A novel feline herpesvirus vaccine

Abstract

The present invention relates to an Equine Herpesvirus (EHV) comprising a feline herpesvirus (FHV) antigen coding sequence inserted into ORF70 (US4) and / or ORF1 / 3. The present invention also relates to a method of immunizing a feline, said method comprising administering to said feline an immunogenic composition of the present invention. Furthermore, the present invention relates to a method for treating or preventing clinical signs of feline herpesvirus in a feline.

Description

[Technical Field]

[0001] A. Field of the Invention The present invention relates to the field of feline herpesvirus vaccines. [Background technology]

[0002] B. Description of Background and Related Art Feline herpesvirus (FHV) FHV is the causative agent of feline viral rhinotracheitis in cats, a disease found worldwide. Feline herpesvirus causes an upper respiratory tract infection accompanied by rhinitis, fever, sneezing, eye and nasal discharge, conjunctivitis (inflammation of the mucous membranes around the eyes and the lining of the eyelids), inflammation of the cornea (keratitis), and lethargy. Modified live and inactivated FHV vaccines have been developed. However, modified live vaccine strains generally have the potential to revert to a pathogenic state resulting in disease in the inoculated animal and the potential for transmission of the pathogen to other animals. Inactivated vaccines generally induce only low levels of immunity (no or only weak stimulation of cell-mediated immunity). In contrast, vector vaccines can induce both neutralizing antibodies against FHV and cellular immunity against FHV. Another problem arises from the fact that FHV culture for vaccine production is performed in feline kidney cell lines. However, vaccines containing residual components of these cells may cause cats to produce autoantibodies that lead to chronic kidney disease or other disorders. Therefore, there is a need for FHV vaccines that are not cultured in feline-derived cell lines.

[0003] This issue of autoantibodies has already been discussed in publications such as Lappin et al. 2005 (AJVR, Vol. 66, No. 3), Whittemore et al. 2010 (J. Vet. Intern. Med. 24:306-313), and Songaksorn et al. 2019 (Vet. Sci. 2019 Nov. 20(6):e73). These publications discuss the use of Crandell feline kidney (CRFK) cell lines to grow feline viruses, such as feline herpesvirus 1 (FHV-1), calicivirus, and panleukopenia virus, and the impossibility of removing any CRFK cell proteins or other cellular components during vaccine production. As a result, during routine vaccination, cats are exposed to CRFK cell proteins, which can lead to immune responses against these proteins. The results of studies presented in Lappin et al. 2005 and Whittemore et al. 2010 demonstrate that administration of a vaccine containing virus grown in CRFK cells induces antibodies, including autoantibodies, against kidney tissue in cats. Furthermore, with currently administered live attenuated or inactivated FHV vaccines, it is not possible to determine whether a particular animal is a carrier of the FHV virus or whether the animal has been vaccinated. Thus, both modified live and inactivated vaccines lack a unique diagnostic signature for distinguishing between vaccinated and infected animals (DIVA). Thus, there is a need for an FHV DIVA vaccine. Summary of the Invention [Means for solving the problem]

[0004] For the reasons stated above, there is a need for new feline herpesvirus vaccines. DETAILED DESCRIPTION OF THE INVENTION

[0005] Detailed Description of the Invention Before describing aspects of the present invention, it should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, a reference to an "antigen" includes a plurality of antigens; a reference to a "virus" is a reference to one or more viruses and equivalents thereof known to those of skill in the art, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 described herein. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the cell lines, vectors, and methods reported in such publications that can be used in connection with the present invention. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0006] The present invention solves the problems inherent in the prior art and provides a clear advance in the state of the art. Generally, the present invention provides an Equine Herpesvirus (EHV) comprising a feline herpesvirus (FHV) antigen coding sequence inserted into ORF70 (US4) and / or ORF1 / 3. The present invention also provides an EHV (equine herpesvirus) comprising a single feline herpesvirus (FHV) antigen coding sequence inserted into ORF70 (US4) and / or a single feline herpesvirus (FHV) antigen coding sequence inserted into ORF1 / 3. The present invention further provides an Equine Herpesvirus (EHV) comprising a feline herpesvirus (FHV) antigen coding sequence encoding 1,400 or fewer amino acids, wherein the FHV antigen coding sequence is inserted into ORF70 (US4) and / or ORF1 / 3.

