WO2025046414A1 - Cdv aiv vectors and uses thereof - Google Patents

Abstract

The present invention provides recombinant canine distemper virus (CDV) 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 animal.

Description

CDV AIV VECTORS AND USES THEREOF

SEQUENCE 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 “23-0048-US-2.xml,” created on July 24, 2024, and 25,475 byte in size, is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to recombinant canine distemper virus (CDV) viral vectors comprising a heterologous polynucleotide encoding a foreign avian influenza (AIV) antigen, for example an AIV hemagglutinin serotype H9 (AIV H9-HA) antigen. The recombinant viral vectors are suitable for use in immunogenic compositions and vaccines, and can provide protection against avian influenza.

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] Canine distemper virus (CDV) belongs to the Paramyxoviridae family and the Morbillivirus genus. Canine distemper, the disease caused by CDV, is a highly infectious, febrile disease of dogs and other mammal families, in particular those of the order Carnivora. The transmission of CDV is usually via the oral or respiratory routes, with sneezing, coughing and sharing of food or water bowls identified as routes of transmission. Initially, CDV replicates in the lymphatic tissue of the respiratory tract, followed by infection of respiratory, gastrointestinal and urogenital epithelium (Whitehouse, CABI Compendium “canine distemper”, 2015; Creevy K.E., Overview of Canine Distemper. In: The Merck Veterinary Manual. 2013). The mortality rate of CDV in canines is high, ranging between 30 and 80 percent. CDV is not known to routinely infect avian populations.

[0005] CDV is an enveloped virus containing a single stranded RNA genome, 15,690 nucleotides in length. 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), hemagglutinin glycoprotein (H), and large protein (L), which are sited between a short 3’ leader region and followed by a short 5’ trailer region. The six genes are separated by intergenic regions of 3 nucleotides. In addition, two non-structural proteins, V and C, are encoded within the P gene (Zhao, et al., Viruses, 12(3):339, 2020)

[0006] Several studies have highlighted the potential of CDV to be used as a vaccine vector. In particular, Wang et al. (Vaccine 30: 5067-5072, 2012), describes a recombinant CDV vaccine strain expressing the rabies virus glycoprotein, generated using reverse genetics. However, a disadvantage, as identified in WO 2019/057859, seen in practice is that recombinant CDV may lose the inserted gene over time. This loss is thought to be mediated by homologous recombination between duplicated non-coding region sequence(s). WO 2019/057859 further describes an expression cassette for insertion between two adjacent essential genes of a Paramyxoviridae virus, creating a virus vector that can accommodate, maintain and express the foreign gene, encoded by the expression cassette, for long periods of time. The disclosed vectors of WO 2019/057859 are designed for mammals, in particular swine, and describe canine, parvovirus VP2, H3 -subtype hemagglutinin of swine influenza virus, a spike protein of porcine epidemic diarrhoea virus, various coronavirus, and avian infectious bronchitis virus S protein. Suitable Paramyxoviridae viruses include CDV, feline morbillivirus, and peste-des-petits-ruminants virus. However, the disclosed studies are limited to the Lederle CDV vaccine strain and vaccination of pigs, resulting in some immunogenicity. Therefore, there is no indication that CDV vectors would be efficacious in other species.

[0007] The development of effective influenza virus vaccines for poultry would be of benefit to both human and veterinary health. The development of effective CDV vaccines would also be of benefit. In particular, it would be of benefit to provide a combined CDV 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 and/or a reduction in shedding. 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 species of vector, i.e. CDV, will be when modified to express an antigen from an avian pathogen such as AIV, which has not previously been tested in that vector context. It is also not always straightforward to predict how successful a particular subtype of AIV HA antigen, such as AIV H9-HA, will be in a CDV vector context.

[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 application describes recombinant CDV 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. The recombinant viral vectors can be used in immunogenic compositions and vaccines to provide animals with protection against avian influenza and/or CDV.

[0010] Thus, in a first aspect, the present invention provides a recombinant canine distemper virus (CDV) vector comprising a heterologous polynucleotide encoding an Avian Influenza Virus (AIV) antigen.

[0011] In a second aspect, the present invention provides one or more nucleic acid molecules comprising the recombinant CDV vector of the invention.

[0012] In a third aspect, the present invention provides a cell comprising the recombinant CDV vector or the nucleic acid molecules of the invention.

[0013] In a fourth aspect, the present invention provides a composition comprising the recombinant CDV vector, the nucleic acid molecules or the cell of the invention. [0014] In a fifth aspect, the present invention provides the recombinant CDV 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 and/or CDV, preferably AIV, in an animal. Alternatively or additionally in a fifth aspect, the present invention provides the recombinant CDV 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.

[0015] In a sixth aspect, the present invention provides a method of inducing a protective immune response against AIV and/or CDV, preferably AIV, in an animal, comprising administering the recombinant CDV vector, the nucleic acid molecules, the cell or the composition of the invention to the animal. Alternatively or additionally 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 CDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian.

[0016] In a seventh aspect, the present invention provides the recombinant CDV 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 and/or CDV, preferably AIV, in an animal. Alternatively or additionally in a seventh aspect, the present invention provides the recombinant CDV 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.

[0017] In an eighth aspect, the present invention provides the recombinant CDV 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.

[0018] In a ninth aspect, the present invention provides a method of reducing AIV shedding in an avian, comprising administering the recombinant CDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian.

[0019] In a tenth aspect, the present invention provides the recombinant CDV 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.

[0020] In an eleventh aspect, the present invention provides a method of manufacturing a recombinant CDV vector, the method comprising: a) providing one or more nucleic acids encoding a CDV vector; b) providing a polynucleotide encoding an AIV antigen; and c) recombinantly combining the nucleic acids encoding the CDV vector and the polynucleotide encoding the AIV antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows a pCG219 plasmid map.

[0022] FIG. 2 shows a pCG171 plasmid map.

[0023] FIG. 3 shows a pCG172 plasmid map.

[0024] FIG. 4 shows a pCG173 plasmid map.

[0025] FIG. 5 shows expression results for vCDV-6.

[0026] FIG. 6 shows kinetics of mean clinical daily scores of different groups vaccinated (Gl) or not (G2) with vCDV6 vaccine candidate expressing H9N2 HA gene after H9N2 challenge at D21.

[0027] FIG. 7 shows dispersions of individual global clinical scores by groups. Gl : vCDV6 and G2: controls. Groups compared to CTL on the “GCS” criterion using a Mann-Whitney- Wilcoxon test. Box plots represent the mini, the lower quartile, the median, the upper quartile and the maxi).

[0028] FIG. 8 shows H9N2 shedding after challenge. Left panel: mean oro-pharyngeal H9N2 RNA excretion by groups expressed as equivalent loglO EID50/mM; the limit of detection was 2.09 loglO equivalent EID50/ml. Right panel: percentage of birds positive for shedding at the three time points. Dpi = day post-infection (challenge).

[0029] FIG. 9 shows dispersions of individual AUC of viral RNA by groups; box plots represent the mini, the lower quartile, the median, the upper quartile and the maxi.

[0030] FIG. 10 shows dispersions of individual HAI titers against antigen IRAK on D20 by groups (loglO HAI titers); box plots represent the mini, the lower quartile, the median, mean (cross), the upper quartile and the maxi. Gl: vCDV6 and G2: controls. CTL group remained negative on D21. Seroconversion occurred in the CDV group. DETAILED DESCRIPTION

[0031] In an embodiment, the present invention provides recombinant CDV vectors comprising a heterologous polynucleotide encoding an avian influenza AIV H9-HA antigen. In one embodiment, the present invention provides a novel recombinant CDV vector comprising a heterologous polynucleotide encoding an AIV H9-HA antigen, for example from a H9N2 Saudi Arabia strain of AIV. In one embodiment, the polynucleotide encoding the AIV H9-HA antigen of the present invention can be defined with reference to SEQ ID NO: 1 (H9 gene of H9N2 Saudi Arabia strain). In one embodiment, the AIV H9-HA antigen of the present invention can be defined with reference to polypeptide sequence of SEQ ID NO: 2 (H9 gene of H9N2 Saudi Arabia strain). [0032] The recombinant CDV viral vectors of the invention can be used in immunogenic compositions and vaccines to provide animals with protection against avian influenza and/or canine distemper. In particular, the recombinant viral vectors of the present invention provide a combined CDV and AIV vector vaccine which is surprisingly effective even in stringent clinical conditions, with an unexpected level of efficacy that can achieve advantageous results such as a significant decrease in viral excretion and a reduction in clinical signs. In one embodiment, the recombinant CDV viral vectors of the invention can be used in immunogenic compositions and vaccines to provide avians with protection against avian influenza. Benefits of the viral vectors of the invention include the capability to induce a protective immune response against AIV infection, including a reduction in viral excretion, even when administered just once and to avians of six or fewer days of age, with demonstrated efficacy via the subcutaneous route of administration.

Recombinant CDV vectors

[0033] In a first aspect, the present invention provides a recombinant canine distemper virus (CDV) vector comprising a heterologous polynucleotide encoding an Avian Influenza Virus (AIV) antigen.

[0034] In an embodiment, the AIV antigen is an AIV haemagglutinin antigen.

[0035] In an embodiment, the AIV antigen is an AIV subtype H9 haemagglutinin (AIV H9- HA) antigen. In a further embodiment, the AIV H9-HA antigen is a Saudi Arabia strain H9-HA antigen.

