OA19476A - Recombinant vectors expressing antigens of avian influenza virus and uses thereof. - Google Patents

Abstract

The present invention provides recombinant viral vectors that contain and express antigens of avian pathogens, compositions comprising the recombinant viral vectors, polyvalent vaccines comprising the recombinant viral vectors. The present invention further provides methods of vaccination against a variety of avian pathogens and method of producing the recombinant viral vectors.

Description

VIRUS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application daims priority to U.S. provisional application 62/410,885 filed on October 21, 2016.

FIELD OF THE INVENTION

The invention relates to recombinant viral vectors for the insertion and expression of foreign genes for use as safe immunization vehicles to protect against a variety of pathogens. It also relates to multivalent composition or vaccine comprising one or more recombinant viral vectors for protection against a variety of pathogens. The présent invention relates to methods of making and using the recombinant viral vectors.

BACKGROUND OF THE INVENTION

Influenza virus is a member of Orthomyxoviridae family (Murphy and Webster, Orthomyxoviruses, Fields Virology, Third Edition, vol. 1, pp. 1397-1445, 1996). There are three types of influenza viruses designated A, B, and C. The influenza virion contains a segmented negative-sense RNA genome. The influenza virion includes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (Ml), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2) proteins. The NP and the matrix protein Ml are used to classify the influenza virus into group A, B or C.

The HA and NA proteins are envelope glycoproteins. The HA protein is responsible for virus attachment and pénétration of the viral particles into the cell and contains the major immunodominant epitopes for virus neutralization and protective immunity. Both HA and NA proteins are considered the most important components for prophylactic influenza vaccines. To date, eighteen different HA subtypes and eleven different NA subtypes hâve been

T identified (Tong et al., 2013, PLoS Pathogens, Vol. 9 (10), New World Bats harbor diverse influenza A viruses).

Globally, influenza is the most economically significant respiratory disease in humans, pigs, horses and poultry. Influenza virus is known for its continuous genetic and antigenic changes, which impede effective control of the virus. Of particular concem for prévention of épidémies and pandémies is the emergence of a new subtype of the virus by genetic reassortment or inter-species transmission.

The highly pathogenic Influenza A virus subtype H5N1 virus is an emerging avian influenza virus (AIV) that has been causing global concern as a potential pandémie threat. H5N1 has killed millions of poultry in a growing number of countries throughout Asia, Europe and Africa. Health experts are concemed that the co-existence of human flu viruses and avian flu viruses (especially H5N1) will provide an opportunity for genetic material to be exchanged between species-specific viruses, possibly creating a new virulent influenza strain that is easily transmissible and léthal to humans (Food Safety Research Information Office. A Focus on Avian Influenza. Created May 2006, Updated November 2007). US8,394,384 reported the making of avian influenza vaccines using plant expression System. US7,910,l 12 disclosed poxvirus vectored vaccines against avian influenza. US8,592,558 disclosed vaccines containing H5 proteins. WO2007019094 studied the immunogenicity of the HA molécule when spécifie HA residues are substituted.

During December 15, 2014 and January 16, 2015, the U.S. Department of Agriculture received 14 reports of birds infected with Asian-origin, highly pathogenic avian influenza A (HPAI), including H5N2 viruses. These reports represent the first reported infections with these viruses in U.S. wild or domestic birds. Although these viruses are not known to hâve caused disease in humans, their appearance in North America might increase the likelihood of human infection in the United States (Morbidity and Mortality Weekly Report, Centers for Disease Control and Prévention, February 6, 2015 / 64(04),111; Bertran et al., 2016, Virology 494, 190-197).

Considering the susceptibility of animais, including humans, to AIV, a method of preventing AIV infection and protecting animais is essential. Accordingly, there is a need for methods to produce effective vaccines against influenza.

SUMMARY OF THE INVENTION

The présent invention relates to a recombinant herpesvirus of turkey (HVT) vector or fowlpox virus (FPV) vector comprising one or more heterologous polynucleotides coding for and expressing at least one antigen of an avian pathogen.

The présent invention provides a composition or vaccine comprising one or more recombinant HVT or FPV vectors comprising one or more heterologous polynucleotides coding for and expressing at least one antigen of an avian pathogen.

The présent invention also provides a polyvalent composition or vaccine comprising one or more recombinant HVT or FPV vectors comprising heterologous polynucleotides coding for and expressing at least one antigen of an avian pathogen and one or more recombinant HVT or FPV vectors comprising heterologous polynucleotides coding for and expressing at least one antigen of an avian pathogen.

The présent invention relates to a method of vaccinating an animal, or inducing an immunogenic or protective response in an animal, comprising at least one administration of the composition or vector of the présent invention.

The présent invention showed surprising resuit that the recombinant viral vectors expressing the modified HA protein provided better protection in avian than the recombinant viral vectors expressing the mutant HA protein. The présent invention also demonstrated that polyvalent compositions or vaccines comprising the viral vectors were effective to protect animais against a variety of avian pathogens without interférence.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, and which is not intended to limit the invention to spécifie embodiments described, may be understood in conjunction with the accompanying figures, incorporated herein by reference, in which:

Figures 1 A and IB depicts a table showing the SEQ ID NO assigned to each DNA and protein sequence.

Figure 2 depicts the genome structure of HVT and its insertion sites. The UL55 insertion site is shown.

Figure 3 depicts pHVTIGISVLPC-HAsyn Sbfl plasmid map.

Figure 4 depicts the dual immunofluorescent staining of recombinant vHVT501 virus expressing LPC-HA H5N2 protein.

Figure 5 depicts the schematic représentation of primer binding sites.

Figure 6 depicts the PCR results to identify rHVT50l.

Figure 7 depicts pHVTIGlHHV3gBroSVLPC-HAsyn Sbfl plasmid map.

Figure 8 depicts the dual immunofluorescent staining of recombinant vHVT502 virus expressing LPC-HA H5N2 protein.

Figure 9 depicts the schematic représentation of primer binding sites.

Figure 10 depicts the PCR results to identify rHVT502.

Figure 11 depicts pHVTIGISVMut-HAsyn Sbfl plasmid map.

Figure 12 depicts the dual immunofluorescent staining of recombinant vHVT503 virus expressing 3 Mut-HA H5N2 protein.

Figure 13 depicts the schematic représentation of primer binding sites.

Figure 14 depicts the PCR results to identify rHVT503.

Figure 15A depicts pCD046-H5N2 HA plasmid map.

Figure 15B depicts the schematic représentation of primer binding sites.

Figure 15C depicts the PCR results to identify rHVT510.

Figure 16 depicts the schematic représentation of the position of the F8 insertion site within the fowlpox virus (TROVAC) genome.

Figure 17 depicts the cloning steps of fowlpox virus donor plasmid pF8 H6pLPC-HA H5N2.

Figure 18 depicts the schematic représentation of primer binding sites for plasmid pF8 H6pLPC-HA H5N2.

Figure 19 depicts the PCT results to identify rFPV3003.

Figure 20 depicts the cloning steps of fowlpox virus donor plasmid pF8 H6p3Mut-HA H5N2.

Figure 21 depicts the schematic représentation of primer binding sites for plasmid plasmid pF8 H6p3Mut-HA H5N2.

Figure 22 depicts the PCT results to identify rFPV3004.

Figure 23A depicts the rHVT-H5 virus shedding data in RNA copy number/10 ul.

Figure 23B depicts the serology results in A/Turkey/Minnesota/125 82/2015 (H5N2) homologous challenge.

Figure 23C depicts the serology results in A/Egypt/N04915/2014 (H5N1) heterologous challenge.

Figure 23D depicts the viral shedding results in A/Turkey/Minnesota/12582/2015 (H5N2) homologous challenge.

Figue 23E depicts the viral shedding results in A/Egypt/N04915/2014 (H5N1) heterologous challenge.

Figures 24A-24G depict the DNA and protein sequence alignments.

DETAILED DESCRIPTION OF THE INVENTION

It is noted that in this disclosure and particularly in thedaims, terms such as “comprises”, “comprised”, “comprising” and the like can hâve the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of ’ and “consists essentially of” hâve the meaning ascribed to them in U.S. Patent law, e.g., they allow for éléments not explicitly recited, but exclude éléments that are found in the prior art or that affect a basic or novel characteristic of the invention.

Unless otherwise noted, technical terms are used according to conventional usage. Définitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V. published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Référencé, published by VCH Publishers, Inc., 1995 (ISBN 1-56081569-8).

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 indicate otherwise. The word “or” means any one member of a particular list and also includes any combination of members of that list.

The term about as used herein, means approximately, in the région 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%. In one aspect, the term about means plus or minus 20% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include ail 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). It is also to be understood that ail numbers and fractions thereof are presumed to be modified by the term about.

The term “animal” is used herein to include ail mammals, birds and fish. The animal as used herein may be selected from the group consisting of 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, bisons), 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), humans, and fish. The term “animal” also includes an individual animal in ail stages of development, including embryonic and fêtai stages.

The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of consecutive amino acid residues.

A particular antigenic polypeptide of interest is hemagglutinin (HA). Influenza hemagglutinin refers to a type of hemagglutinin found on the surface of the influenza viruses. It is an antigenic glycoprotein and is responsible for binding the virus to the cell that is being infected. There are different HA antigens, any of which can be used in the practice of the invention. Of interest is the HA from H5N2, a highly pathogenic avian flu virus. However, HA from other influenza viruses (i.e. H1 -H 16) may be used in the practice of the invention including Hl, H3, H5, H6, H7, H9 and the like. It is further recognized that HA precursors of any of the HA proteins can be used.

HA is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem région. Both régions carry N-linked oligosaccharides, which plays an important rôle in the biological function of HA (Schulze, I.T., J Infect Dis, 1997. 176 Suppl 1: p. S24-8; Deshpande, K.L., et al., PNAS USA, 1987, 84(1): p. 36-40). Among different subtypes of influenza A viruses, there is signifîcant variation in the glycosylation sites of the head région, whereas the stem oligosaccharides are more conserved and required for fusion activity (Ohuchi, R., et al., J Virol, 1997, 71(5): p. 3719-25). Glycans near antigenic peptide eptiopes interfère with antibody récognition (Skehel, J.J., et al., PNAS USA, 1984, 81(6): p. 1779-83), and glycans near the proteolytic site modulate cleavage and influence the infectivity of influenza virus (Deshpande, K.L., et al., 1987). Nucléotide sequence analysis of 62 H5 genes supported the hypothesis that additional glycosylation near the receptor binding site within the HA globular head is an adaptation of the virus following interspecies transmission from wild birds, particularly waterfowl, to poultry (Banks, J., et al., Avian Dis, 2003, 47(3 Suppl): p. 942-50).

The term “nucleic acid”, “nucléotide”, and “polynucleotide” are used interchangeably and refer to RNA, DNA, cDNA, or cRNA and dérivatives thereof, such as those containing modified backbones. It should be appreciated that the invention provides polynucleotides comprising sequences complementary to those described herein. The “polynucleotide” contemplated in the présent invention includes both the forward strand (5’ to 3’) and reverse complementary strand (3’ to 5’). Polynucleotides according to the invention can be prepared in different ways (e.g. by Chemical synthesis, by gene cloning etc.) and can take various forms (e.g. linear or branched, single or double stranded, or a hybrid thereof, primers, probes etc.).

