgatacggcccagactaacttgtacactcgcacggatacaaccaccagtgtcactacggaa aacctcgatcgcactttcaaaccactgattggtccaaggcctttggttaacggactgattggccgtatcaattactattggagcgtactgaagc ctggccaaaccctgcgcgtgagatccaacggtaatctgatagccccttggtatggccatgttctgtctggagagtctcacggcaggatactc aagaccgacctgaactcagggaattgcgtggtccagtgccagaccgaaaagggcggcctcaactctactcttcccttccacaatatcagca aatacgccttcggaacatgccccaaatatattggcgtgaaaagcctgaagctggctattgggctgcgaaacgtcccggctagaagttctagg ggcctgtttggtgcgatagccgggttcatcgaaggaggatggccagggttggtagcaggatggtacggttttcaacatagcaatgatcagg gagttggcatggcagctgaccgggactccacccagaaagcggtcgacaaaatcacctccaaggttaacaatatagtcgacaaaatgaaca aacagtacgagataatcgaccacgaattctctgaagtggaaacacggctcaatatgatcaacaacaaaattgatgatcagatccaggatgtat gggcgtataacgctgagctgttggtgcttcttgaaaaccagaaaaccctggatgagcacgacgctaatgtgaataacctgtacaacaaggtg aagcgggcactcggttctaacgctatggaagatggcaaagggtgttttgagctgtatcacaaatgtgacgaccaatgcatggagactatccg gaatgggacctacaatagacgcaagtacaaggaggagtcccgacttgagaggcaaaagattgaaggcgttaagcttgagtccgaaggaa catacaagatcctgacgatttacagcacagtagccagcagtctggtgctggcaatgggatttgccgcatttctgttttgggctatgagcaacg gttcatgtcggtgcaacatttgcatttgataaacgtSEQ ID NO: 3 (H9 COBRA protein sequence) (vAVW68)METISLMTILLVVTTSNADKICIGHQSTNSTETVDTLTETNVPVTHAKELLHTEHNGMLC ATNLGHPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGTCYPGNVENLEELRTLFSSSSSYQRIQIFPDTIWNVTYTGTSKSCSDSFYRNMRWLTQKNGLYPVQDAQYTNNR GKDILFVWGIHHPPTDTAQTNLYTRTDTTTSVTTENLDRTFKPLIGPRPLVNGLIGRINYY WSVLKPGQTLRVRSNGNLIAPWYGHVLSGESHGRILKTDLNSGNCWQCQTEKGGLNS TLPFHNISKYAFGTCPKYIGVKSLKLAIGLRNVPARSSRGLFGAIAGFIEGGWPGLVAGW YGFQHSNDQGVGMAADRDSTQKAVDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNM INNKIDDQIQDVWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAMEDGKG CFELYHKCDDQCMETIRNGTYNRRKYKEESRLERQKIEGVKLESEGTYKILTIYSTVASS LVLAMGFAAFLFWAMSNGSCRCNICISEQ ID NO: 4 (H9 of H9N2 Saudi Arabia strain protein sequence) (vAVW48)MEPISLMIILLLVTTSNADKICIGHQSTNSTETVDTLTETNVPVTHAKELLHTEHNGMLCA TNLGHPLILNTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGTCYPGNVENLEELRTL FSSSSSYQRIQIFPDTIWNVTYTGTSKSCSDSFYRNMRWLTQKNGLYPVQDAQFTNNRG KDILFVWGIHHPPTDTAQTNLYTRTDTTTSVTTENLDRTFKPLIGPRPLVNGLIGRINYYW SVLKPGQTLRVRSNGNLIAPWFGHVLSGESHGRILKTDLNSGNCWQCQTEKGGLNSTL PFHNISKYAFGTCPKYIGVKSLKLAIGLRNVPARSSRGLFGAIAGFIEGGWPGLVAGWYG FQHSNDQGVGMAADRDSTQI<AVDI<ITSI<VNNIVDI<MNI<QYEIIDHEFSEVETRLNMIN NKIDDQIQDVWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAMEDGKGCF ELYHKCDDQCMETIRNGTYNRGKYKEESRLERQKIEGVKLESEGTYKILTIYSTVASSLV LAMGLAAFLFWAMSNGSCRCNICISEQ ID NO: 5 (NDV AVLNEW vector sequence) ggccgctaatacgactcactataggaccaaacagagaatccgtgaggtacgatagaaggcgaaggagcaatcgaagtcgtacgggtaga aggtgtgaatctcgagtgcgagcccgaagctcaaactcgagagagccttctgccaaaatgtcttctgtattcgatgagtacgagcagctcctc gcggctcagactcgccccaatggagctcatggcggaggagagaaggggagcaccttaaaggtagaagtcccggtattcactctcaacag tgatgacccagaagatagatggaactttgcagtgttttgtcttcggattgctgttagcgaggatgccaacaaaccacttaggcaaggtgctctc atatctctcttatgttcccactctcaagtgatgaggaaccatgttgcccttgcggggaaacagaatgaggccacactggctgttcttgagatcg