WO2026013162A1

WO2026013162A1 - Herpes virus of turkey vector based h5ha vaccine for the treatment and prevention of avian influenza infections in turkeys

Info

Publication number: WO2026013162A1
Application number: PCT/EP2025/069655
Authority: WO
Inventors: Francesco PRANDINI; Francesco Bonfante
Original assignee: Boehringer Ingelheim Vetmedica GmbH; Boehringer Ingelheim Animal Health Italy SpA
Current assignee: Boehringer Ingelheim Vetmedica GmbH; Boehringer Ingelheim Animal Health Italy SpA
Priority date: 2024-07-10
Filing date: 2025-07-10
Publication date: 2026-01-15
Anticipated expiration: 2027-01-10

Abstract

The present invention relates i.a. to a method for treating or preventing avian influenza virus infections in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA.

Description

HERPES VIRUS OF TURKEY VECTOR BASED H5HA VACCINE FOR THE TREATMENT AND PREVENTION OF AVIAN INFLUENZA INFECTIONS IN TURKEYS

SEQUENCE LISTING

This application contains a sequence listing in accordance with 37 C.F.R. 1.821 - 1.825. The sequence listing accompanying this application is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

With the increased incidence of highly pathogenic avian influenza H5N1 outbreaks in many parts of the world, avian influenza virus (AIV) is an important threat to the poultry industry worldwide.

Hemagglutinin protein is the receptor-binding and membrane fusion glycoprotein of influenza virus and the target for infectivity-neutralizing antibodies. The entire hemagglutinin protein (HA) from the H5N1 is composed of about 567 amino acids (including the signal peptide, amino acids 1 to 16), with a molecular weight of approximately 65-75 kDa.

In recent years, different types of vaccines have been developed for preventing avian influenza (Al). Commonly, inactivated (killed) Al vaccines have been used in the field.

Further, a baculovirus-based vaccine expressing a H5HA protein has been developed as well as exemplarily described in WQ2008/052173 A2.

Meanwhile, numerous genetically engineered vaccines have been developed. Live recombinant turkey herpes virus avian influenza vaccines (rHVT-H5) have been developed expressing HA proteins such as a H5HA protein as disclosed in Rauw et al. 2012 (Avian Dis. :56;913-22). Recently, WQ2021/257706 described another rHVT-H5 encoding a COBRA (“Computationally Optimized Broadly Reactive Antigen”) H5 protein. However, there is still a need for improved treatment regimes against AIV (avian influenza virus) in turkey.

DESCRIPTION OF THE INVENTION

Before the aspects of the present invention are described, it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a plurality of antigens, reference to the "virus" is a reference to one or more viruses and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies as reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The present invention solves the problems inherent in the prior art and provides a distinct advance in the state of the art.

Generally, the present invention provides a method for treating or preventing avian influenza virus infections in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

Generally, the present invention provides a method for treating or preventing avian influenza virus infections in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA consists of or comprises the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

Advantageously, the data as provided herein show that using the rHVT-H5 COBRA results in much better results in turkeys than using the conventional rHVT-H5 of the prior art.

The term ‘THVT-H5HA COBRA” means a recombinant herpesvirus of turkeys (HVT) vector comprising a polynucleotide encoding a computationally optimized broadly reactive antigen (COBRA) version of the hemagglutinin (HA) protein of AIV subtype H5. The rHVT-H5HA COBRA has already been described in WO2021/257706 or in Criado et al 2023 (Vaccine 41 : 2893-2904). VAXXITEK® HVT+IBD+H5 is a commercial vaccine having the H5HA COBRA sequence.

One advantage of using the rHVT-H5HA COBRA is that it is compatible with a “DIVA” (Differentiation of Infected and Vaccinated Animals) concept with specific ELISA Kits for differentiating between avians vaccinated with the rHVT-H5HA COBRA and avians infected with an avian influenza virus or H5N1 virus.

The term "avian influenza virus" is known by the person skilled in the art. Briefly, the genome of the influenza A virus consists of eight single-stranded segments, and the viral particles have two major glycoproteins on its surface: hemagglutinin (H) and neuraminidase (N). With at least 16 different hemagglutinin (H1 to H16) and 9 different neuraminidase (N1 to N9) subtypes, there is a considerable antigenic variation among influenza viruses.

Further, there are nine known subtypes of H5 viruses (H5N1 , H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, herein referred as "H5Nx").

The term “turkey” is well known to the person skilled in the art.

The term “treating or preventing” refers to the lessening of the incidence of the particular avian influenza virus infection in a flock or the reduction in the severity of clinical signs caused by or associated with the particular avian influenza virus infection. Thus, the term “treating or preventing” also refers to the reduction of the number of subjects in a flock that become infected with the particular avian influenza virus (= lessening of the incidence of the particular avian influenza infection) or to the reduction of the severity of clinical signs normally associated with or caused by the particular avian influenza virus infection (or the reduction of shedding of avian influenza virus after infection with the particular avian influenza virus or prevention or reduction of mortality after infection with the particular avian influenza virus) in a group of subjects which have received an effective amount of the rHVT- H5HA COBRA protein as provided herein in comparison to a group of subjects which have not received such treatment.

The “preventing or treating” generally involves the administration of an effective amount of the rHVT-H5HA COBRA of the present invention to a subject or flock of subjects that could benefit from such a treatment/prophylaxis. The term “treatment” refers to the administration of the effective amount of the rHVT-H5HA COBRA once the subject or at least some subjects of the flock is/are already infected with such avian influenza virus and wherein such subjects already show some clinical signs caused by or associated with such avian influenza virus infection. The terms “preventing” and “prophylaxis” refer to the administration of a subject prior to any infection of such subject with such avian influenza virus or at least where such subject or none of the subjects in a group of subjects do not show any clinical signs caused by or associated with the infection by such avian influenza virus. The terms “prophylaxis” and “preventing” are used interchangeable in this application.

The term "an effective amount" as used herein means, but is not limited to an amount of antigen, that elicits or is able to elicit an immune response in a subject. Such effective amount is able to lessen the incidence of the particular avian influenza virus infection in a flock or to reduce the severity of clinical signs of the particular avian influenza virus infection.

Preferably, clinical signs are lessened in incidence or severity by at least 10%, more preferably by at least 20%, still more preferably by at least 30%, even more preferably by at least 40%, still more preferably by at least 50%, even more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, still more preferably by at least 90%, still more preferably by at least 95% and most preferably by 100% in comparison to subjects that are not treated. The term “identity” or “sequence identity” is known in the art and refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by- position basis, e.g., the sequences are "identical" at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% "sequence identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%, preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity. The terms “identity”, "sequence identity" and "percent identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences are of the same length.

A sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragments of the two sequences. Typically, the comparison will be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

The skilled person will be aware of the fact that different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

The protein sequences or nucleic acid sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTP programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the BLASTP program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTP and BLASTN) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

As used herein, it is in particular understood that the term "identical to the sequence of SEQ ID NO: X" is equivalent to the term "identical to the sequence of SEQ ID NO: X over the length of SEQ ID NO: X" or to the term "identical to the sequence of SEQ ID NO: X over the whole length of SEQ ID NO: X", respectively. In this context, "X" is any integer selected from 1 to 4 so that "SEQ ID NO: X" represents any of the SEQ ID NOs mentioned herein.

The term “protein”, “amino acid” and “polypeptide” are used interchangeably. The term “protein” refers to a sequence of amino acids composed of the naturally occurring amino acids as well as derivatives thereof. The naturally occurring amino acids are well known in the art and are described in standard text books of biochemistry. Within the amino acid sequence the amino acids are connected by peptide bonds. Further, the two ends of the amino acid sequence are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). The term “protein” encompasses essentially purified proteins or protein preparations comprising other proteins in addition. Further, the term also relates to protein fragments. Moreover, it includes chemically modified proteins. Such modifications may be artificial modifications or naturally occurring modifications such as phosphorylation, glycosylation, myristylation and the like.