[0007] Advantageously, experimental data provided by the present invention demonstrate that feline herpesvirus (FHV) antigens can be successfully expressed when inserted into EHV using the ORF70(US4) and / or ORF1 / 3 insertion sites. Thus, EHV using the ORF70(US4) and / or ORF1 / 3 insertion sites is suitable for expressing FHV antigens.

[0008] The term "EHV" refers to equine herpesvirus or equine abortion virus, a pathogen of horses, belonging to the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae, order Herpesvirales. The term "ORF70" or "US4" defines an insertion site within EHV that is well known to those skilled in the art and has already been described in the prior art, such as in WO 2018 / 054837. Insertion into ORF70 means that a DNA fragment has been inserted into the genomic DNA at the position encoding open reading frame 70 of equine herpesvirus 1, thereby silencing expression of the ORF70 gene product, glycoprotein G. Preferably, however, ORF71 remains functional or intact. The term "ORF1 / 3" defines another insertion site in EHV that is well known to those skilled in the art and has already been described in the prior art, such as WO 2018 / 054837 or Said et al. 2013 (Virus Research 173: 371-376). The use of this insertion site affects ORF1 / ORF2, and therefore this insertion site is also referred to as the ORF1 / ORF2 insertion site. This insertion site was initially identified by chance, when a 1283 bp fragment containing 90% of ORF1 and the entire ORF2 was lost due to accidental deletion during passaging of the vaccine strain EHV-1 RacH during attenuation.

[0009] Generally, an "open reading frame" or "ORF" refers to a length of nucleic acid sequence (DNA or RNA) that includes a translation initiation signal or start codon (e.g., ATG or AUG) and a stop codon, and that can be translated into a polypeptide sequence. As used herein, "antigen" refers to, but is not limited to, a component that elicits an immunological response in a host to an immunological composition or vaccine of interest containing the antigen or its immunologically active components. The term "antigen" encompasses full-length proteins and peptide fragments thereof that comprise or consist of one or more epitopes. The term "antigen-coding sequence" also refers to a sequence that encodes an antigen. Preferably, the antigen-coding sequence is a nucleic acid sequence, such as a cDNA sequence or a DNA sequence. However, the term "nucleic acid sequence" is defined elsewhere herein. The term "feline herpesvirus" or "FHV" is well known to those skilled in the art. FHV is the causative agent of feline viral rhinotracheitis in cats, causing upper respiratory tract infection. FHV belongs to the Alphaherpesvirinae subfamily of the Herpesviridae family. The entire genomes of several FHVs have already been sequenced (e.g., the wild-type strain C-27, the sequence of which is publicly available (ACCN: FJ478159)). Preferably, the FHV is FHV-1.

[0010] Insertion site In one particular aspect of the EHV of the invention, a feline herpesvirus (FHV) antigen coding sequence is inserted into ORF70 (US4). In another particular aspect of the EHV of the invention, a feline herpesvirus (FHV) antigen coding sequence is inserted into ORF1 / 3. In another particular aspect of the EHV of the invention, feline herpesvirus (FHV) antigen coding sequences are inserted into ORF70 (US4) and ORF1 / 3. Feline herpesvirus (FHV) antigen length In another particular aspect of the EHV of the invention, the FHV antigen coding sequence encodes 1400 or fewer amino acids. Advantageously, experimental data provided by the present invention demonstrate that feline herpesvirus (FHV) antigens can be successfully expressed when inserted into EHV using the ORF70 (US4) and / or ORF1 / 3 insertion sites. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence encodes 1200 or fewer amino acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence encodes 1000 or fewer amino acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence encodes between 200 and 1200 amino acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence encodes between 200 and 1000 amino acids.