[0036] In an embodiment, the polynucleotide encoding the AIV H9-HA antigen is a Saudi Arabia strain H9-HA polynucleotide. [0037] In an embodiment, the polynucleotide encoding the AIV 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 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: 2 or any fragments thereof. In a preferred embodiment, the polynucleotide encoding the AIV antigen comprises a sequence having at least 80% identity to SEQ ID NO: 1, and/or the AIV antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2.

[0038] In an embodiment, the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 75%, 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 a preferred embodiment, the polynucleotide encoding the AIV H9-HA antigen comprises a sequence having at least 80% identity to SEQ ID NO: 1. 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: 2 or any fragments thereof. In a preferred embodiment the AIV H9-HA antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2. [0039] In an embodiment of the recombinant CDV vector of the invention, 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: 2.

[0040] In an embodiment of the recombinant CDV vector of the invention, 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: 2.

[0041] Thus, it will be understood that in an embodiment the present invention relates to particular AIV antigens, which may be defined with reference to SEQ ID NOs 1 and 2, which are advantageous in a CDV vector context for the reasons outlined herein.

[0042] In some embodiments, the avian influenza H9 HA polynucleotide is from strain A/avian/Saudi Arabia/910135/2006 (H9N2), and encodes a polypeptide defined by GenBank No: ACY80655.1, which is incorporated by reference herein in its entirety.

[0043] In a preferred embodiment of the recombinant CDV vector of the invention, the genome of the CDV vector comprises a sequence having at least 80% identity to SEQ ID NO: 3, into which the heterologous polynucleotide encoding the AIV antigen has been inserted. In an embodiment of this type, the genome of the CDV vector comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3, into which the heterologous polynucleotide encoding the AIV antigen has been inserted. Thus, in an embodiment, it will be understood that the recombinant CDV vector of the invention comprises a CDV vector genome of SEQ ID NO: 3 (or a sequence having % identity to SEQ ID NO: 3 as defined herein), that a heterologous polynucleotide encoding an AIV antigen of the invention has been inserted into. In an embodiment, the recombinant CDV vector of the invention comprises a CDV vector genome sequence defined in relation to SEQ ID NO: 3 that is interrupted by the polynucleotide encoding the AIV antigen of the invention as well as any other recombinant elements included in the AIV antigen expression cassette. Preferably, the AIV antigen is an AIV H9-HA antigen.

[0044] In embodiments, the CDV vector is an Onderstepoort strain CDV vector. In embodiments, the CDV vector is an Onderstepoort strain CDV vector comprising a genome sequence having at least 80% identity to SEQ ID NO: 3. In a preferred embodiment, the recombinant CDV vector of the invention is a CDV Onderstepoort strain vector comprising a genome sequence having at least 80% identity to SEQ ID NO: 3, into which the heterologous polynucleotide encoding the AIV antigen has been inserted. In an embodiment of this type, the recombinant CDV vector of the invention is a CDV Onderstepoort strain vector comprising a genome sequence having at least at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3, into which the heterologous polynucleotide encoding the AIV antigen has been inserted. Thus, in an embodiment, it will be understood that the recombinant Onderstepoort strain CDV vector of the invention comprises an Onderstepoort strain CDV vector genome of SEQ ID NO: 3 (or a sequence having % identity to SEQ ID NO: 3 as defined herein), that a heterologous polynucleotide encoding an AIV antigen of the invention has been inserted into. In embodiments, the CDV vector is an Onderstepoort strain CDV vector comprising the genome sequence set forth in ENA/EBI accession no. AF014953.1 (which is incorporated by reference herein). In an embodiment, the recombinant Onderstepoort strain CDV vector of the invention comprises an Onderstepoort strain CDV vector genome sequence defined in relation to SEQ ID NO: 3 that is interrupted by the AIV heterologous polynucleotide of the invention as well as any other recombinant elements included in the AIV antigen expression cassette. Preferably, the AIV antigen is an AIV H9-HA antigen.

[0045] SEQ ID NO: 3 is the sequence of pCGl 77, a transfer vector comprising transfer vector elements (e.g. a T7 promoter, terminator, rop (repressor of primer) and ori (origin of replication) sequences) as well as a CDV Onderstepoort genome. It will be understood by the skilled person that transfer vector elements may be present in the recombinant CDV vector of the invention or may be absent from the recombinant CDV vector of the invention. In embodiments, the recombinant CDV vector of the invention comprises transfer vector elements. In an embodiment, the recombinant CDV vector of the invention does not comprise transfer vector elements. In a specific embodiment, the polynucleotide sequence of the recombinant CDV vector of the invention consists of one or more CDV genomic sequences and an AIV H9-HA sequence. Thus, it will be understood that embodiments of the present invention relate to a CDV vector comprising SEQ ID NO: 3 or a sequence with at least 80% identity thereto, into which a polynucleotide encoding an AIV H9-HA antigen has been inserted, preferably between the CDV P and M genes. In essence, this results in the provision of pCG219 as illustrated in Fig. 1. In some embodiments of the present invention, the transfer vector elements are not present in SEQ ID NO: 3. In some embodiments of the present invention, non-CDV elements are not present in SEQ ID NO: 3. In some embodiments, the sequence of the invention defined in respect of SEQ ID NO: 3 or a sequence with identity thereto is defined in respect of positions 3914-19603 of SEQ ID NO: 3 or a sequence with identity to positions 3914-19603 of SEQ ID NO: 3. Thus, in some embodiments herein, the reference to SEQ ID NO: 3 is understood as solely referring to positions 3914-19603 of SEQ ID NO: 3.

[0046] In an aspect, the present invention relates to a recombinant CDV Onderstepoort vector comprising a heterologous polynucleotide. Thus, in an embodiment of this type, the heterologous polynucleotide in such a vector is not limited, e.g. to AIV. In an embodiment of this type, the recombinant CDV Onderstepoort vector is as defined herein, e.g. with reference to SEQ ID NO: 3. In an embodiment of this type, the heterologous polynucleotide encodes a polypeptide. In an embodiment of this type, the heterologous polynucleotide encodes a heterologous antigen. In an embodiment of this type, the heterologous antigen is derived from a pathogen other than CDV. In an embodiment of this type, the heterologous polynucleotide is expressible or expressed. In an embodiment of this type, the recombinant CDV vector is capable of eliciting heterologous protection against the pathogen from which the heterologous antigen is derived.

[0047] In a preferred embodiment of the recombinant CDV vector of the invention, the heterologous polynucleotide is inserted between the P gene and the M gene in the CDV genome. In an embodiment, the heterologous polynucleotide encoding the AIV antigen is inserted between the P and M genes of the CDV vector genome. In a preferred embodiment, the heterologous polynucleotide encoding the AIV antigen is inserted between the P gene and the M gene of the CDV vector genome defined in relation to SEQ ID NO: 3. Preferably, the AIV antigen is an AIV H9-HA antigen.

[0048] In embodiments, the recombinant CDV 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 CDV genome promoter. In embodiments, the promoter driving the AIV antigen expression is a CDV genome promoter. In embodiments, the expression of the AIV antigen is regulated by the promoter. In a preferred embodiment, the expression of the AIV antigen is regulated by the CDV genome promoter. Preferably, the AIV antigen is an AIV H9-HA antigen. [0049] In embodiments, the transcription of the entire CDV genome is under the control of the CDV genome promoter. Each gene in the CDV 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 CDV virus that is under the control of the CDV genome promoter and it does not mean that the heterologous polynucleotide encoding the AIV antigen is flanked by the CDV genome promoter. Alternatively, a recombinant promoter, e.g. a heterologous non-CDV 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.

[0050] In a preferred embodiment, the heterologous polynucleotide encoding the AIV antigen is inserted between the P and M genes and the expression of the AIV antigen is regulated by the CDV genome promoter. In a preferred embodiment, the heterologous polynucleotide encoding the AIV antigen is inserted between the P and M genes of the CDV vector genome of SEQ ID NO: 3 (or a sequence having % identity to SEQ ID NO: 3 as defined herein) and the expression of the AIV antigen is regulated by the CDV genome promoter. Preferably, the AIV antigen is an AIV H9-HA antigen.

[0051] In embodiments, the recombinant CDV vector comprises a polyadenylation (poly A) signal. In embodiments, the polyA signal is a simian virus 40 (SV40) polyA tail. In embodiments, expression of the AIV antigen is influenced by the polyA signal. In embodiments, the polyA signal is located downstream of the heterologous polynucleotide encoding the AIV antigen. Preferably, the AIV antigen is an AIV H9-HA antigen.

[0052] The recombinant CDV vector of the invention is useful as a vaccine against CDV and/or AIV, which can be administered to an animal to induce heterologous protection against CDV and/or AIV. In an embodiment of the recombinant CDV vector of the invention, the recombinant CDV vector is capable of inducing a protective immune response against CDV. In an embodiment, the recombinant CDV vector is capable of inducing a protective immune response against AIV. In an embodiment, the recombinant CDV vector is capable of inducing a protective immune response against CDV and AIV. In a preferred embodiment, the CDV vector is capable of eliciting an immune response against AIV in an avian. In a preferred embodiment, the CDV vector is capable of eliciting a protective immune response against AIV in an avian. In an embodiment, the protective immune response comprises a reduction of clinical signs caused by CDV and/or AIV. 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 CDV vector of the invention is capable of reducing shedding caused by AIV in an avian. In an embodiment, the recombinant CDV 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 CDV 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 animal or avian, such as an animal or avian that has not received a CDV and/or AIV vaccine and has been infected or challenged with CDV and/or AIV.