The term “genomic DNA” or “genome” is used interchangeably and refers to the heritable genetic information of a host organism. The genomic DNA comprises the DNA of the nucléus (also referred to as chromosomal DNA) but also the DNA of the plastids (e.g., chloroplasts) and other cellular organelles (e.g., mitochondria). The genomic DNA or genome contemplated in the présent invention also refers to the RNA of a virus. The RNA may be a positive strand or a négative strand RNA. The term “genomic DNA” contemplated in the présent invention includes the genomic DNA containing sequences complementary to those described herein. The term “genomic DNA” also refers to messenger RNA (mRNA), complementary DNA (cDNA), and complementary RNA (cRNA).

The term “gene” is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes or polynucleotides include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs , such as an open reading frame (ORF), starting from the start codon (méthionine codon) and ending with a termination signal (stop codon). Genes and polynucleotides can also include régions that regulate their expression, such as transcription initiation, translation and transcription termination. Thus, also included are promoters and ribosome binding régions (in general these regulatory éléments lie approximately between 60 and 250 nucléotides upstream of the start codon of the coding sequence or gene), transcription terminators (in general the terminator is located within approximately 50 nucléotides downstream of the stop codon of the coding sequence or gene). Gene or polynucleotide also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a spécifie protein, and which includes regulatory sequences.

The term “heterologous DNA” as used herein refers to the DNA derived from a different organism, such as a different cell type or a different species from the récipient. The term also refers to a DNA or fragment thereof on the same genome of the host DNA wherein the heterologous DNA is inserted into a région of the genome which is different from its original location.

As used herein, the term “antigen” or “immunogen” means a substance that induces a spécifie immune response in a host animal. The antigen may comprise 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 présentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof. Altemately, the immunogen or antigen may comprise a toxin or antitoxin.

The term “immunogenic protein or peptide” as used herein includes polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. Preferably the protein fragment is such that it has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the invention comprises or consists essentially of or consists of at least one epitope or antigenic déterminant. An immunogenic protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. By immunogenic fragment is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molécule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic résonance.

The term “immunogenic protein or peptide” further contemplâtes délétions, additions and substitutions to the sequence, so long as the polypeptide fonctions to produce an immunological response as defined herein. The term conservative variation dénotés the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucléotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, méthionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the substitution of one hydrophobie residue such as isoleucine, valine, leucine or méthionine for another hydrophobie residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid that will not hâve a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molécule but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the définition of the reference polypeptide. Ail of the polypeptides produced by these modifications are included herein. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

The term epitope refers to the site on an antigen or hapten to which spécifie B cells and/or T cells respond. The term is also used interchangeably with antigenic déterminant or antigenic déterminant site. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

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 either a therapeutic or protective immunological response such that résistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a réduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

The terms “recombinant” and “genetically modified” are used interchangeably and refer to any modification, alteration or engineering of a polynucleotide or protein in its native form or structure, or any modification, alteration or engineering of a polynucleotide or protein in its native environment or surrounding. The modification, alteration or engineering of a polynucleotide or protein may include, but is not limited to, délétion of one or more nucléotides or amino acids, délétion of an entire gene, codon-optimization of a gene, conservative substitution of amino acids, insertion of one or more heterologous polynucleotides.

The terms “polyvalent vaccine or composition”, “combination or combo vaccine or composition” and “multivalent vaccine or composition” are used interchangeably to refer to a composition or vaccine containing more than one composition or vaccines. The polyvalent vaccine or composition may contain two, three, four or more compositions or vaccines. The polyvalent vaccine or composition may comprise recombinant viral vectors, active or attenuated or killed wild-type viruses, or a mixture of recombinant viral vectors and wild-type viruses in active or attenuated or killed forms.

One embodiment of the invention provides a recombinant HVT viral vector comprising one or more heterologous polynucleotides coding for and expressing at least one antigen or polypeptide of an avian pathogen. The HVT strains used for the recombinant viral vector may be any HVT strains, including, but not limited to, the HVT strain FC 126 (Igarashi T. et al., J. Gen. ViroL 70, 1789-1804, 1989).

Another embodiment of the invention provides a recombinant poxvirus viral vector comprising one or more heterologous polynucleotides coding for and expressing at least one antigen or polypeptide of an avian pathogen. The poxvirus may be a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. The canarypox virus or fowlpox virus strains may be attenuated strains, such as an attenuated recombinant canarypox virus, for instance ALVAC (U.S. Pat. No. 5,756,103), or an attenuated fowlpox virus, for instance TROVAC (U.S. Pat. No. 5,766,599, 5,174,993, 5,505,941). Both ALVAC and TROVAC include dérivatives that hâve been passaged from the parental strains of ALVAC or TROVAC, and/or the progenies or descendants of ALVAC or TROVAC. In one embodiment, the recombinant TROVAC vaccine may be used as an avian influenza vaccine. In the case of fowlpox, the insertion site or sites are ORFs F7 and/or F8. The F8 insertion locus corresponds to the fowlpox gene encoding photolyase described by Srinivasan and Tripathy (2005, Veterinary Microbiology 108: 215-223). This gene is also described under the name FPV158 in the complété sequence of the fowlpox genome (GenBank accession No. AF198100.1). In the case of canarypox, the insertion site or sites are ORF(s) C3, C5 and/or C6; see also documents cited herein, especially those pertaining to canarypox virus.

Thus, the viral vector in the invention can be any suitable recombinant virus or virus vector, such as a poxvirus (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., human adenovirus, canine adenovirus), herpesvirus (e.g. canine herpesvirus), baculovirus, retrovirus, etc.

Another embodiment of the invention provides a recombinant NDV viral vector comprising one or more heterologous polynucleotides coding for and expressing at least one antigen or polypeptide of an avian pathogen. The NDV strains may be any NDV strains, including, but not limited to, Ulster 2C, Queensland V4, Hitchner Bl, F (e.g., Asplin), La Sota, strain H, Mukteswar, Roakin, Beaudette C, Texas GB, NY parrot 70181, Italien, Milano, Herts 33/56, and AVINEW®.

The genes coding for antigen or polypeptide may be those coding for avian influenza virus HA protein. The antigen or polypeptide may be any antigen from the poultry pathogen of avian influenza virus. The avian influenza virus may be any subtype AIV, including, but not limited to, H5, H7 and H9.

Moreover, homologs of aforementioned antigens or polynucleotides are intended to be within the scope of the présent invention. As used herein, the term “homologs” includes orthologs, analogs and paralogs. The term “analogs” refers to two polynucleotides or polypeptides that hâve the same or similar function, but that hâve evolved separately in unrelated organisms. The term “orthologs” refers to two polynucleotides or polypeptides from different species, but that hâve evolved from a common ancestral gene by spéciation. Normally, orthologs encode polypeptides having the same or similar functions. The term “paralogs” refers to two polynucleotides or polypeptides that are related by duplication within a genome. Paralogs usually hâve different functions, but these functions may be related. Analogs, orthologs, and paralogs of a wild-type polypeptide can differ from the wild-type polypeptide by post-translational modifications, by amino acid sequence différences, or by both. In particular, homologs of the invention will generally exhibit at least 80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98%, 99% sequence identity, with ail or part of the polynucleotide or polypeptide sequences of antigens described above, and will exhibit a similar function.

In one embodiment, the présent invention provides a recombinant HVT or FPV viral vector comprising one or more heterologous polynucleotides coding for and expressing the AIV-HA antigen or polypeptide. In one aspect of the embodiment, the AIV-HA antigen or polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having the sequence as set forth in SEQ ID NO:2, 4, 20, 21, 22, 23,

24, 25, 26, 27, and 28 or a conservative variant, an allelic variant, a homolog or an immunogenic fragment comprising at least eight or at least ten consecutive amino acids of one of these polypeptides, or a combination of these polypeptides. The AIV-HA antigen or polypeptide may be modified at the cleavage site between HAÏ and HA2 from a highly pathogenic avian influenza sequence (multiple basic amino acids: RERRRKR- SEQ ID NO: 14) to a low pathogenic avian influenza sequence (RETR - SEQ ID NO: 15). In another aspect of the embodiment, the heterologous polynucleotide encoding an AIV-HA antigen or polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having the sequence as set forth in SEQ ID NO:2, 4, 20, 21, 22, 23, 24, 25, 26, 27, and 28. In yet another aspect of the embodiment, the heterologous polynucleotide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a polynucleotide having the sequence as set forth in SEQ ID NO: 1, 3, 8, 9, 10, 12, 13 17, or 19.

Variants include allelic variants. The term allelic variant refers to a polynucleotide or a polypeptide containing polymorphisms that lead to changes in the amino acid sequences of a protein and that exist within a natural population (e.g., a virus species or variety). Such natural allelic variations can typically resuit in 1- 5% variance in a polynucleotide or a polypeptide. Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different species, which can be readily carried out by using hybridization probes to identify the same gene genetic locus in those species. Any and ail such nucleic acid variations and resulting amino acid polymorphisms or variations that are the resuit of natural allelic variation and that do not alter the functional activity of gene of interest, are intended to be within the scope of the invention.

The term “identity” with respect to sequences can refer to, for example, the number of positions with identical nucléotides or amino acids divided by the number of nucléotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman). The sequence identity or sequence similarity of two amino acid sequences, or the sequence identity between two nucléotide sequences can be determined using Vector NTI software package (Invitrogen, 1600 Faraday Ave., Carlsbad, CA). When RNA sequences are said to be similar, or hâve a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.

The polynucleotides of the disclosure include sequences that are degenerate as a resuit of the genetic code, e.g., optimized codon usage for a spécifie host. As used herein, “optimized” refers to a polynucleotide that is genetically engineered to increase its expression in a given species. To provide optimized polynucleotides coding for AIV-HA polypeptides, the DNA sequence of the AIV-HA protein gene can be modified to 1) comprise codons preferred by highly expressed genes in a particular species; 2) comprise an A+T or G+C content in nucléotide base composition to that substantially found in said species; 3) form an initiation sequence of said species; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, dégradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites. Increased expression of AIV-HA protein in said species can be achieved by utilizing the distribution frequency of codon usage in eukaryotes and prokaryotes, or in a particular species. The term “frequency of preferred codon usage” refers to the preference exhibited by a spécifie host cell in usage of nucléotide codons to specify a given amino acid. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, ail degenerate nucléotide sequences are included in the disclosure as long as the amino acid sequence of the AIV-HA polypeptide encoded by the nucléotide sequence is functionally unchanged.

Successful expression of the heterologous polynucleotides by the recombinant/modified infectious virus requires two conditions. First, the heterologous polynucleotides must be inserted or introduced into a région of the genome of the virus in order that the modified virus remains viable. The second condition for expression of inserted heterologous polynucleotides is the presence of a regulatory sequences allowing expression of the gene in the viral background (for instance: promoter, enhancer, donor and acceptor splicing sites and intron, Kozak translation initiation consensus sequence, polyadenylation signais, untranslated sequence éléments).