atggttttaccaacggcgtgccccagttcaacaacaggagtggagtgtctgaagagagagcacagagatttatgatgatagcagggtctctc cctcgggcatgcagcaacggtaccccgttcgtcacagctggggttgaagatgatgcaccagaagacattactgataccctggagaggatc ctctctatccaggctcaagtatgggtcacggtggcaaaggccatgactgcatatgagacagcagatgagtcagaaacaagaagaatcaata agtacatgcagcaaggcagggtccagaagaagtacatcctccaccccgtatgcaggagcgcaatccaactcacaatcagacagtctctgg cggtccgcatctttttggttagcgagcttaagagaggccgcaacacggcaggtgggacctccacctattacaacttggtgggggatgtagac tcatacatcaggaacactgggctaactgcattcttcctgacacttaaatatggaattaacaccaagacatcagcccttgcacttagcagcctctc aggcgatatccagaaaatgaagcagctcatgcgcttgtatcggatgaaaggagataatgcgccgtacatgacattgctcggtgacagtgac cagatgagctttgcacctgccgagtatgcacaactttactcctttgccatgggtatggcatcagtcctagataaaggaactagcaaataccaat ttgccagggactttatgagcacatcattctggagacttggagtagagtacgctcaggctcaaggaagtagcatcaatgaggatatggccgcc gagctaaagctaaccccagcagcaaggagaggcctggcagctgctgcccaaagagtgtctgaggagaccagcagcatggacatgccca cccaacaagccggggtcctcactggactcagcgacggaggctcccaagccccccaaggtgcactgaacagatcacaagggcaaccggacaccggggatggggagacccaatttctggatctgatgagagcggtggcaaatagcatgagagaagcgccaaactctgcgcagggcac ccctcaaccggggcctcccccaacccctgggccctctcaagacaatgacaccgactgggggtactgaccgacagcacccagtttgcttct atgaggtcatcccaattcctctgcccacaccccacccctcaatccgcaatcccgcatggccaaacccacaaacgaacccccctgtctccctc ctctcccccagccccacaaccccacctgcccagggcaacataggtacaatgcgacccactaataatcaatacagggccaaagaaattaga aaaaagtacgggtagaagggagacattcagagatcagggcgagtcacccgggtctctgctctcccttctacctagtggattaggatggaga tggccacctttacagatgcggagatcgacgagctatttgagaccagtggaactgtcattgacagcataattacggcccagggaaaaccagt agagactgttggaaggagtgcaatcccacaaggcaaaactaaggctttgagcgcagcatgggagaagcatgggagcatccagtcaccag ccagccaagacacccctgatcgacaggacagatcagataaacaactgtccacacccgagcaagcgagtccaaacgacagccccccagc cacatccactgaccagcctcccactcaggctgcagatgaggccggcgatacacagctcaagaccggagcaagcaactctctgctgtcgat gcttgataaactcagcaataagtcatctaatgctaaaaagggcccagggtcgagccctcaagaaaggcatcatcaacgtctgactcaacaa caggggagtcaacaaagccgcggaaacagccaagagagaccgcagaaccaggccaaggccatccctggaaaccaggtcacagacgc gaacacagcatatcatggacaatgggaggagtcacaactatcagctggtgcaacccatcatgctctccgatcagagcagagccaagacaa tactcctgcacctgtggatcatgtccagctacctgtcgactttgtgcaggcgatgatgtctatgatggaggcgatatcacagagggtaagtaa agtgactatcagctggacctgtcttgaaacagacatcttctatccccatgatgcggtctgaaatccagcagctgaaaacgtctgttgcggtc atggaagccaatttgggcatgatgaagatcctggaccctggttgtgccaacgtttcatctctaagtgatctacgggcagttgcccgatcccac ccggttttaatttctggccccggagacccatctccttatgtgacccaagggggcgaaatggcactcaataaactttcgcaaccggtgcaacac ccctctgaattgattaaacccgccacggcaagcgggcctgatataggagtggagaaagacactgtccgtgcattgatcatgtcacgccctat gcatccgagctcttcagctaggctcttgagcaaactggacgcagccggatcgattgaggaaatcagaaaaatcaagcgccttgcactgaat ggctaatcaccaccgcaacccgcagcagatccctgtccacccagcaccacacggtatctgcaccaagctcctctctgcaaatccaaggtcc aacaccctaattaagtctgtctggccggcccgagcgacaaccctgtcctgcttcctctgccccactaaatgatcgcgcagctgcaatcaatt cagctatattaaggattaagaaaaaatacgggtagaatcggagtgccccgattgtgccaagatggactcatctaggacaatcgggctgtactt