[0053] In an embodiment of the recombinant CDV vector of the invention, one dose of the recombinant CDV vector is capable of inducing a protective immune response against AIV and/or CDV. In an embodiment, one dose of the recombinant CDV vector is capable of inducing a protective immune response against AIV. In an embodiment, one dose of the recombinant CDV vector is capable of inducing a protective immune response against CDV. In an embodiment, this means that a protective immune response against AIV and/or CDV will be induced in an animal that has received (only) a single administration of the recombinant CDV vector of the invention. In an embodiment, this means that a protective immune response against AIV and/or CDV will be induced in an animal that has received (only) a single administration of any AIV and/or CDV vaccine or antigen, wherein the single administration is of the recombinant CDV 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 CDV. In an embodiment, it will be understood that the protective immune response is induced when (only) a single dose of the recombinant CDV vector of the invention has been administered. In an embodiment, “one dose” means that a second or further dose of the recombinant CDV 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 CDV 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 animal has not been primed against the same pathogen(s) to which the invention relates. For example, the animal 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 7.7 log 10 PFU. [0054] In an embodiment of the recombinant CDV vector of the invention, the recombinant CDV vector is capable of inducing a protective immune response against AIV and/or CDV 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 day of age, preferably one day of age. In an embodiment, the recombinant CDV vector of the invention is capable of inducing a protective immune response against AIV and/or CDV even when administered to an avian of one day of age.

[0055] In a second aspect, the present invention provides one or more nucleic acid molecules comprising the recombinant CDV vector of the invention.

[0056] In a third aspect, the present invention provides a cell comprising the recombinant CDV vector or the nucleic acid molecules of the invention.

Compositions

[0057] In a fourth aspect, the present invention provides a composition comprising the recombinant CDV vector, the nucleic acid molecules or the cell of the invention.

[0058] 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 avian influenza. In embodiments, the recombinant viral vectors of the present invention can be used in immunogenic compositions and vaccines to provide animals, such as avians with protection against avian influenza. In an embodiment, the recombinant viral vectors of the present invention can be used in immunogenic compositions and vaccines to elicit an immune response against CDV. In embodiments, the recombinant viral vectors of the present invention can be used in immunogenic compositions and vaccines to provide animals with protection against CDV.

[0059] In some embodiments, the immunogenic compositions are effective to elicit an immune response in an animal, such as an avian, when administered to the animal. In some embodiments, the immunogenic compositions are effective to induce an immune response in an animal, such as an avian, when administered to the animal. In some embodiments, the immunogenic compositions are effective to stimulate an immune response in an animal, such as an avian, when administered to the animal. 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 canine distemper when administered to an animal. In embodiments, the immunogenic compositions of the present invention are formulated such that they are safe and effective to elicit immunity against avian influenza when administered to an avian.

[0060] In embodiments, the vaccines are effective to elicit a protective immune response in an animal, such as an avian, when administered to the animal. In embodiments, the vaccines are effective to induce a protective immune response in an animal, such as an avian, when administered to the animal. In embodiments, the vaccines are effective to stimulate a protective immune response in an animal, such as an avian, when administered to the animal. In embodiments, the vaccines of the present invention are formulated such that they are safe and effective to elicit protective immunity against avian influenza when administered to an avian.

[0061] In embodiments, the immunogenic compositions and vaccines comprise a recombinant CDV vector comprising an avian influenza H9 heterologous polynucleotide that encodes an H9 antigen.

[0062] In embodiments, the immunogenic compositions and vaccines comprise a pharmaceutically or veterinarily acceptable carrier, excipient, and/or adjuvant. Examples of suitable 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.

[0063] In embodiments, the immunogenic compositions and vaccines are formulated for administration to an animal, such as 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. In a preferred embodiment, the immunogenic compositions and vaccines are formulated for administration by subcutaneous injection. [0064] In embodiments, the immunogenic compositions and vaccines can contain a titer of recombinant vector from 10² to 10³, 10³ to 10⁴, 10⁴ to 10⁵, 10⁵ to 10⁶, 10⁶ to 10⁷, 10⁷ to 10⁸, 10⁸ to 10⁹, 10⁹ to IO¹⁰, 10³ to 10⁸, 10⁴ to 10⁸, or 10⁵ to 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), TCID50 (50% Tissue Culture Infective Dose), EID50 (50% Egg Infective Dose), PFU (Plaque Forming Units), and FAID50 (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, applications and uses

[0065] In an embodiment, it is understood that the recombinant CDV vector of the present invention surprisingly induces a protective immune response against AIV due to the presence of the AIV (H9-HA) antigen. In an embodiment, it is understood that the recombinant CDV vector of the present invention will induce a protective immune response against CDV due to the presence of the CDV vector genome (e.g. the backbone of the recombinant CDV vector of the invention), which is preferably SEQ ID NO: 3 (or a sequence with % identity to SEQ ID NO: 3 as defined herein).

[0066] In a fifth aspect, the present invention provides the recombinant CDV 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 and/or CDV, preferably AIV, in an animal. Alternatively or additionally in a fifth aspect, the present invention provides the recombinant CDV 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.

[0067] In a sixth aspect, the present invention provides a method of inducing a protective immune response against AIV and/or CDV, preferably AIV, in an avian, comprising administering the recombinant CDV vector, the nucleic acid molecules, the cell or the composition of the invention to the animal. Alternatively or additionally 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 CDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian. [0068] In a seventh aspect, the present invention provides the recombinant CDV 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 and/or CDV, preferably AIV, in an animal. Alternatively or additionally in a seventh aspect, the present invention provides the recombinant CDV 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.

[0069] In an embodiment, inducing a protective immune response comprises reducing clinical signs associated with infection or challenge by AIV and/or CDV. In an embodiment, the clinical signs are respiratory clinical signs.

[0070] In an eighth aspect, the present invention provides the recombinant CDV 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.

[0071] In a ninth aspect, the present invention provides a method of reducing AIV shedding in an avian, comprising administering the recombinant CDV vector, the nucleic acid molecules, the cell or the composition of the invention to the avian.

[0072] In a tenth aspect, the present invention provides the recombinant CDV 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.

[0073] In an embodiment of the medical uses/methods of the present invention, the recombinant CDV vector is administered once. In an embodiment, this means that the recombinant CDV vector is only administered once. In an embodiment, this means that second/further administrations of the recombinant CDV vector are not carried out. In an embodiment, the recombinant CDV vector is administered once and the protective immune response and/or reduction in shedding is obtained after such single administration. In another embodiment, this means that the initial administration of the recombinant CDV 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 7.7 log 10 PFU.

[0074] In an embodiment of the medical uses/methods of the present invention, the recombinant CDV 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, one or fewer days of age, or one day of age. In a preferred embodiment, the recombinant CDV vector is administered to an avian at six or fewer days of age, preferably one day of age. In an embodiment, the recombinant CDV vector of the invention is administered to an avian of one day of age and induces a protective immune response against AIV.

[0075] In a preferred embodiment of the medical uses/methods of the present invention, the recombinant CDV vector is administered subcutaneously.

[0076] The present invention also provides methods of immunizing, methods for eliciting an immune response and methods for eliciting a protective immune response in an animal using any of the recombinant vectors, immunogenic compositions, and/or vaccines described herein. In embodiments, the animal is an avian. In embodiments, the animal is a chicken, for example a one day old chick.

[0077] In embodiments, the methods comprise administering to an animal, such as an avian, a recombinant vector according to the present invention.

[0078] In embodiments, the methods comprise administering to an animal, such as 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 canine distemper in an animal. In embodiments, the methods are effective to elicit, induce, and/or stimulate an immune response against avian influenza in an avian.

[0079] In embodiments, the methods comprise administering to an animal a vaccine comprising an effective amount of a recombinant vector according to the present invention. In embodiments, the animal is vaccinated/immunized against avian influenza and/or CDV. In embodiments, the methods are effective to elicit, induce, and/or stimulate a protective immune response against avian influenza and/or canine distemper in an animal, and thereby reduce and/or prevent clinical signs associated with subsequent avian influenza and/or CDV exposure, infection, challenge and/or disease in the animal, relative to a non-vaccinated control animal of the same species. In embodiments, the methods are effective to elicit, induce, and/or stimulate a protective immune response against avian influenza in an avian, and thereby reduce and/or prevent clinical signs associated with subsequent avian influenza 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 animal with protection against subsequent avian influenza virus and/or CDV infection or challenge, and clinical disease and signs associated therewith. In embodiments, the protective immune response is effective to provide the avian with protection against subsequent avian influenza virus infection or challenge, and clinical disease and signs associated therewith.

[0080] 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.

[0081] In an embodiment, the animals that the recombinant CDV vectors of the invention are administered to are chickens. In an embodiment, the animals are SPF chickens. In an embodiment, the animals are 1 day old SPF chickens.

Methods of manufacture

[0082] In an eleventh aspect, the present invention provides a method of manufacturing a recombinant CDV vector, the method comprising: a) providing one or more nucleic acids encoding a CDV vector; b) providing a polynucleotide encoding an AIV antigen; and c) recombinantly combining the nucleic acids encoding the CDV vector and the polynucleotide encoding the AIV antigen.