[0011] The terms "protein," "amino acid," and "polypeptide" are used interchangeably. The term "protein" refers to a sequence of amino acids consisting of naturally occurring amino acids and their derivatives. Naturally occurring amino acids are well known in the art and are described in standard biochemistry textbooks. Within an amino acid sequence, the amino acids are joined by peptide bonds. The ends of an amino acid sequence are also referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). The term "protein" encompasses essentially purified proteins or protein preparations that further comprise other proteins. The term also relates to protein fragments. Furthermore, the term includes chemically modified proteins. Such modifications may be artificial or naturally occurring (e.g., phosphorylation, glycosylation, myristylation, etc.). In another particular aspect of the EHV of the invention, the FHV antigen coding sequence comprises no more than 4200 nucleic acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence comprises no more than 3600 nucleic acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence comprises no more than 3000 nucleic acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence encodes between 600 and 3600 nucleic acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence encodes between 600 and 3000 nucleic acids.

[0012] The terms "nucleic acid," "nucleic acid sequence," or "nucleotide sequence" refer to polynucleotides, including DNA molecules, RNA molecules, cDNA molecules, or derivatives thereof. The terms encompass single-stranded and double-stranded polynucleotides. The nucleic acids of the present invention encompass isolated polynucleotides (i.e., isolated from their natural state) and genetically modified forms. Chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides. The terms "nucleic acid" and "polynucleotide" are interchangeable and refer to any nucleic acid. The terms "nucleic acid" and "polynucleotide" also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).

[0013] Antigens and definitions of gD, gB, and gC In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is an FHV antigen coding sequence. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is a single FHV antigen coding sequence. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is not two or more FHV antigen coding sequences. Advantageously, experimental data provided by the present invention demonstrate that feline herpesvirus (FHV) antigens can be successfully expressed when inserted into EHV using the ORF70 (US4) and / or ORF1 / 3 insertion sites, demonstrating superior expression when only one FHV antigen is inserted per insertion site compared to gD and gB flanked by F2A inserted into the ORF1 / 3 site (as shown in Example 6, Tables 5, 7, and 8). Preferably, the FHV antigen is a glycoprotein. The herpesvirus antigen may be the glycoprotein B (gB), glycoprotein C (gC) or glycoprotein D (gD) antigen from feline herpesvirus. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is a glycoprotein coding sequence or a fragment thereof. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is an FHV gD (glycoprotein D) coding sequence or a fragment thereof, and / or an FHV gB (glycoprotein B) coding sequence or a fragment thereof, and / or an FHV gC (glycoprotein C) coding sequence or a fragment thereof.

[0014] Herpesvirus glycoprotein D (gD) is essential for FHV-1 (feline herpesvirus-1) entry and is involved in the interaction with host cells (binding to receptors). The gD protein has hemagglutination activity against feline red blood cells. Herpesvirus glycoprotein B (gB) is essential for FHV entry and is involved in the fusion process. Both glycoproteins can induce neutralizing antibodies. Herpesvirus glycoprotein C (gC) is involved in virus attachment to cells through interaction with heparin sulfate. Glycoprotein gC can also induce neutralizing antibodies. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is an FHV gD coding sequence or a fragment thereof. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is an FHV gB coding sequence or a fragment thereof. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is an FHV gC coding sequence or a fragment thereof.