tgattctacccttccttctagcaacctgctagcattcccgatagtcctacaagacacaggggacgggaagaagcaaatcgccccgcaataca ggatccagcgtcttgactcgtggacagacagcaaagaagactcggtattcatcaccacctatggattcatctttcaggttgggaatgaagaag ccactgtcggcatgatcaatgataatcccaagcgcgagttactttccactgccatgctatgcctagggagtgtaccaaatgtcggagatcttgt tgagctggcaagggcctgcctcactatggtggtaacatgcaagaagagtgcaactaacaccgagagaatggtcttctcagtagtgcaggca ccccaggtgctgcaaagctgtagggttgtggcaaacaaatactcgtcggtgaatgcagtcaagcacgtgaaagcaccagagaagattcct gggagcggaaccctagagtacaaagtgaactttgtctctctgaccgtggtgccaagaaaggacgtctacaagataccaactgcagcactta aggtctctggctcaagtctgtacaatcttgcgctcaatgtcactattgatgtggaggtagacccgaagagcccgttggtcaaatccctttccaa gtccgacagtgggtactatgctaatctcttcttacatattgggcttatgtccactgtagataagaaggggaagaaagtgacatttgacaagctg gaaaggaagataaggagacttgatctatctgtagggcttagtgacgtgctcggaccttccgtgcttgtaaaggcgagaggtgcacggacta agctgctggcacctttcttctctagcagtgggacagcctgctatcccatagcaaatgcctctcctcaggtggccaagatactctggagccaaaccgcgtacctgcggagtgtaaaagtcattatccaagcgggcacccagcgtgctgtcgcagtgaccgccgaccacgaggttacctctactaa gctggagaaggggcataccattgccaaatacaatcccttcaagaaataggctgcatctctgagattgcactccgcccatcttcccggatcac catgacactaaataatgatctgtcttgattacttatagttagttcgcctgtctatcaaattagaaaaaacacgggtagaagattctggatcccggtt ggcgccttcaaggtgcaagatgggctccagatcttctaccaggatcccagtacctcttatgctgaccgtccgagtcatgttggcactgagttg cgtctgtccgaccagcgcccttgatggcaggcctcttgcagctgcagggattgtggtaacaggagacaaagcagtcaacatatacacctca tctcagacagggtcaatcataatcaagttactcccaaatatgcccaaggataaagaggcgtgtgcaaaagccccgttggaggcatacaaca ggacattgactactttgctcaccccccttggtgattctatccgtaggatacaagagtctgtgaccacgtccggaggagggaaacagggacgt cttataggcgccattatcggtggtgtagctctcggggttgcaaccgctgcacagataacagcagcctcggctctgatacaagccaatcaaaa tgctgccaacatactccggctaaaagagagcattgctgcaaccaatgaggctgtgcacgaggtcactaatggattatcacaactagcagtgg cagttgggaagatgcagcaatttgttaatgaccagtttaataaaacagctcaggaattggactgtataaaaattacacagcaggttggtgtaga actcaacctgtacctaactgaattgactacagtattcgggccacaaatcacttcccctgccttaactcagctgactatccaggcgctttacaatc tagctggtgggaatatggattacttgttgactaagttaggtgtggggaacaaccaactcagctcattaattagtagtggcctgatcaccggcaa ccctattctgtacgactcacagactcaactcttgggtatacaggtaaccctaccctcagtcgggaacctaaataatatgcgtgccacctacctg gaaaccttgtctgtaagtacaaccaaaggatttgcctcagcacttgtcccaaaagtagtgacacaggtcggttccgtgatagaagagcttgac acctcgtactgtatagagaccgatttggatctatattgtacaagaatagtgacattccctatgtctcctggtatttattcctgtttgagtggcaatac atctgcttgcatgtactcaaagactgaaggcgcactcactacgccgtatatgaccctcaaaggctcagttattgctaactgtaagatgacaaca tgtagatgtgcagaccccccgggtatcatatcgcaaaattatggagaagctgtgtctctaatagataggcaatcatgcaatatcttatccttaga cgggataactttgaggctcagtggggaatttgatgcaacttatcaaaagaatatctcaatacaagattctcaagtaatagtgacaggcaatctt gatatctcgactgagcttgggaatgtcaacaactcgataagtaatgctttggataagttagaggaaagcaacagcaaactagataaggtcaat gtcaaactgaccagcacatccgctcttattacctatatcgttaactgtcatatctcttgtatgtggtatacttagcctggttctagcatgctacctg atgtacaagcaaaaggcgcaacagaagaccttgttgtggcttgggaataataccctagaccagatgagggccactacaaaaatgtgaatgc