The present invention also provides a method for preventing avian influenza virus infections in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The present invention also provides a rHVT-H5HA COBRA for use in a method for treating or preventing avian influenza virus infections in turkeys comprising administering an effective amount of the rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The present invention also provides a method for reducing or eliminating the mortality in turkeys (due to avian influenza virus infections) comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The present invention also provides a method for reducing or eliminating the mortality in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence consists of or comprises the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The present invention also provides a method for reducing the mortality in turkeys (due to avian influenza virus infections) comprising administering an effective amount of a rHVT- H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The term “reducing” means, that the mortality is reduced by at least 10%, more preferably by at least 20%, still more preferably by at least 30%, even more preferably by at least 40%, still more preferably by at least 50%, even more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, still more preferably by at least 90%, and most preferably by at least 95% and most preferably by 100% in comparison to a subject of a non-immunized control group of the same species. The present invention also provides a rHVT-H5HA COBRA for use in a method for reducing or eliminating the mortality in turkeys (due to avian influenza virus infections) comprising administering an effective amount of the rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The present invention also provides a method for reducing or eliminating the shedding or transmission of avian influenza viruses in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The present invention also provides a method for reducing the shedding or transmission of avian influenza viruses in turkeys comprising administering an effective amount of a rHVT- H5HA COBRA, wherein the H5HA COBRA sequence consists of or comprises the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The present invention also provides a method for reducing the shedding or transmission of avian influenza viruses in turkeys comprising administering an effective amount of a rHVT- H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto. The term “shedding” refers to (influenza) virus that is shed in nasal and oropharyngeal secretions and dispersed through sneezing and coughing. Thus, shedding may be determined by examining the virus titer in trachea, choana or cloacal swabs. The term “shedding” further encompasses the transfer of virus to susceptible animals (i.e. sentinels). It is in the general knowledge of a person skilled in the art how to measure the viral shedding.

The term “reducing” means, that the shedding is reduced by at least 10%, more preferably by at least 20%, still more preferably by at least 30%, even more preferably by at least 40%, still more preferably by at least 50%, even more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, even more preferably by at least 90%, still more preferably by at least 95% most preferably by 100% in comparison to subjects that are not treated (not immunized) but subsequently infected by the particular AIV.

The present invention also provides a rHVT-H5HA COBRA for use in a method for reducing or eliminating the shedding or transmission of avian influenza viruses in turkeys comprising administering an effective amount of the rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto

The present invention also provides a method for reducing the the viral load of avian influenza viruses in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto. The present invention also provides a method for reducing the the viral load of avian influenza viruses in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence consists of or comprises the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

The term “viral load” or “virus load” is well known to the person skilled in that art. The term viral load is interchangeable used with the term viral titer herein. The virus load or virus titer is a measure of the severity of an active viral infection, and can be determined by methods known to the person skilled in the art. The determination can be based on the detection of viral proteins such as by antibody binding to the viral proteins and further detection or, alternatively, by detection of viral DNA or RNA by amplification methods such as RT-PCR (Real Time PCR) or qPCR (Real Time Quantitative PCR). Monitoring of virion associated viral DNA orRNA in plasma by nucleic acid amplification methods is a widely used parameter to assess the status and progression of viral disease, and to evaluate the effectiveness of prophylactic and therapeutic interventions. Exemplary, the viral load or virus titer can be calculated by estimating the live amount of virus in an involved body fluid such as a number of DNA or RNA copies per milliliter of blood plasma.

The present invention also provides a rHVT-H5HA COBRA for use in a method for reducing the viral load of avian influenza viruses in turkeys comprising administering an effective amount of the rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

Antigens

From all the viruses mentioned herein much genetic information is available digitally in public sequence databases such as NCBI’s GenBank™, UniProt, and EMBL’s EBI. The NDV-F (Newcastle Disease Fusion Protein), IBD VP2 (Infectious Bursal Disease Viral Protein 2) and AIV HA (Avian Influenza Virus Hemagglutinin) are all Virus Antigens well known in the art.

The “NDV F” as used herein is encoding the NDV’s fusion-glycoprotein. US 5965138 and WO 2018/112051 exemplarily are describing HVT’s expressing an NDV F and are describing NDV-F sequence(s).

The “IBDV VP2” as used herein is encoding the IBDV’s capsid protein. US5,980,906 and WO 2018/112051 exemplarily are describing HVT’s expressing an IBDV VP2 and are describing IBDV VP2 sequence(s).

The “HA” or “AIV HA’as used herein is encoding the hemagglutinin of AIV (avian influenza). HA is a surface glycoprotein in influenza A viruses. WO2021257706 exemplarily is describing HVT’s expressing an HA and are describing H5HA sequence(s).

In one specific aspect of the use or method according to the present invention said IBD VP2 is selected from the group of genotypes consisting of: G1 , G2, G3, G4, G5, G6 and G7.

Thai et al 2021 (Avian Pathol.: 50, 174-181) and Tomas et al 2020 (Transbound. Emerg. Dis.: 67, 1198-1212) developed a classification of IBDVs with seven genogroups (G1-G7) based on the fast genetic variation in the hypervariable (hv) VP2 area, which is considered a major protective antigen that elicits neutralizing antibodies to protect chickens from IBDV infection. The classical virulent and attenuated IBDV, antigenic variation IBDV, and very virulent IBDV strains correspond to G1 , G2, and G3, respectively; G4 includes the ‘distinct’ IBDV strains; G5 contains strains isolated from Mexico; and G6 and G7 contain strains mainly from Italy and Australia, respectively (Michel & Jackwood 2017, Arch Virol. ; 162(12): 3661-3670).

In one specific aspect of the use or method according to the present invention said IBD VP2 is selected from the group of genotypes consisting of: G1 , G2 and G3.

In one specific aspect of the use or method according to the present invention said IBD VP2 is selected from the group of genotypes consisting of: G1 and G2. In one specific aspect of the use or method according to the present invention said IBD VP2 is selected from the group of strains consisting of: Delaware variant E strain, Faragher 52/70, STC and D78.

Sequence information is readily available in the prior art, GenBank acc.nr: D00869 and AY321953 (strain Faragher 52/70), D00499 (strain STC), or AF499929 (strain D78) (see exemplarily Michel & Jackwood 2017, .Arch Virol. ;162(12):3661-3670). Alternatively, this gene can be obtained from the genome of a classic IBDV isolated from nature, using routine techniques for manipulating a Birnavirus. Classic type IBDV's can be readily identified using serology, or molecular biology.

In one specific aspect of the use or method according to the present invention said NDV F is selected from the group of genotypes consisting of: genotype II (Clone 30), genotype I (D26), genotype IV (Texas), genotype VII.

Tirumurugaan et al 2011 (PLoS ONE 6(12): e28414. doi:10.1371), Kim et al 2007 (JOURNAL OF VIROLOGY: 81 (22), p. 12641-12653) and Naguib et al 2021 (Transboundary and Emerging Diseases: 69, 849-863) describre that NDV divides into class I (with 9 genotypes) and class II (with 11 genotypes). However, class I viruses are generally avirulent to chickens and have also been isolated from live bird samples. The class II viruses include most virulent and some avirulent and vaccine viruses. The mentioned genotypes herein belong to class II.

In one specific aspect of the use or method according to the present invention said NDV F is of genotype VII.

In one specific aspect of the use or method according to the present invention said NDV F is of genotype Vlld.

In one specific aspect of the use or method according to the present invention said NDV F is selected from the group of strains consisting of: Clone 30, D26 strain, Texas strain, JSG0210 and Ch/SD883/13 strain. rHVT-H5HA COBRA Construct

The term “HVT” means Herpesvirus of turkeys and is well known to the person skilled in the art. HVT is also called MDV3, Meleagrid herpesvirus 1 , or turkey herpesvirus. HVT was first described in 1970 (Witter et al., 1970, Am. J. Vet. Res., vol. 31 , p. 525). Well-known strains of HVT such as PB1 or FC-126 have for a long time been used as live vaccines for poultry against Marek's disease caused by MDV1 or MDV2.