The insertion site may be any non-essential région of the HVT genome, including, but not limited to, the région between the STOP codon of ORF UL55 and the junction of UL with the adjacent repeat région (intergenic région 1, the IG1 locus, US5,980,906), the IG2 (intergenic région 2) locus, the IG3 (intergenic région 3) locus, the UL43 locus, the US 10 locus, the US2 locus, the SORF3/US2 locus (see FIG. 2)

In general, it is advantageous to employ a strong promoter functional in eukaryotic cells. The promoters include, but are not limited to, an immédiate early cytomégalovirus (CMV) promoter, mouse CMV IE promoter, guinea pig CMV promoter, an SV40 promoter, Human Herpesvirus Type III glycoprotein B (HHV3gB) promoter, Pseudorabies Virus promoters such as that of glycoprotein X promoter, Herpes Simplex Virus-1 such as the alpha 4 promoter, Marek's Disease Viruses (including MDV-1, MDV-2 and HVT) promoters such as those driving glycoproteins gC, gB, gE, or gl expression, Infectious Laryngotracheitis Virus promoters such as those of glycoprotein gB, gE, gl, gD genes, or other herpesvirus promoters.

One embodiment of the invention provides a recombinant HVT vector comprising a heterologous polynucleotide coding for and expressing the AIV-HA antigen or polypeptide. In one aspect of the embodiment, the polynucleotide encoding the AIV-HA polypeptide is operably linked to the SV40 promoter having the sequence as set forth in SEQ ID NO:5 and therefore the expression of the AIV-HA antigen or polypeptide is regulated by the SV40 promoter. In another aspect of the embodiment, the polynucleotide encoding the AIV-HA polypeptide is operably linked to the mCMV promoter having the sequence as set forth in SEQ ID NO: 16 and therefore the expression of the AIV-HA antigen or polypeptide is regulated by the mCMV promoter. In yet another aspect of the embodiment, the expression of AIV-HA antigen or polypeptide is regulated by the synthetic polyA signal having the sequence as set forth in SEQ ID NO:7. In yet another aspect of the embodiment, the expression of AIV-HA antigen or polypeptide is regulated by the SV40 PolyA having the sequence as set forth in SEQ ID NO: 18. In yet another aspect of the embodiment, the polynucleotide encoding the AIV-HA polypeptide is operably linked to the HHV3gB promoter in reverse orientation having the sequence as set forth in SEQ ID NO:6 and therefore the expression of the NDV-F antigen or polypeptide is regulated by the HHV3gB promoter and/or SV40 promoter. In one aspect, the polynucleotide encoding the AIV-HA polypeptide is codon-optimized. In yet another aspect, the polynucleotide encoding the AIVHA polypeptide is wild-type DNA.

Another embodiment of the invention provides a recombinant FPV vector comprising a heterologous polynucleotide coding for and expressing the AIV-HA antigen or polypeptide. In one aspect of the embodiment, the polynucleotide encoding the AIV-HA polypeptide is operably linked to linked to vaccinia H6 (H6) promoter having the sequence as set forth in SEQ ID NO:11 and therefore the expression of the AIV-HA antigen or polypeptide is regulated by the H6 promoter.

In another embodiment, the présent invention provides a pharmaceutical composition or vaccine comprising an HVT or FPV viral vector comprising a polynucleotide encoding an AIV-HA antigen, and optionally a pharmaceutically or veterinarily acceptable carrier, excipient, vehicle or adjuvant. In another embodiment, the présent invention provides a pharmaceutical polyvalent composition or vaccine comprising two or more viral vectors comprising polynucleotides encoding AIV-HA antigens, and optionally a pharmaceutically or veterinarily acceptable carrier, excipient, vehicle or adjuvant. The viral vectors may be HVT vector or FPV vector.

The pharmaceutically or veterinarily acceptable carriers or adjuvants or vehicles or excipients are well known to the one skilled in the art. Other pharmaceutically or veterinarily acceptable carrier or adjuvant or vehicle or excipients that can be used for methods of this invention include, but are not limited to, 0.9% NaCl (e.g., saline) solution or a phosphate buffer, poly-(L-glutamate) or polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable carrier or vehicle or adjuvant or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro), or facilitating transfection or infection and/or improve préservation of the vector (or protein). Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue expérimentation.

Optionally other compounds may be added as pharmaceutically or veterinarily acceptable carriers or adjuvants or vehicles or excipients, including, but not limited to, alum;

CpG oligonucleotides (ODN), in particular ODN 2006, 2007, 2059, or 2135 (Pontarollo R.A. et al., Vet. Immunol. Immunopath, 2002, 84: 43-59; Wernette C.M. et al., Vet. Immunol. Immunopath, 2002, 84: 223-236; Mutwiri G. et al., Vet. Immunol. Immunopath, 2003, 91: 89103); polyA-polyU, dimethyldioctadecylammonium bromide (DDA) (“Vaccine Design The Subunit and Adjuvant Approach”, edited by Michael F. Powell and Mark J. Newman, Pharmaceutical Biotechnology, 6: p.03, p. 157); N,N-dioctadecyl-N’,N’-bis(2-hydroxyethyl) propanediamine (such as AVRIDINE®) {Ibid, p. 148); carbomer, chitosan {see US Patent Serial No. 5,980.912 for example).

The pharmaceutical compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants. Suitable adjuvants for use in the practice of the présent invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl dérivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units (Klinman et al., 1996; WO98/16247), (3) an oil in water émulsion, such as the SPT émulsion described on p 147 of “Vaccine Design, The Subunit and Adjuvant Approach” published by M. Powell, M. Newman, Plénum Press 1995, and the émulsion MF59 described on p 183 of the same work, (4) cation lipids containing a quatemary ammonium sait, e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (9) any combinations or mixtures thereof.

In one embodiment, the adjuvant may include TS6 TS7, TS8 and TS9 (US7,371,395), LR2, LR3 and LR4 (US7,691,368), TSAP (US20110129494), TRIGEN™ (Newport Labs), synthetic dsRNAs (e.g. poly-IC, poly-ICLC [HILTONOL®]), and MONTANIDE™ adjuvants (W/O, W/O/W, O/W, IMS and Gel; ail produced by SEPPIC).

In one embodiment, the invention provides for the administration of a therapeutically effective amount of a vaccine or composition for the delivery of recombinant HVT and FPV vectors in a target cell. Détermination of the therapeutically effective amount is routine expérimentation for one of ordinary skill in the art.

Another aspect of the invention relates to a method for inducing an immunological response in an animal against one or more antigens or a protective response in an animal against one or more avian pathogens, which method comprises inoculating the animal at least once with the vaccine or pharmaceutical composition of the présent invention. Yet another aspect of the invention relates to a method for inducing an immunological response in an animal to one or more antigens or a protective response in an animal against one or more avian pathogens in a prime-boost administration regimen, which is comprised of at least one primary administration and at least one booster administration using at least one common polypeptide, antigen, epitope or immunogen. The immunological composition or vaccine used in primary administration may be same, may be different in nature from those used as a booster. The prime-boost protocol according to the invention comprises a primary administration followed by at least a second administration to boost the response.

In one aspect of the prime-boost protocol of the invention, a composition or vaccine comprising a recombinant viral vector that contains and expresses an avian influenza antigen in vivo is administered followed by the administration of a composition or vaccine comprising a recombinant viral vector that contains and expresses an avian influenza antigen in vivo. The recombinant viral vector used in prime-administration may be different in nature from those used as a later booster recombinant vector. The viral vector used in the prime-administration may be selected from HVT or FPV, and the viral vector used in the boost-administration may be selected from HVT or FPV that is different from the viral vector used in the prime administration. It is noted however that the prime and boost composition or vaccine may contain the same recombinant vector. It is also noted that the prime and boost composition or vaccine may be polyvalent position or vaccine which may contain two or more viral vectors.

A prime-boost regimen may comprise at least one prime-administration and at least one boost administration using at least one common polypeptide or antigen. A prime-boost regimen may also comprise at least one prime-administration and at least one boost administration using different polypeptides or antigens. The prime-administration may comprise one or more administrations. Similarly, the boost administration may comprise one or more administrations.

The avian pathogens may be Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (i.e., IBDV or Gumboro Disease virus), Marek’s Disease Virus (MDV), Infectious Laryngotracheitis Virus (ILTV), avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian metapneumovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, avian parvovirus, avian astrovirus and chick anémia virus coccidiosis (Eimeria sp), Campylobacter sp., Salmonella sp., Mycoplasma gallisepticum, Mycoplasma synoviae, Pasteurella sp., Avibacterium sp., E. coli or Clostridium sp.

Usually, one administration of the vaccine is performed either at one day-of-age by the subcutaneous or intramuscular route or in ovo in 17-19 day-old embryo. A second administration can be done within 5-30 days after the First administration.

A variety of administration routes in day-old chicks may be used such as subcutaneously or intramuscularly, intradermally, transdermally. The in ovo vaccination can be performed in the amniotic sac and/or the embryo. Commercially available in ovo and SC administration devices can be used for vaccination.

The composition or vaccine may contain a dose from about 10² to about 10²⁰, about 10³ to about 10¹⁸, about 10⁴ to about 10¹⁶, about 10⁵to about 10¹² VLPs (virus like particles) produced in vitro or in vivo from a viral vector, a plasmid, or baculovirus. The viral 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), PFU (Plaque Forming Units), and FAID₅₀ (50% Fluorescent Antibody Infectious Dose), and the VLPs produced in vitro can be titrated by hémagglutination assay, ELISA, and électron microscopy. Other methods may also be applicable depending on the type of VLP. The composition or vaccine may contain from about 10^{2 0} to about 10^{10 0} pfu/dose of viral vector.

The dose volumes can be between about 0.1 and about 10 ml, between about 0.1 and about 5 ml.

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

EXAMPLES

Construction of DNA inserts, plasmids and recombinant viral vectors was carried out using the standard molecular biology techniques described by J. Sambrook et al. (Molecular

Cloning: A Laboratory Manual, 4th édition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2014).

Example 1 Construction of recombinant HVT vectors expressing H5N2-HA Example 1.1 Construction of recombinant ElVT5 01 expressing E15N2-HA The objective of the study is to construct a recombinant HVT virus in which an expression cassette containing Simian Virus 40 (SV40) promoter, Avian Influenza Virus Hemagglutinin (HA) glycoprotein, and a synthetic poly A tail is inserted in the intergenic site of UL55 in HVT virus (Fig. 2).

The parental virus used in the construct is HVT FC 126. An Avian Influenza Virus Heagglutinin (HA) glycoprotein (named LPC-HA) corresponding to H5N2-HA sequence (SEQ ID NO:2 encoded by SEQ ID NO:1, codon-optimized) was chemically synthesized (GenScript). The H5N2-HA (LPC-HA) was derived from a highly pathogenic avian influenza A virus (A/chicken/Washington/61-9/2014(H5N2) isolate (GenBank accession No. KP739381.1). The synthetic HA (LPC-HA) gene was modified at the cleavage site between HAÏ and HA2 from a highly pathogenic avian influenza sequence (multiple basic amino acids: RERRRKR - SEQ ID NO: 14) to a low pathogenic avian influenza sequence (RETR SEQ ID NO: 15).

The promoter is Simian Virus 40 (SV40) promoter (SEQ ID NO:5). The insertion locus is intergenic site of UL55 (IG1) in HVT (Fig. 2). Donor plasmid pHVTIGlSVLPCHAsyn Sbfl (an insertion plasmid containing the UL55 flanking régions of HVT virus+SV40+synthetic poly A) was constructed as described below. Chicken embryo fibroblast cells (CEF) were used for in vitro recombination.