[0083] In an embodiment, the AIV antigen is an AIV haemagglutinin antigen. In an embodiment, the AIV antigen is an AIV subtype H9 haemagglutinin (AIV H9-HA) antigen.

[0084] In an embodiment, the polynucleotide encoding an AIV antigen is a polynucleotide of the invention as defined herein as SEQ ID NO: 1 (or a sequence with % identity to any of SEQ ID NO: 1 as defined herein, such as 80% identity to SEQ ID NO: 1). In an embodiment, the one or more nucleic acids encoding a CDV vector encode the CDV vector of SEQ ID NO: 3 (or a sequence with % identity to SEQ ID NO: 3 as defined herein, such as 80% identity to SEQ ID NO: 3)- [0085] In an embodiment, the method of manufacture further comprises step d) generating recombinant CDV particles from the recombinant nucleic acid obtained in step c). In an embodiment, the CDV particles are infectious CDV particles. In an embodiment, step d) of the method of manufacture involves a reverse genetics system for CDV. 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 CDV N, P and L proteins. For the avoidance of doubt, in an embodiment, the method steps a), b), c) (and d)) are typically in this order (step a) followed by step b) followed by step c) followed by step d) when present).

[0086] The present invention also provides methods of making recombinant CDV vectors comprising an avian influenza H9 heterologous polynucleotide that encodes a hemagglutinin antigen (AIV H9-HA).

[0087] In embodiments, the methods comprise inserting a polynucleotide encoding an avian influenza H9 antigen into a CDV backbone vector. In embodiments, the H9 antigen is from strain A/avian/Saudi Arabia/910135/2006(H9N2).

[0088] In embodiments, the methods further comprise inserting a promoter into the backbone vector. In embodiments, the avian influenza H9 heterologous polynucleotide is operably linked to a promoter, and expression of the H9 antigen is regulated by the promoter.

[0089] 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 heterologous polynucleotide.

General definitions

[0090] 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.

[0091] 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.

[0092] As used herein, the term “animal” includes all mammals and birds. The animal may be selected from equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), bovine (e.g., cattle), swine (e.g., pig), ovine (e.g., sheep, goats, lamas, bison), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), and humans. The term “animal” also includes an individual animal in all stages of development, including embryonic and fetal stages. In one embodiment, the animal referred to herein is a dog. In a preferred embodiment, the animals referred to herein are avians. In a most preferred embodiment, an avian is a chicken.

[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 herein by 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] 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 2. In an embodiment, 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.

[0099] As used herein, the term “gene” is used broadly to refer to any segment of polynucleotide associated with a biological function.

[0100] 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.

[0101] 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 different source, 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.

[0102] 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.

[0103] 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.

[0104] As used herein, a “protective immune response” comprises an “immunological response” to a composition or vaccine 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 to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display a 1 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.

[0105] As used herein, the terms “nucleic acid” and “polynucleotide” refer to RNA, DNA, and derivatives thereof. It is to be understood that unless otherwise specified each reference to a “heterologous polynucleotide”, “polynucleotide sequence”, “nucleotide sequence”, “nucleic acid” or equivalent term encompasses not only the nucleotide sequence per se, but also the complement of that nucleotide sequence, the reverse of that nucleotide sequence, and the reverse complement of that nucleotide sequence. Such references further encompass the RNA equivalent of that nucleotide sequence, the RNA complement (or transcript) of that nucleotide sequence, the reverse of the RNA equivalent of that nucleotide sequence and the reverse of the RNA complement (or transcript) of that nucleotide sequence. Unless otherwise indicated, such references further encompass any modifications or routine or non- substantive variations to any of those sequences, which are well-known in the art and further described herein.

[0106] 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.

[0107] As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of consecutive amino acid residues.

[0108] 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. [0109] 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.

[0110] As used herein, a “vaccine” is an immunogenic composition that is suitable for administration to an animal which, upon administration to the animal, 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).

[0111] The invention will now be further described by way of the following non-limiting examples.

EXAMPLES

EXAMPLE 1: Construction of vCDV-6 expressing AIV H9

[0112] This Example details the construction and characterization of a canine distemper virus (CDV) vector expressing avian influenza virus H9 gene from an H9N2 Saudi Arabia strain (vCDV-6).

A. Objective

[0113] A plasmid harboring the CDV Onderstepoort Haig genome was generated in which was inserted the avian influenza virus H9 gene which comes from an H9N2 Saudi Arabia strain.

B. CDV genome and insert genes characteristics

[0114] Vector: CDV transcription plasmid pCG177 (comprising a CDV Onderstepoort vaccine strain; SEQ ID NO: 3).

[0115] Insert: H9 gene from Influenza A virus (A/avian/Saudi Arabia/910135/2006(H9N2)) strain (Genbank: ACY80655.1). The gene has SEQ ID NO: 1. [0116] The H9 Saudi Arabia gene was synthesized by GeneArt (no. 1166890) and sub cloned in pIV29. Then the H9 Saudi Arabia gene was amplified by PCR and cloned into pCG177 to replace the existing CPIV2 HA gene using the Gibson Assembly Master Mix (NEB, France).

C. Construction of transcription plasmid

[0117] Plasmid name: pCG 219.

[0118] Promoter: T7.

[0119] Polyadenylation signal: none.

[0120] Resistance gene: Ampicillin.

[0121] Cloning: The H9 Saudi Arabia gene was inserted in the pCG177 by cloning using the Gibson Assembly Master Mix (NEB, France).

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

[0123] Plasmid Map: A map of plasmid pCG 219 is shown in FIG. 1.

1). Generation of a CDV vector expressing an avian influenza virus H9 gene from an H9N2 Saudi Arabia strain using reverse genetics methodology (vCDV-6).

[0124] The CDV is a negative RNA virus and the generation of genetically modified CDV virus can be accomplished with a reverse genetics system. The transcription of a full length genomic viral RNA and the simultaneous expression of N, 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 CDV virus and permit the generation of infectious particles. To generate modified CDV particles, the following reagents and conditions were used:

[0125] Transcription plasmid pCG219: The plasmid map is shown in FIG. 1. The amount transfected was 5 pg.

[0126] Expression N plasmid pCG171 : The plasmid map is shown in FIG. 2. The amount transfected was 0.5 pg.

[0127] Expression P plasmid pCG172: The plasmid map is shown in FIG. 3. The amount transfected was 0.5 pg.

[0128] Expression L plasmid pCG173: The plasmid map is shown in FIG. 4. The amount transfected was 0.5 pg.

[0129] T7 RNA polymerase plasmid pNS151: The amount transfected was 0.7 pg.

[0130] Cells and transfection: (a) Name and quantity of cells: CHO cells, 1.10⁶ cells/well (6- well plate); (b) Medium used : MEM glutamax + 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; (g) Incubation time: 48h; (h) Co-culture on VERO cells (0.7 x 10⁶ cells/well (6-well plate)); (i) Incubation 7 days; (j) Harvest: supernatant and cells.

[0131] Cell passages: VERO cells. Cell passage 2 had a titer of 5.28 loglO DICC50/ml. Cell passage 3 had a titer of 5.27 loglO DICC50/ml.

E. Characterization

[0132] Expression of the inserted gene: (a) Cells: VERO cells; (b) MOI: not calculated; (c) Incubation time: 7 days; (d) Antibody: chicken sera H9N2000919; (e) Ascites anti-CDV antibody Mab 319-E4-JB; (f) Techniques: IFI.

[0133] Expression results can be seen in FIG. 5.

EXAMPLE 2: AIV challenge study

A. Objective, study design, vaccines and vaccination

[0134] The objective of this study was to assess the efficacy of a recombinant CDV candidate vaccine expressing the H9 of Avian Influenza H9N2, when administered to SPF chicks at one day of age, against a H9N2 virulent challenge (strain Saudi Arabia 2010) carried out 21 days postvaccination. To reach this objective, the following parameters were monitored after challenge: (i) respiratory and ocular symptoms (clinical monitoring); and (ii) oro-pharyngeal viral excretion (assessed by qRT-PCR).

[0135] On DO, 24 SPF one-day-old chicks were identified, randomly allocated to two different groups (12 birds per group). G1 was treated with the CDV vaccine candidate expressing the H9 gene from the A/avian/Saudi Arabia/910135/2006 strain, and G2 was the unvaccinated control group, as indicated in Table 1 below:

Table 1 : Group constitution and treatment

[0136] Generation of vCDV6 is described in Example 1. The dose of the vaccine is shown in Table 1. The vCDV6 vaccine candidate was thawed, but not diluted before administration and was administered by the SC route.

B. Serology

[0137] Blood sample was taken at D20 to evaluate the serological response induced by the vaccine candidate. Hemagglutination-inhibition (HI) test (based on e.g. Kaufmann, L etal., JoVE 130: e55833 (2017)) was used to evaluate the anti-H9 antibodies using an H9N2 antigen (A/chicken/Irak/AV1342/2011 H9N2).

[0138] Dispersions of individual HAI titers on D20 by groups are presented in FIG. 10.

[0139] On D20, all control animals were seronegative. At the same date, seroconversion was observed in G1 (six animals positive out of twelve).