It is in the general knowledge of a person skilled in the art where to obtain a HVT. Exemplarily, a FC-126 HVT can be purchased as VR-584B from the American Type Culture Collection (ATCC). Further, the genomic sequences of HVT are known as well such as FC- 126 is available as GenBank accession number AF291866 as described exemplarily in Davison 2002 (Veterinary Microbiology 86 (1): 69-88) or Alfonso et al 2001 (Journal of Virology 75 (2); 971-978). Furthermore, the genotyping and characterization of HVT by exemplarily PCR (polymerase chain reaction), real time PCR and LAMP (loop-mediated isothermal amplification) has been described by Cortes et al 2011 (Avian Diseases, 55(2):302-310), Zelnik, V. 2004 (Diagnosis of Marek’s disease In: Marek’s Disease an Evolving Problem. Elsevier Ltd, London. p156-167) and Adedeji et al 2017 (Veterinary World, EISSN: 2231-0916), therefore, HVT can be isolated from the field as well. Commercially available real-time PCR kits are available as well, exemplarily from KYLT (Kylt® HVT LD 100, Kylt® HVT LD 25). The genomic organization of HVT is exemparily described in Alfonso et al 2001 (Journal of Virology 75 (2); 971-978)).

In one specific aspect of the use or method according to the present invention said HVT is strain FC- 126.

In one specific aspect of the use or method according to the present invention said H5HA COBRA sequence is codon optimized.

In one specific aspect of the use or method according to the present invention said H5HA COBRA sequence is a non-naturally occurring H5HA sequence.

In one specific aspect of the use or method according to the present invention said H5HA COBRA encoding sequence is operably linked to a promoter selected from the group comprising a PRV gpX promoter, a Rous sarcoma virus LTR promoter, an SV40 early gene promoter, a chicken beta-actin gene promoter, an immediate early 1 gene promoter from human cytomegalovirus (hCMV IE1 or hCMV) and an immediate early 1 gene promoter from murine cytomegalovirus (mCMV IE1 or mCMV). Preferably the promoter is a mCMV promoter.

In one specific aspect of the use or method according to the present invention said H5HA COBRA encoding sequence is operably linked to an hCMV or mCMV promoter.

As used herein, the term "operably linked" means that the promoter effects transcription or expression of the H5HA coding sequence. This can be achieved in that the promoter is directly linked to the H5HA encoding sequence (such as promoter-H5HA) or indirectly linked to the H5HA encoding sequence by using exemplarily polycistronic transcription elements such as IRES (internal ribosome-entry sites) elements (such as promoter-IBDV VP2-IRES -H5HA or promoter-NDV F- IRES- H5HA) as described in WO2021/257706 or Criado et al 2023 (Vaccine 41 : 2893-2904).

An “internal ribosome entry site” or “IRES” as used herein means a sequence which functionally promotes translation initiation independent from the gene 5'of the IRES and allows two cistrons (open reading frames) to be translated from a single transcript in an animal cell. The IRES provides an independent ribosome entry site for translation of the open reading frame immediately downstream of it. In a eukaryotic cell, a polycistronic transcript thaving an IRES operably linked to the second open reading frame in the transcript allows the sequential translation of that downstream open reading frame to produce the two polypeptides encoded by the same transcript. The IRES can be of varying length and from various sources, e.g. Encephalomyocarditis virus (EMCV), picornaviruses (e.g. Foot-and-mouth disease virus, FMDVor Polio virus (PV), or Hepatitis C virus (HCV). WO 2018/112051 describes the use of an IRES element in HVT

In one specific aspect of the use or method according to the present invention said H5HA COBRA expression is driven by a promoter selected from the group comprising a PRV gpX promoter, a Rous sarcoma virus LTR promoter, an SV40 early gene promoter, a chicken beta-actin gene promoter, an immediate early 1 gene promoter from human cytomegalovirus (hCMV IE1 or hCMV) and an immediate early 1 gene promoter from murine cytomegalovirus (mCMV IE1 or mCMV). Preferably the promoter is a mCMV promoter. In one specific aspect of the use or method according to the present invention said H5HA COBRA expression is driven by an hCMV or mCMV promoter.

The “hCMV” or “human CMV” or “hCMV IE” promoter as used herein is well known in the art. Said promoter has already been described exemplarily in US 5965138 or Koedood et al. (1995, J. of Virol., vol. 69, p. 2194-2207). The nucleotide sequence of the complete hCMV IE gene locus is available e.g. from GenBank under accession number K03104. Said promoter can also be obtained from a variety of commercial sources, such as from suppliers of commercial plasmids for cloning and expression such as from pBK-CMV (exemplarily STRATAGENE, Cat. #212209). The promoter can also be derived for example from the pCMV-MCS series (Stratagene; GenBank acc. nr. AF369966). The hCMV virus itself is available e.g. from the ATCC under accession number VR-1780 or VR-1788 or VR-1590 and, thus, the promoter could be isolated. The genome sequence of hCMV is for example available from GenBank accession number X17403. Additional GenBank accession number information is to be found exemplarily in Sijmons et al 2014 (Viruses (6), 1049- 1072).

The “mCMV” or “mouse CMV” or “mCMV IE” promoter as used herein is well known in the art. Said promoter has already been described exemplarily in US5,980,906, WO 2018/112051 and Dorsch-Hasler et al 1985 (PNAS, vol. 82, p. 8325). The nucleotide sequence of the complete mCMV IE gene locus is available e.g. from GenBank under accession number L06816.1. Said promoter can also be obtained from a variety of commercial sources, such as from suppliers of commercial plasmids for cloning and expression (exemplarily pZIP-mCMV-RFP-Puro). The mCMV virus itself is available e.g. from the ATCC under accession number VR-1399 or VR-194 and, thus, the promoter could be isolated.

In one specific aspect of the use or method according to the present invention said H5HA COBRA encoding sequence is inserted into the HVT Intergenic region.

In one specific aspect of the use or method according to the present invention said H5HA COBRA encoding sequence is inserted into the HVT Intergenic 1 (IG1) site or Intergenic 2 (IG2) site. The intergenic regions 1 and 2 are well known by the person skilled in the art and have already been described exemplarily in US5,980,906.

The intergenic region 1 is between HVT065 (UL55) and HVT066 (homologous to EHV-4 gene 3). The intergenic region 2 is between HVT066 (homologous to EHV-4 gene 3) and HVT067.

In one specific aspect of the use or method according to the present invention said H5HA COBRA encoding sequence is inserted into the HVT Intergenic 1 (IG1) site.

In one specific aspect of the use or method according to the present invention an expression cassette comprising said H5HA COBRA encoding sequence operably linked to a promoter is inserted into the HVT Intergenic region.

In one specific aspect of the use or method according to the present invention an expression cassette comprising said H5HA COBRA encoding sequence operably linked to a promoter is inserted into the HVT Intergenic 1 (IG1) site or Intergenic 2 (IG2) site.

In one specific aspect of the use or method according to the present invention an expression cassette comprising said H5HA COBRA encoding sequence operably linked to a promoter is inserted into the HVT Intergenic 1 (IG1) site.

The term “expression cassette” defines a region within the HVT or vector or construct that contains one or more heterologous polynucleotides (preferably genes) to be transcribed, wherein said polynucleotide sequences (preferably genes) to be transcribed as well as the polynucleotide sequences containing the regulatory elements contained within an expression cassette are operably linked to each other. The heterologous polynucleotides (preferably genes) to be transcribed are transcribed from a promoter and transcription is terminated by at least one polyadenylation signal. Each expression cassette will comprise the regulatory elements necessary for the transcription and translation of any of the selected polynucleotide sequences that are contained within the unit.

The term “intergenic region” is well known by the person skilled in the art. The term encompasses a region between two genes. By using an intergenic region for the insertion of a heterologous polynucleotide, no essential gene (gene essential for example for viability, infectivity or replication) of the virus is inactivated. Accordingly, an intergenic region can be used for the insertion of heterologous polynucleotide(s) such as antigen encoding sequence(s).