Donor plasmid construction

To construct the donor plasmid pHVTIGISVLPC-HAsyn Sbfl, a fragment encompassing the synthetic H5N2-HA gene was excised from LPC-HA H5N2 in pUC57 (synthesized by GeneScript) using Notl and inserted into the same site as the pHVTIG2SVCaFsyn Sbfl plasmid containing SV40 promoter, NDV-F gene, and synthetic polyA tail which was also Notl digested and CIP treated, to replacing the NDV-F gene with the LPC-HA. The Notl digested insert (LPC-HA) and vector (pHVTIG2) was gel extracted using Qiagens Gel Extraction Kit and then ligated. Ligated material was transformed using Top 10 Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp broth and plasmid was extracted using Qiagens MiniSpin Prep kit. The plasmids were screened for insert orientation using Smal digestion. The correct donor plasmid was designated pHVTIG2SVLPC-HAsyn Sbfl. pHVTIG2SVLPC-HAsyn Sbfl mini-prep plasmid was transfected onto one well of a 6-well plate and an IFA was performed using chicken antisera against Avian Influenza H5N2 (Charles Rivers Laboratories, Lot# J0210) and antichicken FITC (Sigma, Cat# F8888). Once transient expression was verified the plasmid was grown in a larger scale culture and plasmid extraction was done by using Qiagens Maxi Prep kit.

An Sbfl digest was performed on the pHVTIG2SVLPC-HAsyn Sbfl maxi prep and pIGlHHV3gBroCaFoptsyn Sbfl maxi prep (a previously constructed and sequence verified plasmid). The SV40 H5N2-HA synthetic poly A tail expression cassette flanked by Sbfl restriction enzymes was gel extracted from the pHVTIG2SVLPC-HAsyn Sbfl plasmid using Qiagens Gel Extraction Kit. The pIG!HHV3gBroCaFoptsyn Sbfl vector flanked by Sbfl restriction enzymes was also gel extracted and CIP treated. The pHVTIG2SVLPC-HAsyn Sbfl expression cassette was ligated to the pIGlHHV3gBCaFsyn Sbfl vector. Ligated material was transformed using Top 10 Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp broth and plasmid was extracted using Qiagens MiniSpin Prep kit. The plasmids were screened for insert orientation using EcoRI+Smal digestion. An anti-genome orientation plasmid was selected, grown in a larger scale culture, and plasmid extraction was done by using Qiagens Maxi Prep kit. This plasmid was sequence verified and designated pHVTIGISVLPC-HAsyn Sbfl (Fig. 3). Recombinant génération

A standard homologous recombination procedure was followed by co-electroporation of secondary CEF cells using pHVTIGISVLPC-HAsyn Sbfl donor plasmid and viral DNA isolated from HVT FC 126 virus. Co-electroporation was performed using 1x10⁷ 2° CEF in 300 μΐ Opti-MEM and shocked at 150 volts with 950 capacitance in a 2 mm electroporation cuvette. The transfected cells were seeded into 96-well plate and incubated for 4 days. The cells grown in the 96-well plate were then duplicated into two 96-well plates and incubated for 3 more days. One set of 96-well plates was used for IFA using chicken polyclonal sera against Avian Influenza H5N2 to identify positive wells containing recombinants and another set of 96-well plates was used for recovering the infected cells from the positive wells.

The recombinant viral purification methods were performed First by 96-well plate duplication and IFA sélection for the wells containing the most IFA positive plaques with the least amount of IFA négative plaques. Wells matching those criteria were then harvested and adjusted to 1ml in DMEM+2% FBS. From the 1ml stock 5-20ul (depending on the number of visible plaques) were removed and mixed with IxlO⁷ CEFs in 10ml DMEM+2% FBS and aliquoted onto a new 96-well plate in an attempt to hâve single HVT plaques per well. The 96-well plates were duplicated after 3 days of incubation and wells that contained plaques were tested for the presence of recombinant HVT and absence of parental virus by IFA and PCR. Again the wells that appeared to hâve more recombinant virus, by comparing the PCR banding results, were harvested and adjusted to 1ml and aliquoted onto new 96-well plates (the same as before). After three rounds of purification of virus infected cells, recombinant HVT expressing LPC-HA protein was isolated and the purity of the recombinant virus was tested by IFA and PCR to confirm the absence of parental virus. Selected recombinant virus was then passed from one well of a 96-well plate (PO) to 2xT-25 flasks (PI), then 2xT-75 flasks (P2), then 2xT-150 flasks (P3), and finally 3x850 cm² roller bottles (pre-MSV stock or P4). Vials with 2 ml aliquot were stored in liquid nitrogen.

Analysis of recombinant by PCR

DNA was extracted from a stock virus by phenol/chloroform extraction; éthanol precipitated, and resuspended in 20mM HEPES. PCR primers were designed to specifically identify the H5N2-HA gene, the promoter, the poly A, as well as, the purity of the recombinant virus from HVT parental virus. PCR was performed using 200 pg of DNA template along with the specified primers pairs indicted in Table 1. PCR cycling conditions are as follows (unless otherwise noted): 94°C - 2 min; 30 cycles of 94°C - 30 sec, 60°C - 45 sec, 68°C - 2 min; 68°C - 3 min. Expression analysis

For immunofluorescence testing, the P4 material was diluted 1:100 in media. Approximately 50 μΐ of the diluted virus was added to 10 ml of DMEM+2% FBS with IxlO⁷

CEFs and then aliquoted onto a 96 well plate (100 μΐ/well). The plates were incubated for 3 days at 37°C+5%CO₂ until viral plaques were visible. The plates were fixed with 95% icecold acetone for three minutes and rinsed gently three times with water. Chicken anti-sera against Avian Influenza H5N2 (lot#J0210, Charles Rivers Laboratory) at 1:500 and HVT Mab L78 (lot#072103, Merial) at 1:3000 was added and the plates were incubated at 37°C for 45 minutes. After the incubation, the plates were washed three times with PBS and FITC antichicken (cat# F8888, Sigma) at 1:500 and TRITC anti-mouse (cat#A10037, Life Technologies) at 1:300 was added. Again the plates were incubated at 37°C for 45 minutes. After the incubation the cells were rinsed three times with PBS and visualized with a fluorescent microscope using fluorescein isothiocyanate (FITC) filter and tetramethyl rhodamine isothiocyanate filter (TRITC).

RESULTS

The nucléotide and amino acid sequences of the donor plasmid pHVTIGl SVLPCHAsyn Sbfl are assigned SEQ ID NOs as shown in Fig. 1.

Recombinant Génération and Expression Analyses

Genomic DNA of HVT FC126 virus was co-electroporated with pHVTIGlSVLPCHAsyn Sbfl donor plasmid to generate recombinant HVT using homologous recombination technique. Recombinant virus was separated from parental HVT virus by immunofluorescent positive well sélection and PCR screening in multiple rounds of plaque purification. A plaque purified recombinant HVT virus expressing the LPC-HA H5N2 protein, designated rHVT501, was scaled up from tissue culture flasks to 3 x 850cm² roller bottles. After about 72 hrs post infection the infected CEFs were harvested. Aliquots were frozen in liquid nitrogen containing 10% FBS and 10% DMSO. Titrations were performed in triplicate on CEFs and a titer of 5.75xl0⁵ pfu/ml was obtained for rHVT501.

Immunofluorescents was performed using chicken anti-sera (lot# J0210, Charles Rivers Laboratories) and a monoclonal antibody spécifie to HVT (Merial) followed by a FITC labeled anti-chicken IgG (cat# F8888, Sigma) and TRITC labeled anti-mouse IgG (cat#A10037, Life Technologies). Ail examined plaques of rHVT501 were found to express LPC-HA H5N2 protein (Fig. 4).

PCR analysis of rHVT501

Purity of recombinant virus was verifîed by PCR using primer pairs that are spécifie to the HVT flanking arms, the SV40 promoter, the LPC-HA H5N2 gene and the synthetic poly A taiL The PCR results demonstrate that recombinant virus rHVT501 carries the intended expression cassette and the virus stock is free from détectable amounts of parental HVT virus (Table 1 and Fig. 5-6).

Table 1 Primer and expected PCR bands

primers	HVT FC 126	pHVTIGlSVLPCHAsyn donor	rHVT501
MB080+MB081	323bp	2593bp	2593bp
SV40PromoterF + syntailR	—	2223bp	2223bp
MB081 +H5N2 LPC F.3		888bp	888bp

PCR reactions with ail primer pairs resulted in the expected PCR products and banding patterns. As shown above, there is no evidence of parental FC 126 in rHVT501. CONCLUSION

Based on PCR testing and immunofluorescence analysis, rHVT501 is a recombinant HVT expressing an H5N2-HA gene under the control of an SV40 promoter. rHVT501 is free of any détectable parental HVT virus.

Example 1.2 Construction of recombinant HVT502 expressing H5N2-HA

The objective of the study is to construct a recombinant HVT virus in which an expression cassette containing a Human Herpesvirus Type III glycoprotein B (HHV3gB) promoter in reverse orientation (ro), a Simian Virus 40 (SV40) promoter, Avian Influenza Virus Hemagglutinin (HA) glycoprotein, and a synthetic poly A tail is inserted in the intergenic site of UL55 in HVT virus (Fig. 2).

The parental virus used in the construct is HVT FC 126. An Avian Influenza Virus Heagglutinin (HA) glycoprotein (named LPC-HA) corresponding to H5N2-HA sequence (SEQ ID NO:2 encoded by SEQ ID NO:1, codon-optimized) was chemically synthesized (GenScript). The promoters used are Human Herpesvirus Type III glycoprotein B (HHV3gB) in reverse orientation (ro) and Simian Virus 40 (SV40) promoter. The insertion locus is intergenic site of UL55 (IG1) in HVT (Fig. 2). Donor plasmid_pHVTIGlHHV3gBroSVLPC

HAsyn Sbfl (an insertion plasmid containing the UL55 flanking régions of HVT virus+HHV3gBro+SV40+synthetic poly A) was constructed as described below. Chicken embryo fibroblast cells (CEF) were used for the in vitro recombination.

Donor plasmid construction

To construct the donor plasmid pHVTIGlHHV3gBroSVLPC-HAsyn Sbfl, a fragment encompassing the synthetic H5N2-HA gene was excised from LPC-HA H5N2 in pUC57 (synthesized by GeneScript) using Notl and inserted into the same site as the pHVTIGlHHV3gBroSVCaFsyn Sbfl plasmid (a previously constructed and sequence verified plasmid) containing HHV3gB promoter in the reverse orientation, SV40 promoter, NDV-F gene, and synthetic polyA tail which was also Notl digested and CIP treated, replacing the NDV-F gene with the LPC-HA. The Notl digested insert (LPC-HA) and vector (pHVTIGl) was gel extracted using Qiagens Gel Extraction Kit and then ligated. Ligated material was transformed using Top 10 Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp broth and plasmid was extracted using Qiagens MiniSpin Prep kit. The plasmids were screened for insert orientation using Smal digestion. The correct donor plasmid was designated pHVTIGlHHV3gBroSVLPC-HAsyn Sbfl. pHVTIGlHHV3gBroSVLPC-HAsyn Sbfl mini-prep plasmid was transfected onto one well of a 6-well plate and an IFA was performed using chicken anti-sera against Avian Influenza H5N2 (Charles Rivers Laboratories, Lot# J0210) and anti-chicken FITC (Sigma, Cat# F8888). Once transient expression was verified the plasmid was grown in a larger scale culture and plasmid extraction was done by using Qiagens Maxi Prep kit. This plasmid was sequence verified and designated _PHVTIGlHHV3gBroSVLPC-HAsyn Sbfl (Fig. 7). Recombinant génération

The homologous recombination procedure as described in Example 1.1 was followed to make recombinant rHVT502.