ggatgagaggcagaaacatccccaatagcagtttgtgtgtaaagtctgacagcctgttaattagaagaattaagaaaaaactaccggatgta gatgaccaaagggcgatatacgggtagaacggtcggggaggccgtccctcaatcgggagccgggcctcacaacatccgttctaccgcat caccaatagcagttttcagtcatggaccgcgcagttagccaagttgcgctagagaatgatgaaagagaggcaaagaatacatggcgcttgg tattccggatcgcaatcctactctcaacggtggtgaccttagccatctctgcagccgcccttgcatatagcatggaggccagcacacctagcg atcttgtaggcataccgactgcgatctctagagcagaggaaaagattacatctgcactcggttccaatcaagatgtagtagataggatatataa gcaggtggccctcgaatctccactggcattgctaaacaccgaatctacaattatgaacgcaataacgtctctctcttatcgaatcaatggggcc gcaaatagcagcggatgtggagcacccattcatgatccagattatattggaggaataggtaaagaacttattgtagatgatgctagcgacgtc acatcatactatccctctgcgttccaagaacacctgaactttatcccggcgcctactacaggatcaggttgcactcggataccctcatttgacat gagcgctacccactactgtatactcacaatgtgatattatctggctgcagagatcactcgcactcacatcaatatttagcacttggtgtgcttcg gacatctgcaacagggagggtattcttttccactctgcgttccatcaatctggatgacacccaaaatcggaagtcttgcagtgtgagtgcaacccccttgggttgtgatatgctgtgctctaaagtcacagagactgaagaagaggattataactcagctatccccacgtcgatggtacatggaagg ttagggttcgacggccaataccacgagaaggacctagatgtcacaacactattcgaggactgggtggcaaactacccaggagtaggggg cgggtcttttattgacaaccgcgtatggttcccagtttacggagggctaaaacccaattcgcccagtgacaccgcacaagaagggaaatatg taatatacaagcgatacaatgacacatgtccagatgagcaagattatcagattcaaatggctaagtcttcatataagcctgggcggtttggagg gaaacgcgtacagcaggccatcttatctatcaaagtgtcaacatccttgggcgaggacccggtactgactgtaccgcccaacacagtaaca ctcatgggggccgaaggcagagttctcacagtagggacatctcatttcctttatcagcgagggtcatcatacttctcccctgccctactatatcc tatgatagtcagcaacaaaacagccactcttcatagtccttatacattcaatgccttcactcgaccaggtagtgtcccttgccaggcttcagcaa gatgccctaactcatgtgttaccggagtctatactgatccatatcccttggtcttctataggaaccacaccttgcgaggggtattcgggacgatg cttgatgataaacaagcaagactcaaccctgtatctgcagtatttgacagcatatcccgcagtcgcataacccgggtgagttcaagcagcac caaggcagcatacacaacatcaacatgttttaaagttgtaaagaccaataaaacctattgtctcagcattgccgaaatatccaataccctcttcg gggaattcagaatcgtccctttactagttgagattctcaaggatgatggggttagagaagccaggtctagccggttgagtcaactgcgagag ggttggaaagatgacattgtatcacctatcttttgcgacgccaagaatcaaactgaataccggcgcgagctcgagtcctacgctgccagttgg ccataatcagctagtgctaatgtgattagattaagtcttgtcggtagtcacttgattaagaaaaaatgtgggtggtagcgggatataaggcaaa acaactcaaggaggatagcacgggtaggacatggcgagctccggtcccgagagggcggagcatcagattatcctaccagagtcacacct gtcttcaccattagtcaagcacaaactactctattactggaaattaactgggctaccactccctgacgagtgtgacttcgaccacctcattctca gccgacaatggaagaaaatacttgaatcggcctcccctgacactgagagaatgataaaacttggaagggcagtgcaccagactctcaacc acaattccaagataaccggagtactccatcccaggtgtttagaagaattggctagtattgaggttcctgactcaaccaacaagtttcggaagat cgagaagaaaatccaaattcacaacacaaggtatggagaactgttcacaagactgtgcacgcatgtagagaagaaattgttgggatcatctt ggtctaataatgtcccccggtcagaagagttcaacagcatccgtacagatccggcattctggtttcactcaaaatggtccacaactaagtttgc atggctccatataaaacagattcaaaggcatctgattgtggcagcaagaacaaggtccgcagccaacaaattggtgacgctgacccataag gtaggccaagtcttgttactcctgagcttgtcattgtgacacatacagatgagaacaagttcacgtgtcttacccaggaacttgtgttgatgtat