The intergenic regions 1 and 2 are well known by the person skilled in the art and have already been described exemplarily in US5,980,906.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA comprises an expression cassette comprising: a) a promoter, and b) i) the H5HA COBRA encoding sequence, or ii) an NDV-F antigen encoding sequence, an Internal Ribosome Entry Site (IRES), the H5HA COBRA encoding sequence, or iii) an IBDV VP2 antigen encoding sequence, an Internal Ribosome Entry Site (IRES), the H5HA COBRA encoding sequence and c) a polyadenylation signal.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA comprises an expression cassette comprising from 5’ to 3’ direction: a) a promoter, and b) i) the H5HA COBRA encoding sequence, or ii) an NDV-F antigen encoding sequence, an Internal Ribosome Entry Site (IRES), the H5HA COBRA encoding sequence, or iii) an IBDV VP2 antigen encoding sequence, an Internal Ribosome Entry Site (IRES), the H5HA COBRA encoding sequence and c) a polyadenylation signal.

Polyadenylation signals are well known to the person skilled in the art such as tk polyA, SV40 late and early polyA, BGH polyA (described for example in U.S. Pat. No. 5,122,458) or hamster growth hormone polyA (described for example in W02010010107). Preferably, the SV40 polyA signal is used.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA or said expression cassette comprises a SV40 polyadenylation signal or wherein said polyadenylation signal is a SV40 polyadenylation signal.

The polyadenylation signal from the simian virus 40 (SV40) late gene is well known to the person skilled in the art. This terminator is available via the commercial 'pCMVB' cloning plasmids (Clontech), since the late 1980's.

In one specific aspect of the use or method according to the present invention said promoter is selected from the group comprising a PRV gpX promoter, a Rous sarcoma virus LTR promoter, an SV40 early gene promoter, a chicken beta-actin gene promoter, an immediate early 1 gene promoter from human cytomegalovirus (hCMV IE1 or hCMV) and an immediate early 1 gene promoter from murine cytomegalovirus (mCMV IE1 or mCMV). Preferably the promoter is a mCMV promoter.

In one specific aspect of the use or method according to the present invention said promoter is a hCMV or mCMV promoter.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA comprises an expression cassette comprising a mCMV promoter, an NDV- F antigen encoding sequence or an IBDV VP2 antigen encoding sequence, an Internal Ribosome Entry Site (IRES), the H5HA COBRA encoding sequence.

In one specific aspect of the use or method according to the present invention said antigen encoding sequence(s) is operably linked to the promoter.

In one specific aspect of the use or method according to the present invention said antigen encoding sequence(s) is operably linked to all the regulatory elements. In one specific aspect of the use or method according to the present invention said antigen encoding sequence(s) is operably linked to the promoter, IRES element and polyadenylation signal.

In one specific aspect of the use or method according to the present invention the expression cassette is inserted into the HVT Intergenic 1 (IG1) site.

Antigen Sequences

In one specific aspect of the use or method according to the present invention said NDV-F antigen sequence consists or comprises the amino acid sequence as shown in SEQ ID NO:2 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

In one specific aspect of the use or method according to the present invention said NDV-F antigen sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in SEQ ID NO:2 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

In one specific aspect of the use or method according to the present invention said NDV-F antigen sequence is encoding the amino acid sequence as shown in SEQ ID NO:2 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

In one specific aspect of the use or method according to the present invention said IBDV VP2 antigen sequence consists or comprises the amino acid sequence as shown in SEQ ID NO:3 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

In one specific aspect of the use or method according to the present invention said IBDV VP2 antigen sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in SEQ ID NO:3 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

In one specific aspect of the use or method according to the present invention said IBDV VP2 antigen sequence is encoding the amino acid sequence as shown in SEQ ID NO:3 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA further is effective in treating or preventing IBDV or NDV infections.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA further is effective in treating or preventing IBDV infections.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA further is effective in preventing IBDV infections.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA further is effective in treating or preventing NDV infections.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA further is effective in preventing NDV infections.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA further comprises a pharmaceutically acceptable carrier.

The term "pharmaceutical-acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immune stimulants, and combinations thereof. “Diluents” can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others.

In one specific aspect of the use or method according to the present invention said pharmaceutically acceptable carrier is a saline solution.

In one specific aspect of the use or method according to the present invention said pharmaceutically acceptable carrier is phosphate buffered saline.

In one specific aspect of the use or method according to the present invention said pharmaceutically acceptable carrier is a Marek's disease vaccine diluent.

Time of Administration and Route of Adminstration

The vaccination with the rHVT-H5HA COBRA can be done in ovo or systemically.

The “in ovo” administration is well known to the person skilled in the art and the person skilled in the art can perform in ovo administration without further ado. The in ovo administration of the vaccine involves the administration of the vaccine to an avian embryo while contained in the egg (for a review on in ovo vaccination see: Ricks et al., Advances in Vet. Med. 495-515, 1999). The vaccine may be administered to any suitable compartment of the egg (e. g. allantois fluid, yolk sac, amnion, air cell or into the embryo) as described in the art (Sharma; Am. J. Vet. Res. 45 1619-1623,1984). Preferably the vaccine is administered below the shell (aircell) membrane and chorioallantoic membrane.

Preferably, the vaccine is injected into embryonated eggs during late stages of the embryonation, generally during the final quarter of the incubation period, preferably 3-4 days prior to hatch. Preferably, the admistration is performed when embryos are between 15 to 19 days old, preferably at day 17, 18 or 19, most preferably at day 18 of age. Subsequently, the vaccinated embryonated eggs are transferred to an incubator for hatch. The process of in ovo administration can be automated using a robotic injection process as described in the prior art. In case an in ovo vaccination is used as first adminstration, preferably the admistration is performed when embryos are between 15 to 20 days old, preferably at day 17, 18 or 19, most preferably at day 18 of age.

The term “systemic administration” is well known to the person skilled in the art and means that the administration is done into muscle tissue (intramuscular), into the dermis (intradermal), underneath the skin (subcutaneous), underneath the mucosa (submucosal), in the veins (intravenous) etc.

In one specific aspect of the use or method according to the present invention said administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are between 15 to 20 days old or systemically within the first week of life.

In one specific aspect of the use or method according to the present invention said administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are between 17 to 19 days old or systemically between day 1 and day 3 of life.

In one specific aspect of the use or method according to the present invention said administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are 18 days old or systemically within day 1 of life.

In one specific aspect of the use or method according to the present invention said administration of an effective amount of said rHVT-H5HA COBRA is done systemically within day 1 of life.

In one specific aspect of the use or method according to the present invention the systemically administration is subcutaneously.

In one specific aspect of the use or method according to the present invention said administration of an effective amount of said rHVT-H5HA COBRA is done subcutaneously within day 1 of life.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA is administered in ovo or systemically. In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA is administered in ovo, subcutaneously or intramuscularly.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA is administered in ovo or subcutaneously.

One or more Doses

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA is administered at two or more doses.

Said rHVT-H5HA COBRA can be administered at two or more doses, with a first dose being administered prior to the administration of a second (booster) dose.

In a preferred aspect of the two-time administration regimen, both the first and second doses of the immunogenic composition are administered in the same amount. Preferably, each dose is in the preferred amounts specified below. In addition to the first and second dose regimen, an alternate embodiment comprises further subsequent doses. For example, a third, fourth, or fifth dose could be administered in these aspects. Preferably, subsequent third, fourth, and fifth dose regimens are administered in the same amount as the first dose, with the time frame between the doses being consistent with the timing between the first and second doses mentioned above.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA is administered once.

Prime and Boost

The prime (first) vaccination with the rHVT-H5HA COBRA can be done in ovo or systemically.

In case a systemic vaccination is used as first adminstration, preferably the admistration is performed within the first week of life of the turkeys, preferably, between day 1 and day 3 of life. In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA is used in a prime and boost vaccination regime.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA is used for the priming in a prime and boost vaccination regime.

In one specific aspect of the use or method according to the present invention said priming with said rHVT-H5HA COBRA is done in ovo when embryos are between 15 to 20 days old or systemically within the first week of life.

In one specific aspect of the use or method according to the present invention said priming with said rHVT-H5HA COBRA is done in ovo when embryos are between 17 to 19 days old or systemically between day 1 and day 3 of life.

In one specific aspect of the use or method according to the present invention a second administration (boost) with an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 40 weeks of age.

The term “H5HA”, as used herein, means a recombinant H5 Hemagglutinin subunit vaccine.