Analysis of recombinant by PCR

The PCR analysis procedure as described in Example 1.1 was performed to verify rHVT502.

Expression analysis

The expression analysis described in Example 1.1 was performed to analyze the expression of rHVT502.

RESULTS

The nucléotide and amino acid sequence of the donor plasmid pHVTIGlHHV3gBroSVLPC-HAsyn Sbfl are assigned SEQ ID NOs as shown in Fig.l. Recombinant Génération and Expression Analyses

Genomic DNA of HVT FC 126 virus was co-electroporated with pHVTIGlHHV3gBroSVLPC-HAsyn Sbfl donor plasmid to generate recombinant HVT using homologous recombination technique. Recombinant virus was separated from parental HVT virus by immunofluorescent positive well sélection and PCR screening in multiple rounds of plaque purification. A plaque purified recombinant HVT virus expressing the LPC-HA H5N2 protein, designated vHVT502, was scaled up from tissue culture flasks to 2 x 850cm roller bottles. After about 72 hrs post infection the infected CEFs were harvested. Aliquots were frozen in liquid nitrogen containing 10% FBS and 10% DMSO. Titrations were performed in triplicate on CEFs and a titer of 8.25xl0⁵ pfu/ml was obtained for vHVT502.

Immunofluorescents was performed using chicken anti-sera (lot# J0210, Charles Rivers Laboratories) and a monoclonal antibody spécifie to HVT followed by a FITC labeled anti-chicken IgG (cat# F8888, Sigma) and TRITC labeled anti-mouse IgG (cat#A10037, Life Technologies). Ail examined plaques of vHVT502 were found to express LPC-HA H5N2 protein (Fig. 8).

PCR analysis of rHVT502

Purity of recombinant virus was verified by PCR using primer pairs that are spécifie to the HVT flanking arms, the promoters, the LPC-HA H5N2 gene and the synthetic polyA tail. The PCR results demonstrate that recombinant virus rHVT502 carries the intended expression cassette and the virus stock is free from détectable amounts of parental HVT virus (Table 2 and Fig. 9-10).

Table 2 Primers and expected PCR bands

primers	HVT FC 126	pHVTIG 1 HH V3 gBroS VLPCHAsyn donor	rHVT501
MB080+MB081	323bp	3086bp	3086bp
SV40PromoterF + syntailR	—	2223bp	2223bp

MB081 +H5N2 LPC F.3	—	888bp	888bp
MB080 + HHV3gBF	—	607bp	607bp

PCR reactions with ail primer pairs resulted in the expected PCR products and banding patterns. As shown in Fig. 10, there is no evidence of parental FC126 in rHVT502. CONCLUSION

Based on PCR testing and immunofluorescence analysis, rHVT502 is a recombinant HVT expressing an H5N2-HA gene under the control of a reverse oriented HHV3gB and SV40 promoter. rHVT502 is free of any détectable parental HVT virus.

Example 1.3 Construction of recombinant HVT503 expressing mutant H5N2-HA The objective of the study is to construct a recombinant HVT virus in which an expression cassette containing Simian Virus 40 (SV40) promoter, Avian Influenza Virus mutant Hemagglutinin (HA) glycoprotein, and a synthetic poly A tail is inserted in the intergenic site of UL55 in HVT virus (Fig. 2).

The parental virus used is HVT FC 126. A mutated Avian Influenza Virus Heagglutinin (HA) glycoprotein (named Mut-HA H5N2) corresponding to H5N2-HA sequence (SEQ ID NO:4 encoded by SEQ ID NO:3) was chemically synthesized (GenScript). The H5N2-HA (Mut-HA H5N2) was derived from a highly pathogenic avian influenza A virus (A/chicken/Washington/61-9/2014(H5N2) isolate (GenBank accession No. KP739381.1). The synthetic HA glycoprotein cleavage site of this synthetic gene was altered to match a low pathogenic cleavage site sequence. The H5N2-HA (Mut-HA H5N2) was further modified to include three amino acid mutations S136N, D171N, S239N (or in mature HA protein without signal peptide, S120N, D155N and S223N, Hoffmann, et al., 2005, PNAS, 102(36), pl2915-12920).

Simian Virus 40 (SV40) promoter was used in the construct. The insertion locus is intergenic site of UL55 in HVT (Refer to Fig. 2). Donor plasmid.pHVTIGISVMut-HAsyn Sbfl (an insertion plasmid containing the UL55 flanking régions of HVT virus+SV40+synthetic polyA) was prepared as described below. Chicken embryo fibroblast cells (CEF) were used for in vitro recombination.

Donor plasmid construction

A fragment encompassing the synthetic H5N2-HA gene was excised from 3 Mut-HA H5N2 in pUC57 (synthesized by GeneScript) using Nofi and inserted into the same site as the pHVTIG2SVCaFsyn Sbfl plasmid containing SV40 promoter, NDV-F gene, and synthetic polyA tail which was also Notl digested and CIP treated, to replacing the NDV-F gene with the 3 Mut-HA. The Notl digested insert (3 Mut-HA) and vector (pHVTIG2) was gel extracted using Qiagens Gel Extraction Kit and then ligated. Ligated material was transformed using Top 10 Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp broth and plasmid was extracted using Qiagens MiniSpin Prep kit. The plasmids were screened for insert orientation using S mal digestion. The correct donor plasmid was designated pHVTIG2SVMut-HAsyn Sbfl. pHVTIG2SVMut-HAsyn Sbfl mini-prep plasmid was transfected onto one well of a 6-well plate and an IFA was performed using chicken antisera against Avian Influenza H5N2 (Charles Rivers Laboratories) and anti-chicken FITC (Sigma, Cat# F8888). Once transient expression was verified the plasmid was grown in a larger scale culture and plasmid extraction was done by using Qiagens Maxi Prep kit.

An Sbfl digest was performed on the pHVTIG2SVMut-HAsyn Sbfl maxi prep and pIGlHHV3gBroCaFoptsyn Sbfl maxi prep (a previously constructed and sequence verified plasmid). The SV40 H5N2-HA synthetic poly A tail expression cassette flanked by Sbfl restriction enzymes was gel extracted from the pHVTIG2SVMut-HAsyn Sbfl plasmid using Qiagens Gel Extraction Kit. The pIG!HHV3gBroCaFoptsyn Sbfl vector flanked by Sbfl restriction enzymes was also gel extracted and CIP treated. The pHVTIG2SVMut-HAsyn Sbfl expression cassette was ligated to the pIGlHHV3gBCaFsyn Sbfl vector. Ligated material was transformed using Top 10 Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp broth and plasmid was extracted using Qiagens MiniSpin Prep kit. The plasmids were screened for insert orientation using EcoR\+Sma\ digestion. An anti-genome orientation plasmid was selected, grown in a larger scale culture, and plasmid extraction was done by using Qiagens Maxi Prep kit. This plasmid was sequence verified and designated pHVTIGl SVMut-HAsyn Sbfl (Fig. 11).

Recombinant génération

The homologous recombination procedure described in Example l.l was followed to make the recombinant rHVT503.

Analysis of recombinant by PCR

The PCR procedure described in Example l.l was performed to verify rHVT503. Expression analysis

The expression analysis procedure described in Example 1.1 was used.

RESULTS

The nucléotide and amino acid sequences of the donor plasmid pHVTIGlSVMutHAsyn Sbfl are assigned SEQ ID NOs as shown in Fig.l.

Recombinant Génération and Expression Analyses

Genomic DNA of HVT FC 126 virus was co-electroporated with pHVTIGlHHV3gBroSVMut-HAsyn Sbfl donor plasmid to generate recombinant HVT using homologous recombination technique. Recombinant virus was separated from parental HVT virus by immunofluorescent positive well sélection and PCR screening in multiple rounds of plaque purification. A plaque purified recombinant HVT virus expressing the 3 Mut-HA

H5N2 protein, designated vHVT503, was scaled up from tissue culture flasks to 2 x 850cm roller bottles. Aller about 72 hrs post infection the infected CEFs were harvested. Aliquots were frozen in liquid nitrogen containing 10% FBS and 10% DMSO. Titrations were performed in triplicate on CEFs and a titer of 3xl0⁵ pfu/ml was obtained for vHVT503.

Immunofluorescents was performed using chicken anti-sera (lot# J0210, Charles Rivers Laboratories) and a monoclonal antibody spécifie to HVT followed by a FITC labeled anti-chicken IgG (cat# F8888, Sigma) and TRITC labeled anti-mouse IgG (cat#A10037, Life Technologies). Ail examined plaques of vHVT503 were found to express LPC-HA H5N2 protein (Fig. 12).

PCR analysis of rHVT503

Purity of recombinant virus was verified by PCR using primer pairs that are spécifie to the HVT flanking arms, the SV40 promoter, the 3 Mut-HA H5N2 gene, and the synthetic polyA tail. The PCR results demonstrate that recombinant virus vHVT503 carries the intended expression cassette and the virus stock is free from détectable amounts of parental HVT virus (Table 3 and Figs 13-14).

Table 3 Primer sequences and expected PCR bands

primers	HVT FC 126	pHVTIGlSVMutHAsyn donor	rHVT501
MBO8O+MBO81	323bp	2593bp	2593bp
SV40PromoterF + syntailR	—	2223bp	2223bp
MB081 +H5N2 LPC F.3	—	888bp	888bp

PCR reactions with ail primer pairs resulted in the expected PCR products and banding patterns. As shown in Fig. 14, there is no evidence of parental FC 126 in rHVT503. CONCLUSION

Based on PCR testing and immunofluorescence analysis, rHVT503 is a recombinant HVT expressing an H5N2-HA gene under the control of an SV40 promoter. rHVT503 is free of any détectable parental HVT virus.

Example 1.4 Construction of recombinant HVT510 expressing mutant H5N2-HA The objective of the study is to construct a recombinant HVT virus in which an expression cassette containing the mCMV promoter, Avian Influenza Virus mutant Hemagglutinin (HA) glycoprotein, and an SV40 polyA tail is inserted in the intergenic site of UL55 in HVT virus (Fig. 2).

The parental virus used in the construct is HVT FC 126. An Avian Influenza Virus Heagglutinin (HA) glycoprotein (named LPC-HA) corresponding to H5N2-HA sequence (SEQ ID NO:2 encoded by SEQ ID NO: 17, wild-type DNA, not codon-optimized) was chemically synthesized (GenScript).

The promoter is mCMV promoter (SEQ ID NO: 16). The insertion locus is intergenic site of UL55 (IG1 ) in HVT (Fig. 2). Donor plasmid pCD046-H5N2 HA, an insertion plasmid containing the UL55 flanking régions of HVT virus+ mCMV promoter + SV40 polyA (SEQ ID NO: 18) was constructed as described below. Chicken embryo fibroblast cells (CEF) were used for in vitro recombination.