C. H9N2 challenge and clinical protection

[0140] From DO to D21, no clinical sign was recorded in groups G1 and G2 as part of daily observation. At D21, birds were challenged with the H9N2 A/chicken/Saudi Arabia/WNB9510/2010, also referred to herein as Saudi Arabia 2010. The challenge strain was diluted in physiological buffered saline (PBS) pH: 7.1 to obtain a challenge suspension with a titer of 9.0 loglO EID50/ml (being 8.54 loglO EID50/0.35 ml). All animals of groups G1 and G2 were challenged with 0.35 ml of the challenge solution by nebulization. In detail, animals from a same group were gathered in a nebulization box and nebulized with 4.2 ml 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. Chickens of all groups were daily monitored from D21 (before challenge) to D31. For this monitoring, particular attention was 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 2.

Table 2: Description and score of respiratory and ocular clinical signs. If an animal was found dead or euthanized on ethical grounds after challenge, it was attributed a score of 6 daily until the end of the study, when clinical signs or necropsy result could be associated to the H9N2 challenge. Clinical signs Score

Type Description

Slight: slight rale, sneezing 1

Respiratory Mild: severe rale, cough, tachypnea (increase of the respiratory frequency) 2

Severe: respiratory distress 3

Slight: conjunctivitis with oedema of the eyelid and excessive weeping 1

Ocular Mild: conjunctivitis with oedema of the eyelid and excessive weeping+ 2 periorbital oedema

Severe: conjunctivitis with oedema of the eyelid and excessive seeping + 3 periorbital oedema + yellow thick exudate

[0141] For each animal, a daily clinical score was calculated by adding the scores associated with respiratory and ocular symptoms (see Table 2) from D22 to D31. For each animal, a global clinical score was calculated by adding all of the daily clinical scores. Each group was compared to the control group on the criterion “Global clinical score” using a Mann- Whitney -Wilcoxon’s test. On D31, all surviving animals were humanely euthanized.

[0142] Evolutions of mean daily clinical scores by groups are presented in FIG. 6. Dispersions of individual global clinical scores by groups are presented in FIG. 7. Following challenge on D21, mainly slight respiratory signs were recorded in the different groups. Rales were sometimes associated with crackling and whistling. The onset of the clinical signs was 2 days post challenge (dpc) in the control group G2 and 3 dpc in Gl. Fewer animals presented clinical signs in group G1 (vCDV6) than in group G2 (controls).

[0143] In addition, one bird from group Gl was found dead during the challenge period. This bird did not present respiratory lesion.

1). H9N2 shedding

[0144] On D25, D27 and D29 (4, 6 and 8 days post-challenge), an oro-pharyngeal swabbing was carried out on all chicks of the six groups. The oro-pharyngeal H9N2 viral load was evaluated by qRT-PCR. When viral excretion was detected in a group, the area under curve (AUC) for the oro-pharyngeal H9N2 excretion from D25 to D29 was determined for each animal according to the trapezium method. [0145] 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). 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.

[0146] Evolutions of mean viral titers in oro-pharyngeal swabs and percentages of positive birds by groups are presented in FIG. 8. Dispersions of individual AUC of viral excretion by groups are presented in FIG. 9.

[0147] On D25 (4 days post-challenge) and D27 (6 days post-challenge), animals excreted virus in all groups with higher viral titers observed on D25 than on D27. On D29 (8 days postchallenge), no viral excretion was observed in the vCDV6 group (Gl), whereas animals carried on excreting virus in control group G2. The vCDV6 vaccinated group had a reduction of percentage of positive swabs at the three time points which was higher at 6 and 8 days post-challenge.

[0148] AUC of viral excretion in the vaccinated group Gl was significantly lower than that of the control group G2 (p = 0.0005).

E. Conclusion

[0149] A significant decrease of the viral excretion was observed in the group vaccinated with vCDV6 (Gl), and as noted above a decrease of the clinical signs was also highlighted.

SEQUENCE LISTING

SEQ ID NO: 1 (H9 gene of H9N2 Saudi Arabia strain) (vCDV-6) atggaacccatcagcctgatgatcatcctgctgctggtgacaaccagcaacgccgacaagatctgcatcggccaccagagcaccaacagc accgagacagtggacaccctgaccgagacaaacgtgcccgtgacccacgccaaagagctgctgcacaccgagcacaacggcatgctgt gcgccaccaacctgggccaccccctgatcctgaacacatgcaccatcgagggcctgatctacggcaaccccagctgcgatctgctgctgg gcggacgcgagtggtcctacatcgtggaaagacccagcgccgtgaacggcacatgctaccccggcaacgtggaaaacctggaagaact gagaaccctgttcagcagcagcagctcctaccagagaatccagatcttccccgacaccatctggaacgtgacctacaccggcaccagcaa gagctgcagcgacagcttctacagaaacatgagatggctgacccagaagaacggcctgtaccccgtgcaggacgcccagttcaccaaca acagaggcaaggacatcctgttcgtgtggggcatccaccacccccccaccgacaccgcccagaccaacctgtacaccagaaccgacac caccaccagcgtgaccaccgagaacctggacagaaccttcaagcccctgatcggccccagacccctggtgaacggcctgatcggcaga atcaactactattggagcgtgctgaagcccggccagaccctgagagtgcgcagcaacggcaacctgatcgccccttggtttggccacgtg ctgagcggcgagagccacggcagaatcctgaaaaccgacctgaacagcggcaactgcgtggtgcagtgccagaccgagaagggcgg cctgaacagcaccctgcccttccacaacatcagcaaatacgccttcggcacatgccccaagtacatcggcgtgaagtccctgaagctggcc atcggcctgaggaacgtgcccgccagaagcagcagaggcctgttcggcgctatcgccggcttcatcgagggcggctggccaggactgg tggccgggtggtacggcttccagcacagcaacgaccagggcgtgggcatggccgccgacagagacagcacccagaaagccgtggac aagatcaccagcaaagtgaacaacatcgtggacaaaatgaacaagcagtacgagatcatcgaccacgagttcagcgaggtggaaaccag actgaacatgatcaacaacaagatcgacgaccagatccaggacgtgtgggcctacaacgccgagctgctggtgctgctggaaaaccaga aaaccctggacgagcacgacgccaacgtgaacaatctgtacaacaaagtgaagagggccctgggcagcaacgccatggaagatggcaa gggctgcttcgagctgtaccacaagtgcgacgaccagtgcatggaaaccatccgcaacggcacctacaaccgcggcaagtacaaagag gaaagcagactggaaaggcagaaaatcgagggcgtgaagctggaaagcgagggcacatacaagatcctgaccatctacagcaccgtgg ccagcagcctggtgctggctatgggactggccgccttcctgttctgggccatgagcaacggcagctgcagatgcaacatctgcatctgatg a

SEQ ID NO: 2 (H9 of H9N2 Saudi Arabia strain protein sequence)(vCDV-6)

MEPISLMIILLLVTTSNADKICIGHQSTNSTETVDTLTETNVPVTHAKELLHTEHNGMLCA TNLGHPLILNTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGTCYPGNVENLEELRTL FSSSSSYQRIQIFPDTIWNVTYTGTSKSCSDSFYRNMRWLTQKNGLYPVQDAQFTNNRG KDILFVWGIHHPPTDTAQTNLYTRTDTTTSVTTENLDRTFKPLIGPRPLVNGLIGRINYYW SVLKPGQTLRVRSNGNLIAPWFGHVLSGESHGRILKTDLNSGNCWQCQTEKGGLNSTL PFHNISKYAFGTCPKYIGVKSLKLAIGLRNVPARSSRGLFGAIAGFIEGGWPGLVAGWYG FQHSNDQGVGMAADRDSTQI<AVDI<ITSI<VNNIVDI<MNI<QYEIIDHEFSEVETRLNMIN NKIDDQIQDVWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAMEDGKGCF

ELYHKCDDQCMETIRNGTYNRGKYKEESRLERQKIEGVKLESEGTYKILTIYSTVASSLV

LAMGLAAFLFWAMSNGSCRCNICI

SEQ ID NO: 3 (CDV Onderstepoort transfer vector sequence) gcgtccgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggagctagcataaccccttggggcctctaaacgggtc ttgaggggttttttgctgaaaggaggaactatatcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgg gatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagcct aacttcgatcactggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgcc ctataccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgctaacg gattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcgcgtcc gccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgcgcatgatcgtgctcctgtcgttgaggacc cggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtct gcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttccgg atctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtc ccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctct cgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggc ccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcac gaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgac ccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaat accgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcgg cgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcca gcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctc aagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctg ccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgt tcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaa gacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtg gcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatcc ggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatc ttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctttta aattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcg atctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaat gataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgca acttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctg caggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtg caaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgct cttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactc tcaaggatcttaccgctgtgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggt gagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattatt gaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccga aaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtcttcaagaattctc atgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatctaacaatg cgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatcgtcc attccgacagcatcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttctatgcgcacccgttctcggagcactgtccga ccgctttggccgccgcccagtcctgctcgcttcgctactggagccactatcgactacgcgatcatggcgaccacacccgtcctgtggatct gctaatacgactcactataggaccagacaaagttggctaaggatagttaaattattgaatattttattaaaaacttagggtcaatgatcctacctta aagaacaaggccagggttcagacctaccaatatggctagccttcttaaaagcctcacactgttcaagaggactcgggaccaaccccctcttg cctctggctccgggggagcaataagaggaataaagcatgtcattatagtcctaatcccgggtgattcaagcattgttacaagatctcgactatt ggatagactgtaggttggtggtgatccaaaaatcaacggccctaaattaactgggagcttaatcagtatcctctccttgtttgtggaatcccc tggacagttgatccagaggatcatagacgaccctgatgtaagcatcaagttagtagaggtaataccaagcatcaactctgtttgcggtcttaca tttgcatccagaggagcaagtctggattctgaggcagatgagttcttcaaaattgtagacgaagggtcgaaagctcaagggcaattaggctg gttagagaataaggatatagtagacatagaagttgataatgctgagcaattcaatatattgctagcttccatcttggctcaaatttggatcctgct agctaaagcggtgactgctcctgatactgcagccgactcggagatgagaaggtggattaagtatacccagcaaagacgtgtggtcggaga attagaatgaacaaaatctggcttgatattgttagaaacaggattgctgaggacctatctttgaggcgattcatggtggcactcatcttggacat caaacgatccccagggaacaagcctagaattgctgaaatgatttgtgatatagataactacattgtggaagctgggttagctagtttcatccta actatcaagtttggcatgaaactatgtatccggctcttgggttgcatgagttttccggagaattaacaactattgaatccctcatgatgctatatc aacagatgggtgaaacagcaccgtacatggttatcttggaaaactctgttcaaaacaaatttagtgcagggtcctacccattgctctggagtta tgctatgggggttggtgttgaacttgaaaactccatgggagggttaaatttcggtcgatcttactttgacccagcttacttcagactcgggcaag aaatggttaggagatctgccggcaaagtaagctctgcacttgccgccgagcttggcatcaccaaggaggaagctcagctagtgtcagaaat agcatccaagacaacagaggaccggacaattcgagctactggtcctaagcaatcccaaatcacttttctgcactcggaaagatccgaagtc gccaatcaacaacccccaaccatcaacaagaggtccgaaaaccagggaggagacaaataccccattcacttcagtgacgaaaggcttcc agggtataccccagatgtcaacagttctgaatggagtgagtcacgctatgacacccaaattatccaagatgatggaaatgacgatgaccgga aatcgatggaagcaatcgccaagatgaggatgcttactaagatgctcagtcaacctgggaccagtgaagatagttctcctgtttataatgata gagagctactcaattaaatattcaagaccagtcttgcatcagtcaacaattatcattctaaactcattataaaaaacttaggacccaggtccaac aaacccgatcaatcattcatccgaccacccgttctatccctaaatggcagaggaacaggcctaccatgtcagcaaagggctggaatgcctc aaagccctcagagagaatcctcctgacattgaggagattcaagaggtcagcagcctcagagaccaaacctgcaacccaggccaagagaa tggaaccacaggcatgcaggaagaggaggactctcagaatctcgatgaatcacacgagccaacaaaaggatcaaactatgtcggccatgt accccaaaataatccgggatgtggagaacgcaatactgcgcttgtggaggcagagcagccccctaaagaggacatccaaccaggacctg gaatacgatgtgatcatgtttatgatcacagcggtgaagaggttaagggaatcgaagatgctgacagtctcgtggtacctgcaggcactgtc ggtaatcgaggattcgagagaggagaaggaagccttgatgatagcactgaggattctggcgaagattattccgaaggaaatgcttcatctaa ctggggatattctttcggccttaaaccggacagagcagctgatgtgagcatgctgatggaagaggaattaagtgctctactcaggacaagca gaaatgtagggattcagaaaagggatgggaagactctgcagttcccacataatcccgaaggtaagacaagggatccggagtgtggatcca ttaaaaagggcacagaagagaggtcagtctcacatggaatggggatagttgctggatcgacaagtggtgcaacccaatctgcactcaagtc aactgggggatcatcagagccaagtgtttctgcggggaatgtccgccaacctgcaatgaatgcaaagatgacccagaaatgcaaactcga gtctggtacgcaactccctcccaggacctcaaatgaggctgagtctgacagtgagtacgatgatgagcttttctctgagatacaagaaattcg atctgccattactaaactaactgaagataatcaagcaatacttactaaactggataccttattactgcttaaaggagagactgattcaattaagaa acaaatcagcaaacaaaatattgctatttccacgattgaggggcatctatcaagcattatgatagctatacctggttttggaaaggacacggga gatcctacggcaaatgtcgacattaacccagagctccgccctatcatagggagagattcaggaagagcactagcagaagttctcaagcagc ccgcatcatcccgcggtaatcggaaggacagtggtattactctgggctcaaaaggtcaactattgagagacctccagctgaaacccattgac aaagagtctagctcggcaatcggatacaaaccgaaggataccgcaccttccaaagctgtacttgcatcattgattagatcaagcagagttgat caaagtcacaaacataacatgctggctctgctcaaaaatatcaagggggatgacaacctaaacgagttctaccaaatggtcaaaagtattact catgcttaatctgtagcgttgactaatctactaaccggcgcaaaactgctttcactatcgcttaaaagcaattataaaaaacttaggacacaaga gcctaagtcctctcctaaaaaatgactgaggtgtacgacttcgatcagtcctcttgggacaccaaaggctcattggcccctattttgcctacca cttatcccgatggtaggctcataccccaagtcagagtaatagatccaggactcggcgatcggaaagatgaatgcttcatgtatatttttttactg ggtataatagaagacaatgatggcctcggacccccaattggaagaacatttggatcgctgcctttaggagttgggcgtactacagccagacc tgaggagttattgaaagaagccaccctgttggatattgtggtaaggcgaactgcaggtgtcaaggaacaactggtattttataataacacccc attgcacatcttaactccgtggaaaaaggtccttacgagtggaagtgtgttcagtgcaaatcaagtctgtaacgcagtcaatctaataccattag acatagcacaaagatttagggtggtatatatgagcatcactcgactatcagacgatggaagttacagaattccccgcgggatgtttgaattcc gctccaggaatgctttagcatttaacattttagtcaccattcaagttgagggagatgtcgattcaagccgaggtaatttgagcatgttcaaagat caccaagcgacattcatggtacatatcggcaatttcagccgcaagaaaaaccaagcctactctgctgattattgtaaactgaaaattgaaaag atgggattagtgtttgctctaggagggataggaggaacgagtcttcacatacgatgtactggtaagatgagcaaggccttgaatgcccagct aggtttcaagaaaatcctgtgttacccgctcatggagatcaatgaagatttgaatcgatttctatggagatcagagtgcaaaatagtaagaatc caagcagtcctgcaaccatcagtcccacaagatttcagagtttataatgatgttatcatcagcgatgatcagggtcttttcaaaattctctaaatc atcagttcatgaactaaaaagcaaacgccttagtagcactgcccaagatcccttgatccccgcaagcgaggattgagggtataagcaccga ccatccagacgttgctcctgcattttgagtgtgtcccataagcctccaaaccgctcactcgtgcccacaactccagtgacgcctcgatacgaa agcagccgaaccaaaacagctcttgcccaagattaggttgatcattataggaccaagaaatgaatggatgcctggggtttttagcttcgcttct aggtatctcacttaacaatatactcccacgcacttgcctgatctcaagctatcactagtagtcctgtttcacggaactatgactatccatctttct atcacagctcattaataattaatcaaaacttagggtccaggacatagcaagccaacaggtcaaccaggtccaccagccaggggccggaca ggaacccccacaaacagccgagccccatgcacaagggaatccccaaaagctccaaaccccaaacacatacccaacaagaccgccccc cacaacccagcaccgaacccgaagagaccaggacctcccgagcacgacacagcataacatcagctcagcgatccacgcactctgatcct cgaacatcggacagacccgtctcctacaccatgaacaggatcaggtcccgcaagcaaactagccacagattgaagaacatcccagttcac ggaaaccacgaggctattatccagcacatgccagagagtgtctcaaaaggagcgagatcccagatcgaaaggcggcaacccaatgcaat caactcaggctctcagtgcacctggttagtcctgtggtgcctcggaatagccagtctctttctgtgttccaaggctcagatacattggaataattt gtcaactattgggattatcgggactgatagtgtccattacaagatcatgactaggcccagtcaccagtacttggtcataaaactgatgcctaat gtttcacttatagataattgtaccaaagcagaattaggtgagtatgagaaattattgaattcagtcctcgaaccaatcaaccaagctttgactcta atgaccaagaatgtgaagcccctgcagtcattagggtcaggtaggagacaaaggcgttttgtaggagtggtacttgcaggtgcagctttagg agtggctacagctgcacaaatcactgcaggaatagctttacatcaatccaacctcaatgctcaagcaatccaatctcttagaaccagccttga acagtctaacaaagctatagaagaaattagggaggccacccaagaaaccgtcattgccgttcagggagtccaggactacgtcaacaacga actcgtccctgccatgcaacatatgtcatgtgaattagttgggcagagattagggttaagactgcttcggtattatactgagttgttgtcaatattt ggcccgagtttacgtgaccctatttcagccgagatatcaattcaggcactgagttatgctcttggaggagaaattcataagatacttgagaagtt gggatattctggaggtgatatgattgcaatcttggagagtcgggggataaaaacaaaaataactcatgttgatctccccgggaaattcatcatc ctaagtatctcatacccaactttatcagaagtcaagggggttatagtccacaggctggaagcagtttcttacaacataggatcacaagagtggt acaccactgtcccgaggtatatgcaactaatggttacttaatatctaattttgatgagtcatcttgtgtattcgtctcaaagtcagccatttgtagc cagaactccctgtatcccatgagcccactcttacaacaatgtataggggcgacacttcatcttgtgctcggaccttggtatctgggactatgg gcaacaaatttattctgtcaaaaggtaatatcgtcgcaaattgtgcttctatactatgtaagtgttatagcacaagcacaattattaatcagagtcc