[0015] Glycoprotein fragments In another particular aspect of the EHV of the invention, the fragment of the glycoprotein coding sequence encodes at least 100, at least 150, at least 200, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids. In another particular aspect of the EHV of the invention, the fragment of the glycoprotein coding sequence comprises at least 300, at least 450, at least 600, at least 900, at least 1050, at least 1200, at least 1500, at least 1800, at least 2100, or at least 2400 nucleic acids. In another particular aspect of the EHV of the invention, the fragment of the glycoprotein coding sequence encodes at least 100, at least 200, at least 300, or at least 350 amino acids. In another particular aspect of the EHV of the invention, the fragment of the glycoprotein coding sequence comprises at least 300, at least 600, at least 900, or at least 1050 nucleic acids. In another particular EHV aspect of the invention, the fragment of the FHV gD coding sequence encodes at least 100, at least 200, at least 250, at least 300, or at least 350 amino acids. In another particular aspect of the EHV of the invention, the fragment of the FHV gD coding sequence comprises at least 300, 600, 750, 900, or 1050 nucleic acids. In another particular EHV aspect of the invention, the fragment of the FHV gB coding sequence encodes at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids. In another particular EHV aspect of the invention, the fragment of the FHV gB coding sequence comprises at least 300, at least 450, at least 600, at least 900, at least 1200, at least 1500, at least 1800, at least 2100, or at least 2400 nucleic acids. In another particular aspect of the EHV of the invention, the fragment of the FHV gC coding sequence encodes at least 100, at least 150, at least 200, at least 300, at least 400, or at least 500 amino acids. In another particular aspect of the EHV of the invention, the fragment of the FHV gC coding sequence comprises at least 300, at least 450, at least 600, at least 900, at least 1200, or at least 1500 nucleic acids.

[0016] Definition of gB, gC, and gD by sequence In another particular aspect of the EHV of the present invention, the FHV antigen coding sequence is an FHV gD coding sequence, and / or an FHV gB coding sequence, and / or an FHV gC coding sequence based on the wild-type feline herpesvirus strain C-27 (ACCN: FJ478159). In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is an FHV gD coding sequence, and the FHV gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in AAB30980.1, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids.

[0017] The term "identity" or "sequence identity" is known in the art and refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., a reference sequence and a given sequence that is compared to the reference sequence. Sequence identity is determined by comparing a given sequence to a reference sequence after optimally aligning the sequences to produce the highest sequence similarity, as determined by the match between strings of sequences. Such alignment determines sequence identity on a position-by-position basis; for example, if the nucleotide or amino acid residue at a position is identical, the sequences are "identical" at that particular position. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give the percentage of sequence identity. Sequence identity can be readily calculated using known methods. Known methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AN, ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York (1993), Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, 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). These teachings are incorporated herein by reference.Preferred methods for determining sequence identity are designed to give the largest match between the sequences tested. Methods for determining sequence identity are codified in publicly available computer programs that 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, SF 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 to achieve the highest level of sequence identity between a given sequence and a reference sequence. By way of example, a polynucleotide having a nucleotide sequence with at least, for example, 85%, preferably 90%, and even more preferably 95% "sequence identity" with a reference nucleotide sequence is intended to indicate that the nucleotide sequence of the given polynucleotide is identical to the reference sequence, except that the given polynucleotide sequence may contain up to 15, preferably up to 10, and even more preferably up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence.In other words, in a polynucleotide having a nucleotide sequence at least 85%, preferably 90%, and even more preferably 95% identical to a reference nucleotide sequence, up to 15%, preferably up to 10%, and even more preferably up to 5% of the nucleotides of the reference sequence may be deleted or substituted with alternative nucleotides, or up to 15%, preferably up to 10%, and even more preferably up to 5% of the total nucleotides of the reference sequence may be inserted into the reference sequence. These variations in the reference sequence can occur at the 5' or 3' terminal position of the reference nucleotide sequence, or at any position between the terminal positions, and may be interspersed individually among nucleotides in the reference sequence or interspersed in one or more contiguous groups within the reference sequence. Similarly, in the case of a polypeptide having a given amino acid sequence that is at least, e.g., 85%, preferably 90%, and even more preferably 95% sequence identity with 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 contain up to 15, preferably up to 10, and even more preferably up to 5 amino acid changes per every 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, and even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, and even more preferably up to 5% of the amino acid residues of the reference sequence may be deleted or substituted with other amino acids, or up to 15%, preferably up to 10%, and even more preferably up to 5% of the total number of amino acid residues of the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may occur at the amino or carboxy terminal position of the reference amino acid sequence, or at any position between the two terminal positions, and may be individually interspersed between residues in the reference sequence or interspersed in one or more adjacent groups within the reference sequence. Preferably, non-identical residue positions differ by conservative amino acid substitutions. However, when determining sequence identity, conservative substitutions are not included as matches.