In this construct, the expression of H5N2-HA is driven by mCMV promoter and the H5N2-HA gene is wild-type DNA, not codon-optimized. A strong promoter like CMV and codon optimization lead to genetic instability of the construct.

Donor plasmid construction

The H5N2 HA gene (GenBank Accession number-KP739381) (1704 bp) of the “pUC57-H5N2 HA” plasmid (4424 bp) was generated by gene synthesis (GenScript) and cloned into the EcoRN site of pUC57. The donor plasmid, pCD046 was digested with Notl and CIP treated, and the 6.6 kb fragment was gel extracted. The pUC57-H5N2 HA was also digested with Notl and a 1.7 kb fragment containing H5N2 HA was gel extracted. The 6.6 kb and 1.7 kb fragments were ligated to create pCD046-H5N2 HA (see FIG. 15). Recombinant génération

The homologous recombination procedure described in Example 1.1 was followed to make the recombinant rHVT510.

PCR analysis ofrHVT510

The PCR procedure described in Example 1.1 was performed to verify rHVT510. Expression analysis

The expression analysis procedure described in Example 1.1 was used.

RESULTS

The nucléotide and amino acid sequences of the donor plasmid pHVTIGl SVMutHAsyn Sbfl are assigned SEQ ID NOs as shown in Fig.l.

Recombinant virus

Aller two rounds of plaque purification, pure recombinant virus (rHVT510) was isolated. The rHVT510 was tested by IFA and PCR to validate the appropriate transgene insertion as well as no remnant parental virus. Genetic stability analysis results showed that rHVT510 is stable after more than 12 passages.

PCR analysis of rHVT510

PCR primers were designed to identify the presence of the AIV H5N2 HA, the mCMV promoter, SV40 polyA tail, the flanking recombination arms of HVT virus. PCR amplifications were preformed using ~200ng of DNA template along with the primer pairs indicated in Table 3.1 and Fig.l5B.

Table 3.1 Primer sequences and expected PCR bands

Primer sets	Expected amplicons	(bp)
	rHVT510	HVT Fc 126

MB080 + MB081	3649	323
mCMV.F + SV40tail.R	3320	none
SV40pro.F + syntail.R	none	none

PCR amplification with varions primers listed in Table 3.1 confirmed that rHVT510 has expected amplification pattern and amplicons (Fig,15C).

It was confirmed that rHVT510 is a recombinant HVT expressing an H5N2-HA gene under the control of an mCMV promoter. rHVT510 is free of any détectable parental HVT virus.

Example 2 Construction of recombinant fowlpox virus vectors expressing H5N2-HA Example 2.1 Construction of recombinant FPV3003 expressing H5N2-HA

The objective of the study is to construct a recombinant Fowlpox virus in which an expression cassette containing a vaccinia H6 promoter and Avian Influenza Virus Hemagglutinin (HA) glycoprotein replacing the FPV 158 CDS (also known as F8) in Fowlpox virus (Fig. 16).

The parental virus used in the construct is attenuated Fowlpox virus (TROVAC). An Avian Influenza Virus Heagglutinin (HA) glycoprotein corresponding to H5N2-HA sequence (SEQ 1D NO:2 encoded by SEQ ID NO:1) was chemically synthesized (GenScript). The HA glycoprotein cleavage site of this synthetic gene was altered to match a low pathogenic cleavage site sequence.

The promoter is vaccinia H6 (H6) promoter (SEQ ID NO:11). The insertion locus is FPV158 CDS (F8) replacement. Donor plasmid_pF8 H6pLPC-HA H5N2 (a plasmid containing the FPV158 (F8) flanking régions of Fowlpox virus+H6) was constructed as described below. Chicken embryo fibroblast cells (CEF) were used for in vitro recombination. Donor plasmid construction

A fragment encompassing the synthetic H5N2-HA gene was excised from LPC-HA H5N2 in pUC57 (synthesized by GeneScript) using Nrul and Xhol and inserted into the same site as the pF8 H6p plasmid (a previously constructed and sequence verified plasmid, Merial) containing H6 promoter (Fig. 17) which was also Nrul and Xhol digested. The Nrul and A7?ol digested insert (LPC-HA) and vector (pF8 H6) were gel extracted using Qiagens Gel Extraction Kit and then ligated. Ligated material was transformed using Top 10 Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp broth and plasmid was extracted using Qiagens MiniSpin Prep kit. Six miniprep plasmids were screened for the insert using Smal digestion. Ail miniprep plasmids had the expected restriction endonuclease pattern. Miniprep #1 was grown in a larger scale culture and plasmid extraction was done by using Qiagens Maxi Prep kit. This plasmid was sequence verified and designated pF8 H6pLPC-HA H5N2.

Recombinant génération

The homologous recombination procedure as described in Example 1.1 was followed to make recombinant rFPV3003.

Analysis of recombinant by PCR

The PCR analysis procedure as described in Example 1.1 was performed to verify rFPV3OO3.

Expression analysis

The expression analysis described in Example 1.1 was performed to analyze the expression of rFPV3003.

RESULTS

The nucléotide and amino acid sequences of the donor plasmid pF8 H6pLPC-HA H5N2 are assigned SEQ ID NOs as shown in Fig.l.

Using immunofluorescence technique as described above, recombinant plaques were found to express the hemagglutinin gene of H5N2.

Purity of recombinant virus was verified by PCR using primer pairs that are spécifie to the fowl pox flanking arms (Fig. 18). The PCR results demonstrate that recombinant virus rFP3003 carries the intended expression cassette and the virus stock is free from détectable amounts of parental fowl pox virus (Fig. 19).

CONCLUSION

Based on PCR testing and immunofluorescence analysis, rFPV3OO3 is a recombinant FPV expressing an H5N2-HA gene under the control of the vaccinia H6 promoter. rFPV3003 is free of any détectable parental FPV.

Example 2.2 Construction of recombinant FP3004 expressing mutant H5N2-HA The objective of the study is to construct a recombinant Fowlpox virus in which an expression cassette containing a vaccinia H6 promoter and Avian Influenza Virus Hemagglutinin (HA) glycoprotein replacing the FPV158 CDS in Fowlpox virus (Fig. 16).

The parental virus used is attenuated Fowlpox virus (TROVAC). An Avian Influenza Virus Heagglutinin (HA) glycoprotein corresponding to mutant H5N2-HA sequence (SEQ ID NO:4 encoded by SEQ ID NO:3) was chemically synthesized (GenScript). The HA glycoprotein cleavage site of this synthetic gene was altered to match a low pathogenic cleavage site sequence.

Vaccinia H6 (H6) promoter was used in the construct. The insertion locus is FPV158 CDS replacement (F8). Donor plasmid pF8 H6p3Mut-HA H5N2 (a plasmid containing the FPV158 (F8) flanking régions of Fowlpox virus+H6) was prepared as described below. Chicken embryo fibroblast cells (CEF) were used for in vitro recombination.

Donor plasmid construction

A fragment encompassing the synthetic mutant H5N2-HA gene was excised from 3 Mut-HA H5N2 in pUC57 (synthesized by GeneScript) using Nru\ anàXhoï and inserted into the same site as the pF8 H6p plasmid (Merial) containing H6 promoter (Fig. 20) which was also Nrul and Xho\ digested. The Nruï and Xhol digested insert (3 Mut-HA) and vector (pF8 H6) were gel extracted using Qiagens Gel Extraction Kit and then ligated. Ligated material was transformed using Top 10 Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp broth and plasmid was extracted using Qiagens MiniSpin Prep kit. Six miniprep plasmids were screened for the insert using Smaï digestion. Ail miniprep plasmids had the expected restriction endonuclease pattern. Miniprep #1 was grown in a larger scale culture and plasmid extraction was done by using Qiagens Maxi Prep kit. This plasmid was sequence verified and designated pF8 H6p3Mut-HA H5N2. Recombinant génération

The homologous recombination procedure as described in Example 1.1 was followed to make recombinant rFP3004.

Analysis of recombinant by PCR

The PCR analysis procedure as described in Example l. I was performed to verify rFP3004.

Expression analysis

The expression analysis described in Example l. I was performed to analyze the expression of rFP3004.

RESULTS

The nucléotide and amino acid sequences of the donor plasmid pF8 H6p3Mut-HA H5N2 are assigned SEQ ID NOs as shown in Fig.l.

Using immunofluorescence technique as described above, recombinant plaques were found to express the hemagglutinin gene of H5N2.

Purity of recombinant virus was verified by PCR using primer pairs that are spécifie to the fowlpox flanking arms (Fig. 21). The PCR results demonstrate that recombinant virus rFP3004 carries the intended expression cassette and the virus stock is free from détectable amounts of parental fowlpox virus (Fig. 22).

CONCLUSION

Based on PCR testing and immunofluorescence analysis, rFPV3004 is a recombinant FPV expressing a mutant H5N2-HA gene under the control of the vaccinia H6 promoter. rFPV3004 is free of any détectable parental FPV.

Example 3 Immunogenicity and challenge studies in SPF chickens

Immunogenicity and challenge studies were conducted in spécifie pathogen free (SPF) chickens vaccinated subeutaneous, 0.2 ml per chick, 3100 pfu/dose, at one day of âge with HVT-AIV recombinant vaccines. Twenty chickens were assigned to each vaccine group (see Table 4). A group vaccinated with diluent only was included as a challenge control.

Table 4 Vaccination scheme

Group	Vaccines
SHAM(l)a	Diluent only
rHVT-501(l)a	rHVT-501 vector
rHVT-502(2)b	rHVT-502 vector
rHVT-503(2)b	rHVT-503 vector

SHAM(2)^b

Diluent only ^arHVT-501: Study 1 ^b rHVT-502; rHVT-503: Study 2

Challenge study

One-day-old chickens were vaccinated according to Table 4. At 4 weeks of âge, chickens were challenged with Tk/MN/12582/15 H5N2 at 1O^6OEID5o per chicken. After challenge, the chickens were observed daily for morbidity and mortality, and the morbid chickens were counted as infected with influenza. Oropharyngeal swabs to détermine challenge virus shedding from respiratory tract were collected at 2 and 4 days post-challenge (DPC) in 1.5 ml of brain heart infusion (BHI) medium (Becton-Dickinson, Sparks, MD) containing antimicrobial compounds (100 pg/mL gentamicin, 100 units/mL penicillin, and 5 pg/mL amphotericin B). Remaining chickens from ail groups were bled for sérum collection at days 42 and 56 of âge. The birds were euthanized with intravenous sodium pentobarbital (100 mg/kg body weight) at 56 days of âge.

As shown in Tables 5 and 6, for the groups challenged with Tk/MN/12582/15 H5N2, surprisingly, rHVT501 expressing the H5N2 HA (LPC-HA H5N2) provided better protection in chickens than rHVT503 which contains the mutant H5N2 HA gene. rHVT501 gave 100% protection against clinical disease, while rHVT502 and rHVT 503 gave 45-50% and 15% protection against clinical disease, respectively. Ail three rHVT recombinant vaccines provided protection against avian influenza virus infections compared to the Controls.