tgataagttgctgacatttattgcctccgatacctgcccactggttgaaatagatggtgttactatccaagttggaggcaggcaataccctgata tggtatacgaaggcaaagttgccttaggccctgctatatcacttgagaggttagatgtaggtacaaatttagggaacgcccttaagaaactgg atgatgctaaggtactgatagactcctctaaccagatccttgagacggttaggcgctcttcctttaattttggcagtctcctcagcgttcctatatt aagttgtacagccctggctttgttgttgctgatttactgttgtaaaagacgctaccaacagacactcaagcagcatactaaggtcgatccggca tttaaacctgatctaactggaacttcgaaatcctatgtgagatcactctgaagtattctggtcatatatctcgcttgattgccaggtttgaaatctatt gaccccgcccaattttcttcaaaagtcactcaactgcaataaacattggaaaagactgaccatgattatcgtgattaaagaaaacttagggctc aggtagtccagcaatgctctcctaccaagacaaggtgggtgccttctacaaggataatgcaagagccaattcaaccaagctgtccttagtga cagaagaacatgggggcaggagaccaccttatttgtgtttgtccttctcatctattggttggaatcctggcctgctgctatcactggagttcg atttcaccaagtatcaactagcaatatggaatttagcagattgctgaaagaggatatggagaaatcagaggccgtacatcaccaagtcataga tgtcttgacaccgctcttcaagattattggagatgagattgggttacggttgccacaaaagctaaacgagatcaaacaatttatccttcaaaaga caaatttcttcaatccgaacagagaattcgacttccgcgatctccactggtgcattaacccgcctagtaaggtcaaggtgaattttactaattact gtgagtcaattgggatcagaaaagctattgcatcggcagcaaatcctatccttttatcagccctatctgggggcagaagtgacatattcccacc acacagatgcagtggagctactacttcagtaggcaaagttttccccctatcagtctcattatccatgtctttgatctcaagaacctcagagataat caatatgctgaccgctatctcagacggcgtgtatggcaaaacttacttgctagtgcctgatgatatagaaagagagttcgacactcaagagatt cgagtctttgaaatagggttcatcaaaaggtggctgaatgacatgccattactccaaacaaccaactatatggtactcccggagaattccaaa gccaaggtatgtactatagcagtgggtgagttgacactggcttccttgtgtgtagaagagagcactgtattattatatcatgacagcagtggttc acaagatggtattctagtagtgacactggggatattttgggcaacacctatggatcacattgaggaagtgatacctgtcgctcacccatcaatg gagaaaatacatataacaaaccaccggggttttataaaagattcaattgcaacctggatggtgcctgccctggcctctgagaaacaagaaga acaaaaaggttgtctggagtcagcttgtcaaagaaaaacctaccccatgtgcaaccaaacgtcatgggaacccttcggaggaagacagttg ccatcttatgggcggttgacattacctctagatgcaagtgttgaccttcaacttaacatatcgttcacatacggtccggttatactgaatggagat ggtatggattattatgaaagcccacttttgaactccggatggcttaccattcctcccaaaaacggaacaatctctggattgataaacaaagcag gtagaggagaccagttcactgtactcccccatgtgttaacatttgcgcccagggaatcaagtggaaattgttatttacctattcaaacatctcaa attatagatagagatgtcctcattgagtccaatatagtggtgttgcctacacagagttttagatatgtcatagcaacgtatgacatatcacgaagt gatcatgcgatgtttattatgtttatgacccaatccggacgatttcttatacgcacccatttagactaactaccaagggtagacctgatttcctaa ggatgaatgttttgtgtgggataacaatttgtggtgtcaccaattttacagattcgaggctgacatcgccaactctacaaccagtgttgagaatt tagtccgtataagattctcatgtaaccgttaaaatccctgacagtatgatgatacacatctcaattggccttaggcatgataactgtggtgagaa atcccttacagacgattgaattaaaccatctctagcattataaaaaaactaaggatccaagatcctttcagccatggactctgtatcagtgaacc agattctataccctgaggtccatctagatagcccaattgtaaccaataagctagtatctattttagaatacgcacgaattagacataactatcagc tccttgatacaacattagtgcgtaatatcaaagagagaatttcagaagggttctcaaaccagatgatcattaactgcatcgaaattgggagcatt attaatcagaccttgtatcttatcccaaacacaaccatgtgatatacccaaattgcaacaaacttctatttcatgcacaggatcgagtcatctctc tgaggctgagaaatatattcaaaagaggaaatagcatctatagcaaaataacagacggggtcaaaaaatgcttaaacgatattaatcttaatat tggtttagggggtgcactggacaagactattgggaccaaaattgatgaagcaggcataattatgcaaagctcacagtggttcgaacctttcctt ctatggtttacaattaaaacagaaatgagatcagtgattaaatcctctactcacaactgtcgcaagcggaggcagaaccctgtctttgtaaaag gtgaatcatgaatgtgtagtctctagggaccttgtatgtattattgacctcaccagtcacattgtttattacctaacatttgaaatggtcctgatgt actgtgatgtaatagaagggaggctaatgactgatactgctatggcaattgatcaacgttactcaactttgcatgtcaggatcaggtatctctgg gatctaattgacggattttcccggatctgggaaattcaacctatcaattggtggctctactggagcctctctcattggcttacttgcaattaaaag acatcaccttctctctcaggggtgcttttctgagtcactgcttgctgaaattcaggagattttacaggacaatggcttctatactgaagagacgtt ccaaacttaacccaagctctagacttcgttttcatcacagaggatatacatataacaggagaaatcttttccttctttagaagtttcggtcaccca aggttagaagcaataacagcagcagagaacgtacggaaacacatgaatcaacccaaagttgtctcctatgagactatgatgaagggacac gctatatctgtgggataatcattaacggttatcgggatagacatggggggacttggcctccgatggatcttcctgttcatgcatctcctatcatc agaaatgctcatgcctcaggggagggaatcacctatagtcaatgtatagaaaattggaaatccttcgcaggaattcgatttaaatgctttatgc ctcttagcctagacagtgatttgaccatgtacctgaaagataaggctttggcagccctaagaaaagagtgggactcagtgtacccaaaagaa tcctcaggtacaatccacctcgctccactgagtctcggagactgttaatgtgtttctagaggactctcagtttgacccttataacatgattatgt acgttatctcaggtcaatatctagaagatcctgatttcaacctatcatacagtctcaaagagaaagagattaaagaggtagggagattattcgct aaaatgacctacaaaatgcgagcctgtcaggtcatagcagaaaacttgatatctaatggaattgggaagtacttcaaggacaatgggatggc aaaggatgaacacgatctcactaaatcattgcacactctggctgtgtccggggttccgaaagacaagaaagattcccatcgtggcctcacta accagcgtaaatccctgaagcctgcaccttatcgaggagcccttcactccgtctcttccccaagtagtagatatatagacccaaacccaaattt ttgcaccagtagaagagaagacaatgacatagagatctatgaaactgtaagtgcatttataactacagatctcaaaaagtactgtctgaattgg cgtatgagaccatcagtatttttgctcagagattaaatgaaatctatggtctcccctcatttttccaatggttgcacagaagattggaacagtcga tctatacgtaagtgacccccactgccctccagatctcgatcgtcatgtggacttgaatacagcccctaactctcaaatattcatcaaataccca atgggaggggtggaggggtattgtcagaagttatggactattagcaccataccttatctgtacttggcggcacatgagagtggtgtcagaatt gcatcacttgtccaaggtgataaccaaactattgctgtcactaaaagagtaccaagcacctggtcctatgccttgaagaaatctgaagccagt cgagtgaccacagaatactttatagccttgagacagaggttacatgatgtcggacatcatttgaaagcaaatgaaacaataatctcttcccactt ttttgtatactcaaaaggaatctattatgatggaatgttaatttcacaatccctgaagagtatagctaggtgtgtattttggtcagaaacaatagtg gatgagacccgagccgcgtgcagcaacatttcaacaacattagcgaaagccattgagaaagggtttgaccgatatttagcctacacgctga acattttaaaaatcatccaacaagtattaatttcattaggattcactatcaattcagcgatgacacgggatgtgatagaacccctcttacaagatc actgtctctgaccaagatggcaattctccccgcacccattggcggttttaattacctcaatatgagtaggctctttgtcaggaatatcggggatc ccgtgacatctctattgctgacctcaaacgaatgatccgatcaggccttcttggagtggagattctacatcaggtcatgactcaatacccaggt gactcttctatttagatgggcaagtgacccttattctgccaatctgccctgtgtccagagcataacccgactccttaaaaatatcacagctagg catgtccttatcaacagtccaaatccgatgctgagaggattgttccatgatgaaagtcaggatgaggatgaagctttagcagctttcttgatgg ataggaaaattattatcccaagggctgcacatgaaattctagataacacgatcacgggtgcaagagaggcaattgccggaatgctagacac cacaaaggggttgataagagcaagcatgaaaagaggaggtctaacccctagaataataacccgtttgtcaacttacgattatgagcaatttag ggcaggtatcagactgttgtcagggaaggggcatgatccgctcatcgatcaagactcatgttccgtccagttagcgagagcattaaggaac cacatgtgggccaagctggcgaagggtcgtcctatttatggtctagaagtcccggatatccttgaatcaatgaagggttatatgatcagaaga catgaatcctgttgctttgcgcatcaggctctcataactatggttggttttttataccagcgaattgccaattggatagtattacagagggaacat ctgcactgagggtgccatacatagggtccacaacagaagaaagaacagacatgaaactagcattcgtcaaatctcctagtaggtctctaaaa tcagcagtgagaatagcaactgtgtactcatgggcctatggtgatgatgacgaatcttggcaagaggcttggaccttggcaaaacagagag cgaacatctcacttgaggaattacggatgattaccccaatttccacttctactaatctagctcaccgactaagagacaagagtactcaagtcaa atactcagggacctctctcatcagagtagcacgttatgcaactatctcgaatgataatctttcttttattatagatgacaagaaagtggacacaaa ttattatcaacaaggtatgctcctgggcctggggatccttgagcacttatttagattgtcttcaaccaccggcgactctaacaccgtgttacat ttacatgttgaaacagattgttgcgtaatacccatgagtgaccatccaagagtcccagggctcagaaaggtcgtcataccaagaaatgtttgta caaatcctttgatttatgacagtaaccctattattgagaaagatgcagtcagactttataaccagagtcacagaaagcacattgtagagtttgtca catggacaacagggcagctttatcatgtgctagctaaatctactgctatgtctatggttgagatgattacaaagtttgaaaaggaccacctaaat gaagtcactgcgttaatggcgatgatgatatcaatagttttatcactgagtttcttctagtgagcctagattatttactgtatatctaggtcaatgt gctgcaatcaactggggctttgaaattcattatcaccgaccttctggaaagtaccaaatgggtgagttgttgttctctttcctgagtagaatgagt aaaggagtcttcaaaattttaaccaatgcattgagccatcctaaagtatatagacggttttgggacagtgggatgattgaacctgttcatggacc ctctcttgactcccaaaacctacatataactgtatgcaacctgatctataactgttacatgatttacctagaccttctgttaaatgatgaattagatg atttctcattcattttatgcgaaagtgacgaggatgtcatacctgaaagatttgacaacatacaagccaggcacctatgcatctatctgaccttta ttgtaaccctcgtgattgtccccagattcgtgggttgacaccaacacagaaatgtgctgtgttgtcggggtacttaaaatcaaaagccctagaa tcccatgttggtctgacatggaatgacaaacctatcttaatagatcaatattcatgttccctgacatatcttagaagaggctcaatcaagcagata agattgagagtggatcccggattcatcactgatgctgttggatgcttagaaaggcgtcctctaagaaataattctacctctaaggcctcagaatt aaagtcagaatttgacccaccgaaagatgacttggctaaacttctgagtcagctgtcaacaaggacacataacttacctattacaggattagg agtccggaactatgaggttcactcattcagaagaattgggatcaactctactgcatgttacaaggccgttgaaatagcttctgtgattaagaac gaatttacgtctgaagaacacggattattcctaggagaaggttcaggtgcaatgttgacagtatataaagagctattaagattgtcaagatgtta ttataacagtggtgtgtcggtagaatccagaactggacaacgagagatttcaccttacccttctgaggtcagtctggtggaacatcaattagga ctcgataaattggtgactgtgcttttcaatgggagaccagaagtaacttgggttgggagtgttgattgttacaagtacatactgagccagatctc tgctagcagtcttgggttgattcactcggatatagagtcactaccggataaagacataattgaaaagttggaagaattgtctgctatattatcaat gactttgatattagggaaggtagggtcagtgttagtaattaagatcatgccagttagtggcgactgggttcaaggatttattttgtatgcactccc acatttcttcgaagttcatagtttacccaagatacagcaattttgtgtcaacagaggcctaccttgtttttaccggtcttagagcagggagacta gtcaatcctgaggggattaaacaacagattttgcgagtcggtattcgaacttcacccgggttggtagggcacatcctttcatcaaagcagaca gcatgtgtgcagtcttgcatggacctccatttcatgctaaatcttcaatcctcaccttcagggtttaacaagtattgagaaggtattaatcaattg tgggcttacaattaatggtcttaaagtatgtaagaacctgcttcaccatgatatttcgtcaggcgaggaagggctgaaaggatctatcacgatc ctttaccgggaactcgcaaggttcaaggataaccaccaatcttcacatggaatgttccatgcataccctgtgttaatcgcaagtcaggaaagg gagctcgtatctatcattgcaaagaagtactgtggctatattttgctttactcgggagacttatacgaaattaccaggattgtccgaaacctgaaa gccaaccacataattttcgacctgcatcgtaatttattcatggataatctgtccagatctgacaggtctctcatcctaacgacaatccccaaaaa gaattggctctttcagctcgagacaaaagagataaaggagtggttcaaattattaggttatagtgcactgattagaaatcactgacaggttagtc tggctcctagcccccttctattcattgctattaaacttagttatacgaaaaaaaacaacggttattaataagttatcatacccagctttgtctggtgg gtcggcatggcatctccacctcctcgcg