[0018] As used herein, the terms "identity," "sequence identity," and "% identity" are used interchangeably. For purposes of the present invention, it is defined herein that to determine the % identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into a first amino acid sequence or nucleic acid sequence for optimal alignment with a second amino acid sequence 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, the molecules are identical at that position. The % identity between both 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) × 100). Preferably, both sequences are the same length.

[0019] Sequence comparisons can be performed over the entire length of both sequences being compared, or over fragments of both sequences. Typically, comparisons will be performed over the entire length of both sequences being compared. However, sequence identity can be performed over a region of, for example, 20, 50, 100 or more contiguous amino acid residues. Those skilled in the art will be aware of the fact that different computer programs can be used to determine the homology between two sequences. For example, the sequence comparison and determination of the percent identity between two sequences can be achieved using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the algorithm by Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)), which is incorporated into the GAP program of the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. Those skilled in the art will understand that although all these different parameters will produce slightly different results, the overall percent identity of two sequences will not change significantly when using different algorithms.

[0020] The protein or nucleic acid sequences of the present invention can further be used as "query sequences" to conduct searches against public databases (e.g., 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, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present 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 National Center for Biotechnology Information homepage at http: / / www.ncbi.nlm.nih.gov / . As used herein, it is specifically understood that the term "identical to the sequence of SEQ ID NO:X" is equivalent to either the term "identical to the sequence of SEQ ID NO:X over the length of SEQ ID NO:X" or the term "identical to the sequence of SEQ ID NO:X over the entire length of SEQ ID NO:X." In this context, "X" is any integer selected from 1 to 24, and "SEQ ID NO:X" represents any of the SEQ ID NOs referred to herein.

[0021] In another particular aspect of the EHV of the present invention, the FHV antigen coding sequence is an FHV gB coding sequence, The gB coding sequence consists of, or comprises, a nucleic acid sequence encoding the amino acid sequence set forth in AAB28559.3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is a glycoprotein coding sequence, which consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300 or at least 350 consecutive amino acids. In another particular aspect of the EHV of the invention, the FHV antigen coding sequence is a gD, gB or gC coding sequence, wherein the gD, gB or gC coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300 or at least 350 consecutive amino acids.

[0022] In another particular aspect of the EHV of the invention, the FHV antigen-coding sequence is a glycoprotein consisting of or comprising the nucleic acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 600, at least 900, or at least 1050 consecutive nucleic acids. In another particular aspect of the EHV of the invention, the FHV antigen-encoding sequence is gD, gB or gC consisting of or comprising the nucleic acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 600, at least 900 or at least 1050 consecutive nucleic acids.

[0023] In another particular aspect of the EHV of the invention, the gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300 or at least 350 consecutive amino acids. In another particular aspect of the EHV of the invention, the gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence.

[0024] In another particular aspect of the EHV of the invention, the gB coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700 or at least 800 consecutive amino acids. In another particular aspect of the EHV of the invention, the gB coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence.

[0025] In another particular aspect of the EHV of the invention, the gC coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400 or at least 500 consecutive amino acids. In another particular aspect of the EHV of the invention, the gC coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:3, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence.

[0026] In another particular aspect of the EHV of the invention, the gD coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 600, at least 900 or at least 1050 consecutive nucleic acids. In another particular aspect of the EHV of the invention, the gD coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence.

[0027] In another particular aspect of the EHV of the invention, the gB coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 450, at least 600, at least 900, at least 1200, at least 1500, at least 1800, at least 2100 or at least 2400 consecutive nucleic acids. In another particular aspect of the EHV of the invention, the gB coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence.