Table 5 Results of rHVT-AIV efficacy - number of birds survived after challenge

Days post inoculation	SHAM(l)	rHVT-501(l)	rHVT-502(2)	rHVT-503(2)	SHAM(2)
0	20	19	20	20	20
1	20	19	20	20	20
2	6	19	11	5	5
3	3	19	10	3	1

4	1	19	10	3	0
5	1	19	10	3	0
6	1	19	9	3	0
7	1	19	9	3	0
8	1	19	9	3	0
9	1	19	9	3	0
10	1	19	9	3	0
11	1	19	9	3	0
12	1	19	9	3	0
13	1	19	9	3	0
14	1	19	9	3	0

Table 6 Results of rHVT-AIV effîcacy - percent survival after challenge

Days post inoculation	SHAM(l)	rHVT-501(l)	rHVT-502(2)	rHVT-503(2)	SHAM(2)
0	100	100	100	100	100
1	100	100	100	100	100
2	30	100	55	25	25
3	15	100	50	15	5
4	5	100	50	15	0
5	5	100	50	15	0
6	5	100	45	15	0
7	5	100	45	15	0
8	5	100	45	15	0

9	5	100	45	15	0
10	5	100	45	15	0
11	5	100	45	15	0
12	5	100	45	15	0
13	5	100	45	15	0
14	5	100	45	15	0

Viral shedding was investigated using quantitative RT-PCR test on oropharynx and cloacal swabs samples taken from survivor birds at 2 and 4 dpc. The swabs were tested by quantitative real time reverse transcriptase polymerase chain reaction (qRRT-PCR) for avian influenza virus, and qRRT-PCR cycle threshold values were converted to équivalent infectious titers in embryonating chicken eggs based on régression line produced using a challenge virus dilution sériés (Lee et al., Journal of Virological Methods 119(2):151-158). Briefly, RNA was extracted from oropharyngeal swab material by adding 250μ1 of swab medium to 750μ1 of Trizol LS (Invitrogen Inc., Carlsbad, CA), followed by mixing via vortexing, incubation at room température for 10 min, and then 200μ1 of chloroform was added. The samples were vortexed again, incubated at room température for 10 min, and then centrifuged for 15 min at approximately 12,000xg. The aqueous phase was collected and RNA isolated with the MagMAX AI/ND viral RNA isolation kit (Ambion, Inc. Austin TX) in accordance with the kit instructions using the KingFisher magnetic particle processing System (Thermo Scientific, Waltham, MA). The avian influenza virus challenge strains were used to produce the RNA for the quantitative standard. Allantoic fluid virus stocks were diluted in BHI broth (Becton-Dickinson) and titrated in embryonating chicken eggs at the time of dilution as per standard methods (Swayne et al., 2008, Avian influenza. In: Isolation and Identification of Avian Pathogens. 5th ed., pp. 128-134). Whole virus RNA was extracted from ten-fold dilutions of titrated virus as described for swab material. qRRT-PCR for the influenza matrix gene was performed as previously described (Lee et al., 2004). Virus titers in samples were calculated based on the standard curves, either calculated by the Smart

Cycler II (Cepheid, Inc. Sunnyvale, CA) software or extrapolation of the standard curve équation.

In addition to providing 100% protection against clinical disease, rHVT501 also reduced virus shedding significantly as determined by the virus copy number in lOul swab material as shown in Figure 23.

Example 4 Immunogenicity and challenge studies in SPF chickens against homologous and heterologous AIV challenges

The goal of the study is to détermine efficacy of rHVT501 and rHVT510 administered to one-day-old SPF chickens, against challenge with two (homologous and heterologous) high-pathogenicity Avian Influenza Virus (HPAIV) strains.

Seventy-two one-day-old chickens were separated into 6 groups (see Table 7). The study was carried out according to the timeline outlined in Table 8.

Table 7 Study groups

Group	Vaccine PFU/dose	Route/ Volume (ml)	Number Of Birds Placed	Number Of Birds at Challenge	H5 challenge*
1	rHVT501 -2,200	0.2 ml / SQ	12	10	[Minnesota/12582]
2	rHVT510 -2,100	0.2 ml / SQ	12	10	[Minnesota/12582]
3	Sham-Vaccinated Négative Controls	0.2 ml / SQ	12	10	[Minnesota/12582]
4	rHVT501 -2,200	0.2 ml / SQ	12	10	Heterologous
5	rHVT510 -2,100	0.2 ml / SQ	12	10	Heterologous
6	Sham-Vaccinated Négative Controls	0.2 ml / SQ	12	10	Heterologous

* The birds were challenged on Study Day 28, with one of two strains of HPAIV H5: “Homologous”

A/turkey/Minnesota/125 82/2015; Clade 2.3.4.4; [Minnesota/12582]; or “Heterologous” (A/Egypt/N04915/2014, H5N1), by the intrachoanal route, with ~10⁶ °EID₅₀ per dose.

Table 8 Study timeline

Study Day or Range	Activity
Day 0	Ail birds were vaccinated or sham-vaccinated by the SQ route.
Days 16-25	Ail groups were reduced to ten (10) birds and neck banded for individual identification with numbered bands.
Days 25-28	Blood was collected via venipuncture from the wing or jugular vein.
Day 28	Ail the birds were challenged with HPAIV by the intrachoanal route, 0.1 ml per bird.
Day 28-42	The birds were observed daily for any unfavorable reactions to the challenge.
Day 30*	Oropharyngeal and/or cloacal swabs were collected from ail birds (including birds found dead on this study day) and stored in brain and heart infusion media (BHI) at -70°C until molecular testing to détermine virus shedding was conducted.
Day 32*	Oropharyngeal and/or cloacal swabs were collected from ail birds (including birds found dead on this study day) and stored in brain and heart infusion media (BHI) at -70°C until molecular testing to détermine virus shedding was conducted.
Day 42	Blood was collected from ail the remaining birds and the birds were terminated.

*Swabs samples were collected only on Study Days 30 and 32, from ail birds alive on these days and also from birds found dead on these particular study days. Any other birds that are found dead or are euthanized on any other study day were not sampled.

Ail birds were observed for typical HPAIV clinical signs, including mortality for 14 days post-challenge. The clinical signs include: severe dépréssion, nervous or respiratory System signs and/or death. At the end of the observation period (Study Day 42), the survivors will be bled for serology and terminated.

The sérum collected pre and post-challenge were used in hémagglutination inhibition 10 (HI) assays to détermine the antibody levels against selected AIV strains. An aliquot of the pre-challenge sérum was also used for cross-neutralization tests.

HPAIV viral load was tested by real time RT-PCR in the collected swabs following routine procedures. Viral RNA was extracted using MagMAX™-96 AI/ND Viral RNA Isolation Kit® (Ambion, Inc.) following the manufacturer’s instructions. The resulting viral

RNA extracts were quantifïed by one-step qRRT-PCR which targets the influenza matrix gene using 7500 FAST Real-time PCR System (Applied Biosystems, Foster City, CA, USA).

The standard curves for viral RNA quantification were established with RNA extracted from dilutions of the same titrated stocks of the challenge viruses. For analysis, ail the négative samples were considered to be lower than the limit of détection established for each virus. Results

The mortality results are shown in Table 9 below. The results demonstrated that both rHVT501 and rHVT510 provided 100% protection against homologous AIV challenges, rHVT510 provided 100% protection against heterologous AIV challenges and rHVT501 provided 90% protection against heterologous AIV challenges.

Table 9 Number of birds positive for HPAIV and percent protection/infection by Group

Group	Vaccine PFU/dose*	H5 challenge**	# Positive/ Total # birds	% Protection (% Infection)
1	rHVT501 2,200	Minnesota/12582	0/10	100
2	rHVT510 2,100	Minnesota/12582	0/10	100
3	Sham-V accinated Négative Controls	Minnesota/12582	10/10	(100%)
4	rHVT501 2,200	Egypt/2014	1/10	90
5	rHVT510 2,100	Egypt/2014	0/10	100
6	Sham-V accinated Négative Controls	Egypt/2014	10/10	(100%)

* Ail birds were challenged on Study Day 28, by the intrachoanal route with A/turkey/Minnesota/12582/2015; Clade 2.3.4.4; [Minnesota/12582], at 1O⁶⁹EID5o per dose (Groups 1-3) or A/Egypt/N04915/2014, H5N1, clade 2.2.1; [Egypt/2014], at 10^{5 7}EID₅₀ per dose (Groups 4-6).

Serology results are shown in Fig.23B and 23C and Tables 10 and 11.

Table 10 Hémagglutination inhibition (HI) assay results A/Turkey/Minnesota/12582/2015 (H5N2) homologous challenge

Group	Bird #	HI Serology-Number Positive* per Total (GMT- includes positive birds)	log2 GMT
		Pre-challenge	Post-challenge	Pre-challenge	Post-challenge
rHVT501	1	16	128	4	7

	2	32	64	5	6
3	32	128	5	7
4	32	16	5	4
5	32	32	5	5
6	32	128	5	7
7	64	64	6	6
8	32	64	5	6
9	64	64	6	6
10	32	32	5	5
	#birds	10/10	10/10
GMT	34.3	59.7
log2 GMT	5.1	5.9
rHVT510	1	32	128	5	7
2	16	16	4	4
3	16	32	4	5
4	16	32	4	5
5	16	8	4	3
6	16	32	4	5
7	8	8	3	3
8	16	32	4	5
9	16	32	4	5
10	32	64	5	6
	#birds	10/10	10/10
GMT	17.1	27.9
log2 GMT	4.1	4.8
sham	1	4		2	-
2	4	-	2	-
3	4	-	2	-
4	4	-	2	-
5	4	-	2	-
6	4	-	2	-
7	4	-	2	-
8	4	-	2	-
9	4	-	2	-
10	4	-	2	-
	#birds
GMT
log2 GMT

Positive*: above 4 log2 GMT are considered positive.

-**: bird not survived the challenge.

Table 11 Hémagglutination inhibition (HI) assay results A/Egypt/N04915/2014 (H5N1) heterologous challenge

Group	Bird #	HI Serology-Number Positive* per Total (GMT- includes positive birds)	Iog2 GMT
		Pre-challenge	Post-challenge	Pre-challenge	Post-challenge
rHVT501	1	8	512	3	9
2	4	64	2	6
3	4	-	2	-
4	4	128	2	7
5	8	8	3	3
6	4	4	2	2
7	4	512	2	9
8	4	256	2	8
9	4	32	2	5
10	4	64	2	6
	#birds	2/10	8/9
GMT	8	69.1
log2 GMT	3	6.1
rHVT510	1	4	4	2	2
2	4	4	2	2
3	4	4	2	2
4	4	4	2	2
5	4	8	2	2
6	4	4	2	2
7	4	8	2	3
8	4	32	2	5
9	4	16	2	4
10	4	4	2	2
	#birds	0/10	4/10
GMT	4	13.5
log2 GMT	2	3.8
sham	1	4	-	2	-
2	4	-	2	-
3	4	-	2	-
4	4	-	2	-
5	4	-	2	-
6	4	-	2	-
7	4	-	2	-
8	4	-	2	-
9	4	-	2	-
10	4	-	2	-

	#birds	0/10
GMT	4
log2 GMT	2

The HI results showed that for the pre-challenge response, a total of 12 birds hâve HI titers above the cut-off in rHVT501 group versus 10 in rHVT510 group, and for the postchallenge response the respective numbers are 18 and 14. The différence in the post5 heterologous-challenge response indicated that fewer birds reacted in rHVT510 group because the vaccine has well controlled the réplication of the challenge virus. Also the lower level of HI titer in the rHVT510 group when compared to the rHVT501 group indicates that the challenge virus was not able to replicate easily.