31

Claims

1. A recombinant Canine Distemper Virus (CDV) vector comprising a heterologous polynucleotide encoding an Avian Influenza Virus (AIV) antigen.

2. The recombinant CDV vector of claim 1, wherein the AIV antigen is an AIV haemagglutinin antigen.

3. The recombinant CDV vector of claim 1 or 2, wherein the AIV antigen is an AIV subtype H9 haemagglutinin (AIV H9-HA) antigen.

4. The recombinant CDV vector of any one of claims 1 to 3, wherein the polynucleotide encoding the AIV antigen comprises a sequence having at least 80% identity to SEQ ID NO: 1, and/or the AIV antigen comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2.

5. The recombinant CDV vector of any one of claims 1 to 4, wherein the heterologous polynucleotide is inserted between the P gene and the M gene in the CDV genome.

6. The recombinant CDV vector of any one of claims 1 to 5, wherein the CDV vector is an Onderstepoort strain CDV vector, such as a vector having at least 80% identity to SEQ ID NO:3.

7. The recombinant CDV vector of any one of claims 1 to 6, wherein the CDV vector is capable of eliciting an immune response against AIV and/or CDV in an animal, preferably capable of eliciting an immune response against AIV in an avian.

8. The recombinant CDV vector of any one of claims 1 to 7, wherein the CDV vector is capable of eliciting a protective immune response against AIV and/or CDV in an animal, preferably capable of eliciting a protective immune response against AIV in an avian.

9. One or more nucleic acid molecules comprising the recombinant CDV vector of any one of claims 1 to 8.

10. A cell comprising the recombinant CDV vector of any one of claims 1 to 8 or the nucleic acid molecules of claim 9.

11. A composition comprising the recombinant CDV vector of any one of claims 1 to 8, the nucleic acid molecules of claim 9 or the cell of claim 10.

12. The recombinant CDV vector of any one of claims 1 to 8, the nucleic acid molecules of claim 9, the cell of claim 10 or the composition of claim 11, for use in a method of inducing a protective immune response against AIV and/or CDV, preferably against AIV, in an animal.

13. A method of inducing a protective immune response against AIV and/or CDV, preferably against AIV, in an animal, comprising administering the recombinant CDV vector of any one of claims 1 to 8, the nucleic acid molecules of claim 9, the cell of claim 10 or the composition of claim 11 to the animal.

14. The recombinant CDV vector of any one of claims 1 to 8, the nucleic acid molecules of claim 9, the cell of claim 10 or the composition of claim 11, for use in the manufacture of a medicament for inducing a protective immune response against AIV and/or CDV, preferably against AIV, in an animal.

15. The use or the method of any one of claims 12 to 14, wherein inducing a protective immune response comprises reducing clinical signs associated with infection by AIV and/or CDV.

16. The use or the method of claim 15, wherein the clinical signs are respiratory clinical signs.

17. The use or the method of any one of claims 12 to 16, wherein the protective immune response is provided against avian influenza and the animal is an avian.

18. The recombinant CDV vector of any one of claims 1 to 8, the nucleic acid molecules of claim 9, the cell of claim 10 or the composition of claim 11 , for use in a method of reducing AIV shedding in an avian.

19. A method of reducing AIV shedding in an avian, comprising administering the recombinant CDV vector of any one of claims 1 to 8, the nucleic acid molecules of claim 9, the cell of claim 10 or the composition of claim 11 to the avian.

20. The recombinant CDV vector of any one of claims 1 to 8, the nucleic acid molecules of claim 9, the cell of claim 10 or the composition of claim 11, for use in the manufacture of a medicament for reducing AIV shedding in an avian.

21. The use or the method of any one of claims 12 to 20, wherein the recombinant CDV vector is administered once and wherein the protective immune response and/or reduction in shedding is obtained after such single administration.

21. The use or the method of any one of claims 12 to 20, wherein the recombinant CDV vector is administered to an avian at six or fewer days of age, preferably at one day of age.

22. The use or the method of any one of claims 12 to 20, wherein the recombinant CDV vector is administered subcutaneously.

23. A method of manufacturing the recombinant CDV vector of any one of claims 1 to 8, the method comprising: a) providing one or more nucleic acids encoding a CDV vector; b) providing a polynucleotide encoding an AIV antigen; and c) recombinantly combining the nucleic acids encoding the CDV vector and the polynucleotide encoding the AIV antigen.