A H5HA protein is commercially available. The Volvac® B.E.S.T. Al + ND oil emulsion vaccine is manufactured by Boehringher Ingelheim Vetmedica® (Guadalajara, Mexico). This vaccine contains H5-HA of the (A/ duck/ China/E319-2Z 2003) HPAI H5N1 virus (clade 2.3.2) which is further modified/optimized in having 120N, 155N, 223N and the modification 328K+ as described further below. The H5HA is generated using Baculo Expression System Technology (B.E.S.T.) in insect cells. In addition, this vaccine contains the inactivated LaSota strain of the Newcastle disease virus.

In one specific aspect of the use or method according to the present invention said H5HA is of H5N1 origin.

In one specific aspect of the use or method according to the present invention said H5HA protein comprises or consists of a H5HA of a H5N1 virus having the amino acid 223N and the modification 328K+ or of a H5HA of a H5N1 virus having the amino acid 120N, 155N, 223N and the modification 328K+.

In another specific aspect of the use or method according to the present invention said H5HA protein is derived from a H5HA of a H5N1 virus of clade 2.3.2 having the amino acid 223N and the modification 328K+ or is derived from a H5HA of a H5N1 virus of clade 2.3.2 having the amino acid 120N, 155N, 223N and the modification 328K+.

In one specific aspect of the use or method according to the present invention said H5HA protein is derived from a H5HA of A/ duck/ China/E319-2/ 2003 having the amino acid 223N and the modification 328K+ or is derived from a H5HA of A/ duck/ China/E319-2/ 2003 having the amino acid 120N, 155N, 223N and the modification 328K+.

The terms “120N”, “155N”, “223N” and “328K+” are known by the person skilled in the art. 120N, 155N and 223N means that at that amino acid position of the H5HA is an Asparagine. Further, 328K+ means that at amino acid position 328 of the H5HA protein an additional (second) Lysine (K+) is inserted. Furthermore, the numbering of the amino acid positions of the H5 protein can be done by the person skilled in the art without further ado. However, WO2013024113 and W02008052173 A2 describe in detail how the numbering of the amino acid positions is to be understood.

In one specific aspect of the use or method according to the present invention the numbering of the amino acid positions of the H5HA protein refers to the amino acid position as given in A/ duck/ China/E319-2/ 2003 and/or wherein the modification 328K+ means that at amino acid position 328 of H5HA protein a second Lysine (K+) is inserted.

In one specific aspect of the use or method according to the present invention said H5HA protein comprises or consists of a polypeptide sequence as shown in SEQ ID NO:4 or a sequence having at least 98% sequence identity thereto.

In one specific aspect of the use or method according to the present invention said H5HA protein comprises or consists of a polypeptide sequence as shown in SEQ ID NO:4 or a sequence having at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at at least 99.9% sequence identity thereto.

WO2013024113 and W02008052173 give detailed information about suitable baculovirus expression and insect cell systems for expressing the H5HA. Further, the document Lin et al. 2008 (J Vet Med Sci.: 70(11):1147-52) discloses the use of a baculovirus/insect cell system for the production of H5 protein of clade 2 H5N1 virus A/duck/China/E319-2/03.

In another specific aspect of the use or method according to the present invention said H5HA protein comprises a mineral oil and/or a water/oil emulsion and/or oil-in-water emulsion and/or a mineral oil-in-water emulsion.

In another specific aspect of the use or method according to the present invention said H5HA protein comprises an oil-in-water emulsion.

In one specific aspect of the use or method according to the present invention said inactivated H5 vaccine is of H5N1 , H5N2, H5N3, H5N6 or H5N8 origin.

Inactivated H5N1 , H5N2, H5N3, H5N6 and H5N8 vaccines are known to the person skilled in the art and can be prepared without further ado. Such vaccines are also commercially available. EgyFlu is an inactivated H5N1 reassortant vaccine prepared from strain RG A/chicken/Egypt/18-H/2009 (H5N1) (produced by the manufacturer Harbin Weike Biotechnology CO., China). Nobilis Influenza H5N2 is an adjuvanted inactivated vaccine based on A/duck/Potsdam/1402-6/1986 (H5N2). CEVac Flukem is an H5N2 Avian Influenza Virus in inactivated form based on A/chicken/Mexico/232/1994 (H5N2), homogenized with an oil adjuvant. Zoetis H5N3 is a reverse genetics inactivated vaccine based on A/chicken/Vietnam/C58/2004 (H5N3). Vaxigen Flu-H5N8 from Avimex is an inactivated H5N8 oil emulsion vaccine.

In one specific aspect of the use or method according to the present invention said inactivated H5 vaccine is an H5N8 vaccine.

In one specific aspect of the use or method according to the present invention said inactivated H5 vaccine comprises an adjuvant. In one specific aspect of the use or method according to the present invention said inactivated H5 vaccine comprises a mineral oil and/or a water/oil emulsion and/or oil-in- water emulsion and/or a mineral oil-in-water emulsion.

In one specific aspect of the use or method according to the present invention said inactivated H5 vaccine is an oil emulsion vaccine.

In one specific aspect of the use or method according to the present invention a second administration (boost) of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 20 weeks of age.

In one specific aspect of the use or method according to the present invention a second administration (boost) of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 4 and 14 weeks of age.

In one specific aspect of the use or method according to the present invention said first administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are between 17 to 19 days old and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 20 weeks of age.

In one specific aspect of the use or method according to the present invention said first administration of an effective amount of said rHVT-H5HA COBRA is done systemically between day 1 and day 3 of life and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 20 weeks of age.

In one specific aspect of the use or method according to the present invention between said first administration of an effective amount of said rHVT-H5HA COBRA and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is a time of at least two weeks. In one specific aspect of the use or method according to the present invention between said first administration of an effective amount of said rHVT-H5HA COBRA and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is a time between two weeks and 40 weeks.

In one specific aspect of the use or method according to the present invention between said first administration of an effective amount of said rHVT-H5HA COBRA and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is a time between two weeks and 20 weeks or between 5 and 12 weeks.

Dosage

In one specific aspect of the use or method according to the present invention the rHVT- H5HA COBRA comprises between 1000 and 9000 PFU’s per dose.

The term “PFU” stands for plaque forming unit which is a technique well known to the person skilled in the art and describes the number of virus particles capable of forming plaques per unit volume.

In one specific aspect of the use or method according to the present invention the rHVT- H5HA COBRA comprises between 3000 and 8000 PFU’s per dose.

In one specific aspect of the use or method according to the present invention the inactivated H5 vaccine or H5HA protein comprises between 200 to 800 HA Units per dose.

The term “HA Unit” is a technique well known to the person skilled in the art. An HA assay (also called HA titration) is performed to quantify the virus particles necessary for hemagglutination and is recorded in HA units.

In one specific aspect of the use or method according to the present invention said rHVT- H5HA COBRA is a vaccine.

The term “vaccine” as used herein means that the host after vaccination displays a protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. CLAUSES

The following clauses are described herein: A method for treating or preventing avian influenza virus infections in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto. A rHVT-H5HA COBRA for use in a method for treating or preventing avian influenza virus infections in turkeys comprising administering an effective amount of the rHVT- H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto. A method for reducing or eliminating the mortality in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto. A rHVT-H5HA COBRA for use in a method for reducing or eliminating the mortality in turkeyscomprising administering an effective amount of the rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

5. A method for reducing or eliminating the shedding or transmission of avian influenza viruses in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

6. A rHVT-H5HA COBRA for use in a method for reducing or eliminating the shedding or transmission of avian influenza viruses in turkeys comprising administering an effective amount of the rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto. rHVT-H5HA COBRA Construct

7. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 6, wherein said HVT is strain FC-126.

8. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 7, wherein said H5HA COBRA sequence is codon optimized.

9. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 8, wherein said H5HA COBRA sequence is a non-naturally occurring H5HA sequence.

10. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 9, wherein said H5HA COBRA encoding sequence is operably linked to a promoter selected from the group comprising a PRV gpX promoter, a Rous sarcoma virus LTR promoter, an SV40 early gene promoter, a chicken beta-actin gene promoter, an immediate early 1 gene promoter from human cytomegalovirus (hCMV IE1 or hCMV) and an immediate early 1 gene promoter from murine cytomegalovirus (mCMV IE1 or mCMV), preferably wherein the promoter is a mCMV promoter. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 10, wherein said H5HA COBRA expression is driven by a promoter selected from the group comprising a PRV gpX promoter, a Rous sarcoma virus LTR promoter, an SV40 early gene promoter, a chicken beta-actin gene promoter, an immediate early 1 gene promoter from human cytomegalovirus (hCMV IE1 or hCMV) and an immediate early 1 gene promoter from murine cytomegalovirus (mCMV IE1 or mCMV), preferably wherein the promoter is a mCMV promoter. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 11 , wherein said H5HA COBRA encoding sequence is inserted into the intergenic region, preferably into the HVT Intergenic 1 (IG1) site or HVT Intergenic 2 (IG2) site. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 12, wherein said rHVT-H5HA COBRA comprises an expression cassette comprising: a) a promoter, and b) i) the H5HA COBRA encoding sequence, or ii) an NDV-F antigen encoding sequence, an Internal Ribosome Entry Site (IRES), the H5HA COBRA encoding sequence, or iii) an IBDV VP2 antigen encoding sequence, an Internal Ribosome Entry Site (IRES), the H5HA COBRA encoding sequence and c) a polyadenylation signal. The method or use of the rHVT-H5HA COBRA of clause 13, wherein the promoter is selected from the group comprising a PRV gpX promoter, a Rous sarcoma virus LTR promoter, an SV40 early gene promoter, a chicken beta-actin gene promoter, an immediate early 1 gene promoter from human cytomegalovirus (hCMV IE1 or hCMV), an immediate early 1 gene promoter from murine cytomegalovirus (mCMV IE1 or mCMV). The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 14, wherein said rHVT-H5HA COBRA comprises an expression cassette comprising a mCMV promoter, an NDV-F antigen encoding sequence or an IBDV VP2 antigen encoding sequence, an Internal Ribosome Entry Site (IRES), the H5HA COBRA encoding sequence. The method or use of the rHVT-H5HA COBRA of any one of clauses 13 to 15, wherein the NDV-F antigen sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in SEQ ID NO:2 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto. The method or use of the rHVT-H5HA COBRA of any one of clauses 13 to 16, wherein the IBDV VP2 antigen sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in SEQ ID NO:3 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

Time of Administration and Route of Adminstration The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 17, wherein said administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are between 15 to 20 days old or systemically within the first week of life. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 18, wherein said administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are between 17 to 19 days old or systemically between day 1 and day 3 of life. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 19, wherein said administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are 18 days old or systemically within day 1 of life. 1. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 20, wherein said administration of an effective amount of said rHVT-H5HA COBRA is done systemically within day 1 of life. 2. The method or use of the rHVT-H5HA COBRA of any one of clauses 18 to 21 , wherein the systemically administration is subcutaneously. 3. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 22, wherein said administration of an effective amount of said rHVT-H5HA COBRA is done subcutaneously within day 1 of life. 4. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 23, wherein said rHVT-H5HA COBRA is administered in ovo or systemically. 5. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 24, wherein said rHVT-H5HA COBRA is administered in ovo, subcutaneously or intramuscularly.

One or more Doses 6. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 25, wherein said rHVT-H5HA COBRA is administered at two or more doses. 7. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 25, wherein said rHVT-H5HA COBRA is administered once.

Prime and Boost 8. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 27, wherein said rHVT-H5HA COBRA is used in a prime and boost vaccination regime. 9. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 28, wherein said rHVT-H5HA COBRA is used for the priming in a prime and boost vaccination regime. 30. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 and 29, wherein said priming with said rHVT-H5HA COBRA is done in ovo when embryos are between 15 to 20 days old or systemically within the first week of life.

31. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 30, wherein said priming with said rHVT-H5HA COBRA is done in ovo when embryos are between 17 to 19 days old or systemically between day 1 and day 3 of life.

32. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 31 , wherein a second administration (boost) with an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 40 weeks of age.

33. The method or use of the rHVT-H5HA COBRA of clause 32, wherein said H5HA is of H5N1 origin.

34. The method or use of the rHVT-H5HA COBRA of any one of clauses 32 or 33, wherein said H5HA protein is derived from a H5HA of A/ duck/ China/E319-2Z 2003 having the amino acid 223N and the modification 328K+ or is derived from a H5HA of A/ duck/ China/E319-2/ 2003 having the amino acid 120N, 155N, 223N and the modification 328K+.

35. The method or use of the rHVT-H5HA COBRA of any one of clauses 32 to 34, wherein said H5HA protein comprises or consists of a polypeptide sequence as shown in SEQ ID NO:4 or a sequence having at least 98% sequence identity thereto.

36. The method or use of the rHVT-H5HA COBRA of any one of clauses 32 to 35, wherein said H5HA protein comprises or consists of a polypeptide sequence as shown in SEQ ID NO:4 or a sequence having at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at at least 99.9% sequence identity thereto.

37. The method or use of the rHVT-H5HA COBRA of any one of clauses 32 to 36, wherein said inactivated H5 vaccine is of H5N1 , H5N2, H5N3, H5N6 or H5N8 origin.

38. The method or use of the rHVT-H5HA COBRA of any one of clauses 32 to 37, wherein said inactivated H5 vaccine is an H5N8 vaccine. 39. The method or use of the rHVT-H5HA COBRA of any one of clauses 32 to 38, wherein said H5HA protein or said inactivated H5 vaccine comprises a mineral oil and/or a water/oil emulsion and/or oil-in-water emulsion and/or a mineral oil-in-water emulsion.

40. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 39, wherein a second administration (boost) of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 20 weeks of age.

41. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 40, wherein a second administration (boost) of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 4 and 14 weeks of age.

42. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 41 , wherein said first administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are between 17 to 19 days old and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 20 weeks of age.

43. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 42, wherein said first administration of an effective amount of said rHVT-H5HA COBRA is done systemically between day 1 and day 3 of life and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 20 weeks of age.

44. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 43, wherein between said first administration of an effective amount of said rHVT-H5HA COBRA and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is a time of at least two weeks.

45. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 44, wherein between said first administration of an effective amount of said rHVT-H5HA COBRA and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is a time between two weeks and 40 weeks. 46. The method or use of the rHVT-H5HA COBRA of any one of clauses 28 to 45, wherein between said first administration of an effective amount of said rHVT-H5HA COBRA and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is a time between two weeks and 20 weeks or between 5 and 12 weeks.

Dosage

47. The method or use of any the rHVT-H5HA COBRA of one of clauses 1 to 46, wherein the rHVT-H5HA COBRA comprises between 1000 and 9000 PFU’s per dose.

48. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 47, wherein the rHVT-H5HA COBRA comprises between 3000 and 8000 PFU’s per dose.

49. The method or use of the rHVT-H5HA COBRA of any one of clauses 1 to 48, wherein said rHVT-H5HA COBRA is a vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The results of the shedding are shown in Figure 1A (Tracheal Shedding) and in Figure 1 B (Cloacal Shedding). Shown is the AUC, the expression of the cumulative shedding throughout the 8 days of testing. The test groups are from left to right as follows: Negative Control, Vectormune® Al, Vectormune® Al + ExactVac® DNA, Vectormune® Al + Vaxigen-Flu® H5N8, Vaxxitek HVT-IBD-H5, Vaxxitek HVT-IBD-H5 + ExactVac DNA, Vaxxitek HVT-IBD-H5 + VaxigenFlu® H5N8, Vaxxitek HVT-IBD-H5 + Volvac B.E.S.T. AI+ND. SEQUENCES OVERVIEW

SEQ ID NO:1 : H5HA COBRA sequence (amino acid sequence)

SEQ ID NO:2: NDV-F antigen sequence (amino acid sequence)

SEQ ID NO:3: IBDV VP2 antigen sequence (amino acid sequence)

SEQ ID NO:4: H5HA protein (amino acid sequence)

SEQ ID NO:1 : H5HA COBRA sequence (amino acid sequence)

MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDL

DGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKH

LLSRINHFEKIQIIPKSSWSNHEASSGVSSACPYQGKSSFFRNWWLIKKNSAYPTIKRSY

NNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSG

RMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGA

INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRETRGLFGAIAGFIEGGWQGMV

DGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRI

ENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGN

GCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVAS SLALAIMVAGLSLWMCSNGSLQCRICI