Viral shedding results are shown in Fig.23D and 23E and Tables 12 and 13.

Table 12 Viral shedding results _________________A/Turkey/Minnesota/125 82/2015 (H5N2) homologous challenge______

Group	Bird #	qRT-PCR (loglO EID50 titer/lml)
		2dpc	4dpc
rHVT501	1	1.9*	1.9
2	1.9	1.9
3	1.9	2.0
4	1.9	2.3
5	1.9	1.9
6	1.9	2.0
7	1.9	1.9
8	4.7	4.8
9	1.9	1.9
10	1.9	1.9
	#birds	1/10	4/10
	Mean	2.2	2.2
	STD	0.9	0.9
rHVT510	1	1.9	1.9
2	1.9	1.9
3	1.9	1.9
4	1.9	1.9
5	1.9	1.9
6	1.9	1.9
7	1.9	1.9
8	1.9	1.9
9	1.9	1.9
10	1.9	1.9

	#birds	0/10	0/10
	Mean	1.9	1.9
	STD	0.0	0.0
sham	1	7.3	-
2	7.9	-
3	7.1	-
4	5.7	-
5	7.9	-
6	6.6	-
7	6.8	-
8	8.4	-
9	7.5	-
10	6.7	-
	#birds	10/10
	Mean	7.2
	STD	0.8

*: 2.0 = Lowest Limit of Détection. Négatives is 1.9

Table 13 Viral shedding results A/Egypt/N04915/2014 (H5N1) heterologous challenge

Group	Bird #	qRT-PCR (logl0 EID50 titer/lml)
		2dpc	4dpc
rHVT501	1	3.5*	3.4
2	3.7	3.3
3	4.1	4.0
4	5.2	4.5
5	1.6	2.5
6	2.5	2.1
7	3.9	3.6
8	5.6	3.7
9	3.5	2.8
10	5.0	3.9
#birds	9/10	10/10
Mean	3.9	3.4
STD	1.2	0.7
rHVT510	1	3.5	3.7
2	2.4	1.6
3	4.6	4.2
4	2.8	1.7
5	5.2	4.4
6	4.4	4.5
7	2.1	1.6
8	4.8	4.1

	9	4.3	4.8
10	2.0	2.1
#birds	10/10	8/10
Mean	3.6	3.3
STD	1.2	1.4
sham	1	5.9	-
2	7.0	-
3	6.7	-
4	6.7	-
5	6.0	-
6	5.2	-
7	5.6	-
8	6.4	-
9	6.2	-
10	6.7	-
#birds	10/10
Mean	6.2
STD	0.6

*: 1.7 = Lowest Limit of Détection. Négatives is 1.6

Tables 12-13 and FIG. 23D-23E demonstrated that both rHVT501 and rHVT510 reduced viral shedding in vaccinated birds. The average viral shedding in rHVT501 and rHVT510 groups is much lower than the viral shedding in the sham group in both homologous and heterologous challenge studies. The viral shedding results showed that in the A/Turkey/Minnesota/125 82/2015 (H5N2) homologous challenge study, none (0/10) ofthe birds in rHVT510 group shed any virus at both 2dpc and 4dpc, and one (1/10) and four (4/10)birds in rHVT501 group shed virus at 2dpc and 4dpc, respectively. Ail birds (10/10) in the sham group shed virus. The viral shedding results confirmed the HI results that rHVT510 has well controlled the réplication of the challenge virus so that the challenge virus was not able to replicate easily.

Example 5 Immunogenicity and challenge studies in broiler chickens with matemally 15 derived antibody (MDA)

The goal of the study is to evaluate the prime-boost administration (two administrations) of two heterologous vaccines or administration at the same time (one administration) of two heterologous vaccines in MDA-positive broiler chickens to overcome

MDA and increase immune response. The heterologous vaccines may be different types of vaccines, such as HVT AIV-HA vaccine or FPV AIV-HA vaccine.

Broiler chickens with AI H5 MDA are vaccinated with rHVT501 alone in ovo or oneday-old and boosted 3 weeks later with a recombinant NDV expressing influenza HA (rNDVH5) to détermine the presence of synergy between these two vaccine candidates. The birds are challenged 3 weeks post-boost (6 weeks of âge) with Tk/MN/12582/15 H5N2 at 1O^6OEID5o per chicken by the intranasal route.

Cloacal and Oropharyngeal swabs are taken at 2 and 4 DPC to evaluate impact on viral shed as described previously.

***

Having thus described in detail preferred embodiments of the présent invention, it is to be understood that the invention defined by the above examples is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the présent invention.

Ail documents cited or referenced herein (“herein cited documents”), and ail documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product spécifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

AMENDED CLAIMS

Claims (15)

1. AMENDED CLAIMS

   1. A composition or vaccine comprising one or more recombinant viral vectors comprising at least one or more heterologous polynucleotides coding for and expressing at least one antigen of an avian influenza virus, wherein the recombinant viral vector comprises a heterologous polynucleotide coding for and expressing an HA antigen having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 20, 21, 22, 23, 24, 25, 26, 27, or 28; preferably wherein the recombinant viral vector is a herpesvirus of turkeys (HVT) or a fowlpox virus (FPV).
2. 2. The composition or vaccine of claim 1, wherein the recombinant viral vector comprises a heterologous polynucleotide having at least 70% sequence identity to the sequence as set forth in SEQ IDNO: 1,3,8, 9, 10, 12, 13, 17 or 19.
3. 3. The composition or vaccine of claim 1 or claim 2, wherein the polynucleotide encoding the HA antigen is operably linked to a promoter selected from the group consisting of an immédiate early cytomégalovirus (CMV) promoter, guinea pig CMV promoter, an SV 40 promoter, Human Herpesvirus Type III glycoprotein B (HHV3gB) promoter, Pseudorabies Virus promoters such as that of glycoprotein X promoter, Herpes Simplex Virus-1 alpha 4 promoter, a Marek's Disease Virus glycoprotein A (or gC) promoter, a Marek's Disease Virus glycoprotein B promoter, a Marek's Disease Virus glycoprotein E promoter, a Marek's Disease Virus glycoprotein I promoter, an Infectious Laryngotracheitis Virus glycoprotein B, an Infectious Laryngotracheitis Virus glycoprotein E promoter, an Infectious Laryngotracheitis Virus glycoprotein D promoter, an Infectious Laryngotracheitis Virus glycoprotein I promoter, vaccinia H6, and a combination thereof.
4. 4. The composition or vaccine of any one of daims 1-3, wherein the polynucleotide encoding the HA antigen is inserted in the région selected from the group consisting of the IG1 locus (UL55), the IG21ocus, the IG3 locus, the UL43 locus, the US 10 locus, the SORF3/US2 locus, the F7 locus, the F8 locus on the HVT or FPV genome.
5. 5. The composition or vaccine of any one of daims 1-4, wherein the avian influenza antigen comprises a mutated HA cleavage région having the sequence as set forth in SEQ ID

   NO:15.
6. 6. The composition or vaccine of any one of daims 1-5, wherein the composition further comprises a second viral vector comprising a heterologous polynucleotide coding for and expressing at least one antigen of an avian influenza virus; preferably wherein the second viral vector is selected from a herpesvirus of turkeys (HVT) or a fowlpox virus (FPV).
7. 7. The composition or vaccine of daim 6, wherein the second viral vector comprises a heterologous polynucleotide coding for and expressing an HA antigen having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:2, 4, 20, 21, 22, 23, 24, 25,26, 27, or 28.
8. 8. The composition or vaccine of daim 6 or 7, wherein the second viral vector comprises a heterologous polynucleotide having at least 70% sequence identity to the sequence as set forth in SEQ ID NO: 1, 3, 8, 9, 10, 12, 13, 17 or 19.
9. 9. The composition or vaccine of any one of daims 1-8, wherein the composition or vaccine further comprises a pharmaceutically or veterinarily acceptable carrier, excipient, vehicle or adjuvant.
10. 10. A recombinant viral vector comprising one or more heterologous polynucleotides coding for and expressing at least one antigen of an avian influenza virus, wherein the recombinant viral vector comprises a heterologous polynucleotide coding for and expressing an HA antigen having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 20, 21, 22, 23, 24, 25, 26, 27, or 28; preferably wherein the recombinant viral vector is a herpesvirus of turkeys (HVT) or a fowlpox virus (FPV).
11. 11. The recombinant viral vector of claim 10, wherein the recombinant viral vector comprises a heterologous polynucleotide having at least 70% sequence identity to the sequence as set forth in SEQ ID NO: 1, 3, 8, 9, 10, 12, 13, 17 or 19.
12. 12. The recombinant viral vector of claim 10 or 11, wherein the polynucleotide encoding the HA antigen is operably linked to a promoter selected from the group consisting of an immédiate early cytomégalovirus (CMV) promoter, mouse CMV IE promoter, guinea pig CMV promoter, an SV 40 promoter, Human Herpesvirus Type III glycoprotein B (HHV3gB) promoter, Pseudorabies Virus promoters such as that of glycoprotein X promoter, Herpes Simplex Virus-1 alpha 4 promoter, a Marek's Disease Virus glycoprotein A (or gC) promoter, a Marek's Disease Virus glycoprotein B promoter, a Marek's Disease Virus glycoprotein E promoter, a Marek's Disease Virus glycoprotein I promoter, an Infectious Laryngotracheitis Virus glycoprotein B, an Infectious Laryngotracheitis Virus glycoprotein E promoter, an Infectious Laryngotracheitis Virus glycoprotein D promoter, an Infectious Laryngotracheitis Virus glycoprotein I promoter, vaccinia H6, and a combination thereof.
13. 13. The recombinant viral vector of any one of daims 10-12, wherein the polynucleotide encoding the HA antigen is inserted in the région selected from the group consisting of the IG 1 locus (UL55), the IG2 locus, the IG3 locus, the UL43 locus, the US 10 locus, the SORF3/US2 locus, the F7 locus, the F8 locus on the HVT or FPV genome.
14. 14. The recombinant viral vector of any one of claims 12-16, wherein the avian influenza antigen comprises a mutated HA cleavage région having the sequence as set forth in SEQ ID NO: 15.
15. 15. The composition of any one of daims 1-9 or vector of any one of claims 10-14 for use in a method of vaccinating an animal or for use in inducing an immunogenic or protective response in an animal against avian influenza pathogens, preferably wherein the animal is avian; further wherein the method comprises at least one administration of the composition or vector ; preferably wherein the administration comprises a prime-boost administration regimen; preferably wherein the prime-boost administration comprises a prime-administration of a composition or vaccine or a polyvalent composition or vaccine comprising one or more viral vectors selected from the group consisting of HVT and FPV, and a boost-administration 5 of a composition or vaccine or a polyvalent composition or vaccine comprising one or more viral vectors selected from the group consisting of HVT and FPV which are the same or different from the viral vectors used in the prime-administration.

    ABSTRACT

    The présent invention provides recombinant viral vectors that contain and express antigens of avian pathogens, compositions comprising the recombinant viral vectors,

    5 polyvalent vaccines comprising the recombinant viral vectors. The présent invention further provides methods of vaccination against a variety of avian pathogens and method of producing the recombinant viral vectors.