gcagatatgatggagggcagagatatggtcaacataatatcatccacggcggcacatctcaggagcctatcagagaaaattgatgacattct gcggttagtagatgccctggcaaaagatctgggtaatcaagtctacgatgttgtagcactcatggagggatttgcatacggcgccgtccagc tgcttgagccgtcaggtacattcgcaggggatttcttcgcattcaacctgcaggagctcaaagacactttgatcggcctccttcctaaggatat agcagaatctgtgactcacgcaatagccactgtattctctggcttagaacaaaatcaagcggctgagatgctgtgcctgttgcgtctatgggg ccacccattacttgagtcccgtattgcggcaaaagcagtaaggagccaaatgtgcgcaccaaaaatggtagactttgatatgatcctccaggt attgtctttctttaaaggaacaatcatcaacggatacagaaagaagaatgcaggtgtttggccacgtgtcaaagtagatacgatatacgggaa ggtcattgggcagctacacgctgattcagcggagatttcacacgatatcatgttgagagagtacaagagtttatctgcgcttgaattcgagcca tgtatagaatacgaccctatcaccaatctgagcatgtttctaaaagacaaggcgatcgcacacccgaaagacaactggctcgccgcgtttag gcgaaaccttctctctgaggaccagaagaaacatgtaaaggaggcaacctctactaaccgtctcttgatagagttcttagagtcaaatgatttt gatccatataaggagatggaatatctgacgacccttgagtacctaagagatgacaatgtggcagtatcatactcgctcaaggagaaggaagt31gaaggttaatgggcggatttttgctaagctaacaaagaaattaaggaactgtcaagtgatggcggaagggatcttagctgaccagattgcac ctttctttcaagggaatggggtcattcaggatagcatatctttaaccaagagtatgctagcgatgagtcaattgtctttcaacagcaataagaaa cgtatcactgactgcaaagaaagagtagcctcaaaccgcaatcacgatcaaaagagcaagaatcgtcggagagttgccacttttataacga ctgacctgcaaaagtactgtctaatggagatatcagacaatcaaactgttcgctcatgccatcaatcagctgatgggcttacctcacttcttcg aatggattcatctaagactaatggatactacgatgtttgtaggagaccctttcaatcccccaagtgacccaactgactgtgatctctcaagagtc ccaaatgatgacatatatattgtcagtgctagagggggtattgagggattatgtcagaagctatggacaatgatctcaattgctgcaatccaact tgctgcagcaagatcacattgtcgcgtcgcctgtatggtacagggtgacaatcaagtaatagctgtaacgagagaggtaaggtcagatgact ccccggaaatggtgttaacacaattgcatcaagccagtgataatttcttcaaggaattgattcatgttaatcatttgattggccataatttgaagga tcgtgaaacaatcagatcagacacattcttcatatacagcaaacgaatattcaaagatggagcaatactcagtcaagtcctcaaaaattcatct aaattagtgctaatatcaggcgaccttagtgaaaacaccgtaatgtcctgtgccaacattgcatctactatagcacggctgtgcgagaacggg cttccaaaggattctgttattacttaaactacctgatgagttgcgtgcagacatactttgattctgagttttccatcactaacagctcgcaccccg attctaaccagtcgtggattgaagacatctcttttgtgcactcatatgtcctgacccctgcccagctagggggactgagcaacctccaatactc aaggctctacacgaggaacatcggtgacccgggaactactgcttttgcagagatcaagcgattagaagcagtggggttactaagtcctagta tatgactaacatcttaactaggccgcctggaaatggagattgggccagtctgtgtaacgacccttactctttcaattttgagactgtcgcgagt ccaaatattgtccttaagaaacatacacaaagagtcctatttgaaacttgttcaaatcccttattatctggcgtgcatacagaggataatgaggc agaagagaaggcgttggctgaatttttactcaatcaagaagtaattcatccacgtgtcgcacatgctatcatggaagcaagctctataggtag gaggaagcagattcaagggcttgttgacacaacaaacaccgtaatcaagattgcattgactaggaggccacttggcatcaagaggctgatg cggatagtaactactcgagcatgcatgcaatgctgttagagacgatgttttctcatctaacaggtctaaccaccccttagtttcctctaatatgt gttctctgacgctagcagactatgcacggaatagaagctggtcaccattgacggggggtagaaagatactgggtgtatctaatcctgatacta tagaacttgtagagggtgagatccttagcgtcagcggaggatgcacaagatgtgacagcggagatgaacaattcacttggttccatcttccg agcaatatagaactgaccgatgacaccagcaagaatcctccgatgagagtgccgtacctcgggtcaaagactcaagagaggagggccgc ctcgcttgcgaaaatagctcatatgtcaccacatgtgaaagctgctctaagggcatcatccgtgttgatctgggcttatggagacaacgaagt