SEQ ID NO:2: NDV-F antigen sequence (amino acid sequence)

MGSKPSTRIPAPLMLITRIMLILGCIRPTSSLDGRPLAAAGIWTGDKAVNVYTSSQTGSIIV

KLLPNMPRDKEACAKAPLEAYNRTLTTLLTPLGDSIRKIQGSVSTSGGGKQGRLIGAVIGS

VALGVATAAQITAAAALIQANQNAANILRLKESIAATNEAVHEVTDGLSQLSVAVGKMQQF

VNDQFNNTARELDCIKITQQVGVELNLYLTELTTVFGPQITSPALTQLTIQALYNLAGGNM

DYLLTKLGIGNNQLSSLIGSGLITGYPILYDSQTQLLGIQVNLPSVGNLNNMRATYLETLSV

STTKGYASALVPKWTQVGSVIEELDTSYCIESDLDLYCTRIVTFPMSPGIYSCLSGNTSA

CMYSKTEGALTTPYMALKGSVIANCKITTCRCTDPPGIISQNYGEAVSLIDRHSCNVLSLD

GITLRLSGEFDATYQKNISILDSQVIVTGNLDISTELGNVNNSISNALDRLAESNSKLEKVN

VRLTSTSALITYIVLTVISLVFGALSLVLACYLMYKQKAQQKTLLWLGNNTLDQMRATTRA SEQ ID NO:3: IBD P2 antigen sequence (amino acid sequence)

MTNLQDQTQQIVPFIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNLTVGDTGSGLIVFFP

GFPGSIVGAHYTLQSNGNYKFDQMLLTAQNLPASYNYCRLVSRSLTVRSSTLPGGVYAL

NGTINAVTFQGSLSELTDVSYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRL

GDPIPAIGLDPKMVATCDSSDRPRVYTITAADDYQFSSQYQPGGVTITLFSANIDAITSLSI

GGELVFQTSVQGLVLGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNLVIPTNEITQP

ITSIKLEIVTSKSGGQAGDQMSWSASGSLAVTIHGGNYPGALRPVTLVAYERVATGS T

VAGVSNFELIPNPELAKNLVTEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFRE

YFMEVADLNSPLKIAGAFGFKDIIRAIRR

SEQ ID NO: 4 H5HA protein (amino acid sequence, 552 amino acids)

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW

LLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNS

WSDHEASSGVSSACPYQGSSSFFRNWWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHH

PNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAIN

FESNGNFIAPEYAYKIVKKGDSAIMKSEVEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGE

CPKYVKSNKLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSN

EQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGF

LDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDN

ECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSL

WMCSNGSLQCRICI

120N, 155N, 223N and the modification 328K+ shown in bold.

EXAMPLES

The following examples are set forth below to illustrate specific embodiments of the present invention. These examples are merely illustrative and are understood not to limit the scope or the underlying principles of the present invention.

Example 1 (Comparison Study in Turkey)

The aim of the current study is to test both single-shot vaccination schedules and primeboosting vaccinations in female meat turkeys of commercial origin.

Vaccines:

VAXXITEK HVT+IBD+H5 is a commercial vaccine manufactured by Boehringer Ingelheim Animal Health USA. It contains a recombinant vector herpesvirus of turkeys (vHVT) expressing computationally optimized broadly reactive antigen (COBRA) H5 of avian influenza virus (AIV) in combination with virus protein 2 (VP2) of infectious bursal disease virus (IBDV) (rHVT-IBD-AI). However, other constructs have been described expressing the H5 COBRA of avian influenza virus alone (rHVT-AI) or in combination with fusion (F) protein of Newcastle disease virus (NDV) (rHVT-ND-AI), but using all other elements (such as the promoter) as in the commercial vaccine product (Criado et al 2023: Vaccine 41 : 2893- 2904).

Volvac® B.E.S.T. Al + ND oil emulsion is a commercial vaccine (manufactured by Boehringer Ingelheim Vetmedica®, Guadalajara, Mexico). This vaccine contains the H5-HA (A/ duck/ China/E319-2/ 2003) H5N1 virus (clade 2.3.2) which is further modified/optimized in having 120N, 155N, 223N and the modification 328K+. The H5HA is generated using the Baculo Expression System Technology (B.E.S.T.) in insect cells. In addition, this vaccine contains the inactivated LaSota strain of the Newcastle disease virus.

ExactVac® is a commercial DNA vaccine (Huvepharma, USA). Vaxigen® Flu H5N8 is a commercial whole-virus inactivated oil emulsion vaccine (Laboratorio Avi-Mex, S.A. de C.V., Mexico) which contains a virus expressing the HA and NA genes of the clade 2.3.4.4b HPAI H5N8 A/green-winged teal/Egypt/877/2016 virus.

Vectormune® Al (CEVA Sante Animale, France) is a rHVT-H5 comprising a H5 protein of a H5N1 strain belonging to clade 2.2 (A/Swan/Hungary/4999/2006).

Single Vaccination or Prime Vaccination:

Birds are vaccinated or primed at 1 day of age subcutaneously with VAXXITEK HVT+IBD+H5 (Boehringer Ingelheim Animal Health USA) carrying a computationally optimized broadly reactive antigen (C.O.B.R.A.) following the instructions of the manufacturer.

Alternatively, birds are primed at 1 day of age subcutaneously with Vectormune® Al (CEVA) which is a rHVT-H5.

Boost Vaccination:

Subgroups of animals (each group containing 8 animals) are boosted once on day 36 or 28 of age subcutaneously. Boosting is performed with either a subunit vaccine (Volvac ® B.E.S.T. AI+ND, Boehringer Ingelheim Animal Health, USA) on day 36, a DNA vaccine (ExactVac, Huvepharma, USA) or a commercial inactivated oil emulsion vaccine (Vaxigen® Flu H5N8) on day 28.

An additional group of 15 birds is sham vaccinated twice, through subcutaneous inoculation of 0.2 and 0.5 ml of phosphate buffer saline (PBS) at 1 and 28 days, respectively. These sham vaccinated groups were housed in a third room to avoid the contamination of animals by the HVT vaccines.

Inoculum:

Birds are challenge at 50 days of age in their productive cycle. The challenge virus is the A/turkey/ltaly/21VIR9520-3/2021 isolate (sequence available EPI_ ISL_1 1007548) belonging to the 2.3.4.4b clade and is administered via the nasal route and through the choanal slit at a dose of 10⁶ EID₅o/2OO microliters. In particular, 150 pl are dosed in the nose and the rest in the choanal split. In previous experiments, the same dose caused 100% mortality in naive turkeys of 8 weeks of age, within 4 days from infection. Study Design:

There are different vaccination groups:

Negative Control

(Vectormune® Al)

Vectormune® Al + Vaxigen-Flu® H5N8

Vectormune® Al + ExactVac® DNA

Vaxxitek HVT-IBD-H5

Vaxxitek HVT-IBD-H5 + VaxigenFlu® H5N8

Vaxxitek HVT-IBD-H5 + ExactVac DNA

Vaxxitek HVT-IBD-H5 + Volvac B.E.S.T. AI+ND.

Blood samples are collected after the challenge on days 7, 14 and 21 post infection (p.i.) to monitor the humoral immune response and to evaluate the sensitivity of commercial ELISA kits (according to standard protocol) as DIVA tools. Tracheal and cloacal swabs are collected on days 2, 4,6, 8 p.i.. Viral RNA is isolated using the MagMAX™ Pathogen RNA/DNA Kit (Applied Biosystems™, USA) and the KingFisher™ extractor (Thermo Scientific™, USA), according to the manufacturer's instructions. Swabs are tested by quantitative rRT-PCR targeting the Matrix gene of influenza type A viruses (according to standard practice, see Spackman et al 2002 in J Clin Microbiol.40(9):3256-60). Quantification relies on in vitro transcribed RNA. Ten-fold serial dilutions (10¹ to 10⁹) of the challenge viruses are processed in triplicate to develop standard curves for virus quantification. Viral load is expressed in terms of EID₅o equivalents. The limit of detection (LoD) of the RRT-qPCR used in this study is 15.00 EID₅o/1OO pl. Cumulative shedding is calculated by the trapezoidal method to infer the area under the curve (AUC) (Brown et al 2009: Journal of Veterinary Diagnostic Investigation: 21 :437-45).