aaattggactgctgctcttaaaattgcaagatctcggtgcaatataaactcagagtatcttcgactattgtcccccttacccacagctgggaatct ccaacatagactggatgacggcataactcagatgacattcacccctgcatctctctacagggtgtcaccttatattcacatatccaatgattctc aaaggttattcacggaagaaggagtcaaagagggaaatgtagtttatcagcaaatcatgctcttgggtttatctctaatcgaatcactcttcccg atgacgacaaccaggacatacgatgagatcacattgcacctccacagtaaatttagctgctgtatcagggaagcaccggttgcagttcctttc gagtactcgggatggcaccagaactaaggacagtgacctcaaataagtttatgtatgatcctagtcctgtatcggagggtgacttgcgaga cttgacttagctatctttaagagttatgagcttaatctagaatcatatcccacaatagagctaatgaacattctttcaatatccagcgggaagttaa tcggccagtctgtggtttctatgatgaagatacctccataaagaatgacgccataatagtgtatgacaacacccggaattggatcagcgaag ctcagaattcagatgtggtccgcctattcgagtatgcagcactgaagtgcttctcgactgttcttatcagctctactatctgagagtaagaggc ctagacaatatcgtgttgtatatgagtgacttatataagaatatgccaggaattctactttccaacattgcagctacaatatctcatcccatcattcattcaagattgcatgcagtaggcctggtcaatcacgacgggtcacaccaacttgcagacacagatttcatcgaaatgtctgcaaaactattagtc tctgcactcgacgcgtggtctcaggtttatatgcagggaataagtatgatctgctgttcccgtctgtcttagatgataacctgagtgagaagat gcttcagctgatatctcggttatgctgcctgtatacggtgctctttgctacaacaagagagatcccgaaaataagaggcttatctgcagaagag aagtgttcagtacttactgagtacctactgtcagatgctgtgaaaccattacttagttctgagcaagtgagctctatcatgtctcctaacatagtta cgtcccagctaatctatattacatgtctcggaagagccttaatttgattagggaaagagaggacagggacactatcttggcattgttgttcccc caagagccactacttgagttccccttagtacaagatattggcgctcgagtgaaagatccattcacccgacaacctgcggcgtttttacaagaat tagatttgagcgctccagcaaggtatgacgcatttacacttagtcaggttcattctgaacacacatcaccaaatccggaggacgactacttagt acgatacctgtcagaggaatagggaccgcgtcctcctcttggtataaggcatctcaccttctttctgtacctgaggtcagatgtgcaaggcac gggaattccttatacttggcagaaggaagcggagccattatgagtcttctcgaactgcatgtgccgcatgagactatctattacaatacgctctt ctcaaacgagatgaaccccccacagcggcatttcggaccgaccccaacacagtttctgaattcagttgtttataggaatctacaggcggagg taccatgtaaggatggatttgtccaggagttccgtccattatggagagagaatacagaagaaagcgatctgacctcagataaagcagtgggt tacatcacatctgcagtgccctaccggtctgtatcattgctgcactgtgacattgagattcctccaggatccaatcaaagcttactggatcaact ggctaccaatctgtctctgattgccatgcattctgtaagggagggcggggtcgtgatcatcaaagtgttgtatgcaatgggatattacttccatc tactcatgaactgttcactccgtgttctacgaaaggatatattctctctaatggctatgcatgtagaggggatatggagtgttacctggtatttgtc atgggctatcgaggtgggcctacatttgtacatgaggtagtgaggatggcaaaaactctagtgcagcggcacggtacacttttgtccaaatca gatgagatcacactgactaggttatttacctcacagcggcagcgtgtaacagacatcctatccagtcctttaccgagactaataaagttcttga gaaagaatatcgatactgcgctaattgaagccgggggacaacccgtccgtccattctgtgcagagagcttggtgaggacactagcggaca caactcagatgacccagatcatcgctagtcacattgacacagtcattcgatctgtgatctacatggaggctgagggtgatctcgccgacaca gtgttcttatttaccccctacaatctctctacagacggtaaaaagagaacatcacttaaacagtgcacaaggcagatcttagaggtcacaatatt gggtcttagagttgaaaatctcaataaagtaggtgatgtagtcagtctagtacttaaaggtatgatttctctggaggacctgatccctctaagaa catactgaagcgtagtacctgccctaagtatttgaagtctgttctaggtattactaaactcaaagaaatgtttacagacacctctttattatacttg actcgtgctcaacaaaaattctacatgaaaactataggcaacgcagtcaagggatactacagtaactgtgactcttaaagataatcacatatta ataggctcctttctagttaactgagccctgttgatttaatgatactatattagaaaaaagttgcactccgatcctttaggactcgtgttcgaattca aataatgtcttagaaaaaagttgcgcgtaattgttcttgaatgtagtcttgtcattcaccaaatctttgtttggtgggtcggcatggcatctccacc tcctcgcggtccgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggagctagcataaccccttggggcctctaaac gggtctgaggggtttttgctgaaaggaggaactatatcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggc atcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttacc agcctaacttcgatcactggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggtggcatggattgtaggcg ccgccctataccttgtctgcctccccgcgtgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgc taacggattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcg cgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaa cgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgtt ccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctct ggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcct ctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacat ggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgctt cacgaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacg gtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagcca tgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtg aaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctg cggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaagg ccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgacc ctgccgcttaccggatacctgtccgccttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtctgagtccaacccgg taagacacgactatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtg gtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtaccttcggaaaaagagtggtagctctga tccggcaaacaaaccaccgctggtagcggtggtttttgtttgcaagcagcagatacgcgcagaaaaaaaggatctcaagaagatccttg atcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctt ttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcag cgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgca atgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgc aacttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgct gcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgt gcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataa tctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttg ctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaac tctcaaggatctaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgg gtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatatt attgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttcccc gaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtcttcaagaattctcatgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatctaacaat gcgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatcgtc cattccgacagcatcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttctatgcgcacccgttctcggagcactgtccg accgcttggccgccgcccagtcctgctcgcttcgctacttggagccactatcgactacgcgatcatggcgaccacacccgtcctgtggatc tgc