Post vaccination monitoring relies on molecular methods for the detection of the HVT vector virus in feather calami and/or the hemagglutination inhibition (HI) assay (according to standard protocol) using both vaccine homologous antigens and the challenge virus heterologous antigens. In more detail, the HI test is carried out according to standard procedures (World Organization for Animal Health, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2021) against the challenge virus and antigens that were either homologous or close surrogates to the vaccines seed strains. Sera are treated with 10% chicken erythrocytes for 30 min to remove non-specific agglutinins. The starting dilution of the sera is 1 :4 (analytical threshold), and negative sera are assigned a value of 1 Iog2 (i.e. 1 :2). The diagnostic threshold for positivity is set at a titer > 1 :8. The HI homologous antigen to the Vaxigen vaccine is provided by the manufacturer. For the other vaccines, surrogate strains are selected based on the amino acid identity at the level of the HA1 protein. In particular, for the B.E.S.T. and the Vaxxigen vaccines, we use the clade 2.3.4 H5N1 A/Anhui/1/2005 virus, a strain sharing amino acid identities with the vaccine seed strains of 96.6% and 95.4%, respectively. For the Vectorumune vaccine, we select the A/Cygnus olor/Croatia/1/2005 strain with an amino acid identity of 99.7%. For the detection of antibodies against the nucleoprotein (NP) of avian influenza viruses, it is adopted the ID Screen® Influenza A Nucleoprotein Indirect (IDvet, France) (FLUNPS). To monitor the kinetic of the humoral response against the H5 hemagglutinin, we use the ID Screen® Influenza H5 Indirect ELISA (IDvet, France) (FLUH5S), according to the manufacturer’s instructions. In particular, for the FLUH5S assay we use a starting dilution of 1 :500 for all groups. For both ELISA assays, serum samples recording a sample-to- positive (S/P) ratio > 0.5 are scored as positive. To assess the potential association between the HI titer and S/P ratios, Spearman's rank correlation coefficient is calculated for the serological data obtained from sera collected at 50 and 64 days from birds primed with Vaxxitek® HVT+IBD+H5 with or without a booster vaccination and from challenged and nonchallenged birds. Additionally, a simple linear regression analysis is performed, with the HI titer obtained against the challenge antigen as the dependent variable and the corresponding S/P ratio as the independent variable. For the detection of antibodies against the N1 protein, the ID Screen® Influenza N1 Antibody Competition (IDvet, France) (FLUACN1) is used and sera with sample-to-negative (S/N) percentages < 50% are scored as positive, between 50% and 60% sera were considered doubtful, while sera with percentages > 60% are recorded as negative.

Results

1 . Serological Response to Vaccination:

The serological response to vaccination is measured by the HI titers against the challenge antigen. The results are shown in Table 2.

Table 2: HI titers against the challenge antigen:

The HI Titer results are confirmed by the S/P ratios (data not shown) measured by the FLUH5S kit.

Conclusion:

The results in Table 2 at day 50 (day of challenge) show that using a rHVT-H5 COBRA for a single vaccination gives good results. Further, using a rHVT-H5 COBRA for priming results in much better results than using Vectormune® Al for priming.

2. Mortality

The results of the survival rate after challenge are shown in Table 3.

Table 3: Survival Rate (challenge at 50 days of age):

Conclusion:

Importantly, the single vaccination with Vaxxitek HVT-IBD-H5 provides 100% protection (full protection from mortality), whereas the single vaccination with Vectormune® Al only provides 62.5% protection (priming alone was not sufficient to achieve complete protection from mortality and disease). Further, excellent protection is achieved with rHVT-H5 COBRA when boosted with either the H5N8 or the subunit vaccine. Thus, Vaxxitek HVT-IBD-H5 provides better protection than Vectormune® Al if given as a single shot vaccination or in a prime and boost regime.

3. Shedding

The results of the shedding are shown in Figure 1A (Tracheal Shedding) and in Figure 1 B (Cloacal Shedding). Shown is the AUC, the expression of the cumulative shedding throughout the 8 days of testing.

The test groups are from left to right as follows:

Negative Control Vectormune® Al Vectormune® Al + ExactVac® DNA Vectormune® Al + Vaxigen-Flu® H5N8 Vaxxitek HVT-IBD-H5

Vaxxitek HVT-IBD-H5 + ExactVac DNA

Vaxxitek HVT-IBD-H5 + VaxigenFlu® H5N8

Vaxxitek HVT-IBD-H5 + Volvac B.E.S.T. AI+ND.

Conclusion:

Importantly, the single vaccination with Vaxxitek HVT-IBD-H5 results in less shedding compared to the single vaccination with Vectormune® Al. Further, better reduction in shedding is achieved when rHVT-H5 COBRA is used for priming compared when Vectormune® Al is used for priming. Thus, Vaxxitek HVT-IBD-H5 results in less shedding than Vectormune® Al if given as a single shot vaccination or in a prime and bosst regime. Priming with the rHVT-H5 COBRA vaccine and boosting with the subunit vaccine confers the best reduction in shedding.

Overal Conclusion from the Turkey Study:

The data show that using a rHVT-H5 COBRA for a single vaccination gives good serological results. Further, rHVT-H5 COBRA for priming results in much better serological results than using Vectormune® Al for priming. Vaxxitek HVT-IBD-H5 provides better protection than Vectormune® Al if given as a single shot vaccination or in a prime and boost regime.

Vaxxitek HVT-IBD-H5 results in less shedding than Vectormune® Al if given as a single shot vaccination or in a prime and bosst regime.

Claims

CLAIMS:

1. A method for treating or preventing avian influenza virus infections in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

2. A method for reducing or eliminating the mortality in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

3. A method for reducing or eliminating the shedding or transmission of avian influenza viruses in turkeys comprising administering an effective amount of a rHVT-H5HA COBRA, wherein the H5HA COBRA sequence is encoding an amino acid sequence consisting of or comprising the amino acid sequence as shown in to SEQ ID NO:1 or a sequence having at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity thereto.

4. The method of any one of claims 1 to 3, wherein said H5HA COBRA is codon optimized and/or wherein the H5HA COBRA is a non-naturally occurring H5HA sequence.

5. The method of any one of claims 1 to 4, wherein said H5HA COBRA encoding sequence is operably linked to a promoter selected from the group comprising a PRV gpX promoter, a Rous sarcoma virus LTR promoter, an SV40 early gene promoter, a chicken beta-actin gene promoter, an immediate early 1 gene promoter from human cytomegalovirus (hCMV IE1 or hCMV), an immediate early 1 gene promoter from murine cytomegalovirus (mCMV IE1 or mCMV).

6. The method of any one of claims 1 to 5, wherein said H5HA COBRA encoding sequence is inserted into the HVT Intergenic 1 (IG1) site or Intergenic 2 (IG2) site.

7. The method of any one of claims 1 to 6, wherein said rHVT-H5HA COBRA is administered in ovo or systemically.

8. The method of any one of claims 1 to 7, wherein said first administration of an effective amount of said rHVT-H5HA COBRA is done in ovo when embryos are between 15 to 20 days old or systemically within the first week of life.

9. The method of any one of claims 1 to 8, wherein said administration of an effective amount of said rHVT-H5HA COBRA is done subcutaneously within day 1 of life.

10. The method of any one of claims 1 to 9, wherein said rHVT-H5HA COBRA is used for the priming in a prime and boost vaccination regime.

11. The method of claim 10, wherein said priming with said rHVT-H5HA COBRA is done in ovo when embryos are between 15 to 20 days old or systemically within the first week of life.

12. The method of any one of claims 10 and 11 , wherein a second administration (boost) of an effective amount of an inactivated H5 vaccine or H5HA protein is done systemically between 2 and 20 weeks of age.

13. The method of any one of claims 10 to 12, wherein between said first administration of an effective amount of said rHVT-H5HA COBRA and said second administration of an effective amount of an inactivated H5 vaccine or H5HA protein is a time of at least two weeks.