Claims

CLAIMS1. A recombinant Newcastle disease virus (NDV) vector comprising a heterologous polynucleotide encoding an AIV subtype H9 haemagglutinin (AIV H9-HA) antigen, wherein:(i) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80% identity to SEQ ID NO: 1, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4; or(ii) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80% identity to SEQ ID NO: 2, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3.

2. The recombinant NDV vector of claim 1, wherein:(i) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 95% identity to SEQ ID NO: 1, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 4; or(ii) the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 85% identity to SEQ ID NO: 2, and / or the AIV H9-HA antigen comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 3.

3. The recombinant NDV vector of claim 1 or 2, wherein:(i) the polynucleotide encoding the AIV H9-HA antigen comprises the sequence of SEQ ID NO: 1, and / or the AIV H9-HA antigen comprises the amino acid sequence of SEQ ID NO: 4; or(ii) the polynucleotide encoding the AIV H9-HA antigen comprises the sequence of SEQ ID NO: 2, and / or the AIV H9-HA antigen comprises the amino acid sequence of SEQ ID NO: 3.

4. The recombinant NDV vector of any one of claims 1 to 3, wherein the genome of the NDV vector comprises a sequence having at least 80% identity to SEQ ID NO: 5, into which the heterologous polynucleotide encoding the AIV H9-HA antigen has been inserted.

5. The recombinant NDV vector of any one of claims 1 to 4, wherein the heterologous polynucleotide encoding the AIV H9-HA antigen is inserted between the P and M genes of the NDV vector genome.

6. The recombinant NDV vector of any one of claims 1 to 5, wherein the recombinant NDV vector is capable of inducing a protective immune response against AIV.

7. One or more nucleic acid molecules comprising the recombinant NDV vector of any one of claims 1 to 6.

8. A cell comprising the recombinant NDV vector of any one of claims 1 to 6 or the nucleic acid molecules of claim 7.

9. A composition comprising the recombinant NDV vector of any one of claims 1 to 6, the nucleic acid molecules of claim 7 or the cell of claim 8.

10. The recombinant NDV vector of any one of claims 1 to 6, the nucleic acid molecules of claim 7, the cell of claim 8 or the composition of claim 9, for use in a method of inducing a protective immune response against AIV in an avian.

11. A method of inducing a protective immune response against AIV in an avian, comprising administering the recombinant NDV vector of any one of claims 1 to 6, the nucleic acid molecules of claim 7, the cell of claim 8 or the composition of claim 9 to the avian.

12. The recombinant NDV vector of any one of claims 1 to 6, the nucleic acid molecules of claim 7, the cell of claim 8 or the composition of claim 9, for use in the manufacture of a medicament for inducing a protective immune response against AIV in an avian.

13. The use or the method of any one of claims 10 to 12, wherein inducing a protective immune response comprises reducing clinical signs associated with infection by AIV.

14. The use or the method of claim 13, wherein the clinical signs are respiratory clinical signs.

15. The recombinant NDV vector of any one of claims 1 to 6, the nucleic acid molecules of claim 7, the cell of claim 8 or the composition of claim 9, for use in a method of reducing AIV shedding in an avian.

16. A method of reducing AIV shedding in an avian, comprising administering the recombinant NDV vector of any one of claims 1 to 6, the nucleic acid molecules of claim 7, the cell of claim 8 or the composition of claim 9 to the avian.

17. The recombinant NDV vector of any one of claims 1 to 6, the nucleic acid molecules of claim 7, the cell of claim 8 or the composition of claim 9, for use in the manufacture of a medicament for reducing AIV shedding in an avian.

18. The use or the method of any one of claims 10 to 17, wherein the recombinant NDV vector is administered once and wherein the protective immune response or reduction in shedding is obtained after such single administration.

19. The use or the method of any one of claims 10 to 18, wherein the recombinant NDV vector is administered to an avian at six or fewer days of age, preferably at one day of age.

20. The use or the method of any one of claims 10 to 19, wherein the recombinant NDV vector is administered by the oculo-nasal route.

21. A method of manufacturing the recombinant NDV vector of any one of claims 1 to 6, wherein the method comprises: a) providing one or more nucleic acids encoding a NDV vector; b) providing a polynucleotide encoding an AIV H9-HA antigen; and c) recombinantly combining the nucleic acids encoding a NDV vector and the polynucleotide encoding an AIV H9-HA antigen.

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