[0028] In another particular aspect of the EHV of the invention, the gC coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 450, at least 600, at least 900, at least 1200 or at least 1500 consecutive nucleic acids. In another particular aspect of the EHV of the invention, the gC coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence.

[0029] EHV In another particular aspect of the EHV of the invention, the EHV is a vector. In another particular aspect of the EHV of the invention, the EHV is an EHV vector. In another particular aspect of the EHV of the invention, the EHV is attenuated. The term "attenuated" refers to a pathogen that has reduced pathogenicity compared to the isolated wild-type. In the present invention, "attenuated" is synonymous with "non-pathogenic." In the present invention, an attenuated virus is one that has reduced pathogenicity so that it does not cause clinical signs of infection but is still able to induce an immune response in the target animal. However, an attenuated virus can also mean that the incidence or severity of clinical signs is reduced in animals infected with the attenuated virus, particularly an EHV-1 RacH viral vector, compared to a "control group" of animals infected with a non-attenuated virus or pathogen and not receiving the attenuated virus. In this context, the term "reduce / reduced" means a reduction of at least 10%, preferably 25%, even more preferably 50%, even more preferably 60%, even more preferably 70%, even more preferably 80%, even more preferably 90%, and most preferably 100% compared to a control group as defined above.

[0030] In another particular aspect of the EHV of the invention, the EHV is genetically engineered. The term "genetically engineered" refers to an EHV that has been mutated by using a "reverse genetics" approach. Preferably, the EHV of the present invention is genetically engineered. However, "reverse genetics" techniques are well known to those skilled in the art. In another particular aspect of the EHV of the invention, the EHV is recombinant. As used herein, the term "recombinant" refers to a viral genome or nucleic acid sequence that has any alteration that does not occur naturally in the corresponding viral genome or nucleic acid sequence. For example, a viral genome or nucleic acid sequence is considered "recombinant" if it contains an insertion, deletion, inversion, rearrangement, or point mutation that has been artificially introduced (e.g., by human intervention). Thus, the viral genome or nucleic acid sequence is not related to all or part of the viral genome or nucleic acid sequence with which it is associated in nature. The term "recombinant virus" encompasses viruses that have been genetically modified. Also, viruses that include exogenous sequences, such as heterologous sequences or exogenous antigen-encoding sequences (e.g., feline antigen-encoding sequences), are recombinant viruses. The terms recombinant virus and non-naturally occurring virus are used interchangeably.

[0031] In another particular aspect of the EHV of the invention, the EHV is selected from the group consisting of EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9. To date, eight different species of equine herpesviruses have been identified, five belonging to the Alphaherpesvirinae subfamily (EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9) and three belonging to the Gammaherpesvirinae subfamily. In another particular aspect of the EHV of the invention, the EHV is EHV-1. The term "EHV-1" is familiar to those skilled in the art, but non-limiting reference sequences for EHV-1 may include, for example, the wild-type EHV-1 strain ab4 (Genbank accession number AY665713.1) or RacH (Hubert 1996. Journal of Veterinary Medicine, Series B, 43: 1-14.). In another particular aspect of the EHV of the invention, the EHV is a RacH or a RacH SE. Two approved modified live (MLV) vaccines against EHV-1 are currently available in the United States and Europe (Rhinomune® (Boehringer Ingelheim) and Prevaccinol® (MSD)). Both vaccines contain the classically attenuated EHV-1 RacH strain, which has been passaged 256 times in porcine epithelial cells for attenuation (Ma et al. 2013. Vet Microbiol. 167(1-2):123-34). The mechanism of attenuation has been investigated at the molecular level. RacH is also a very safe vaccine strain because the chance of reversion to virulence is very low, if at all, due to passage in vaccinated animals.

[0032] ORF70 insertion site In another particular aspect of the EHV of the invention, the insertion into ORF70 (US4) is characterized by a partial deletion, truncation, substitution or modification in ORF70 (US4), while US5 (ORF71) remains functional. Preferably, the insertion results in a deletion of the 5' 801 base pairs of ORF70, abolishing expression of the orf70 gene product, glycoprotein G, while leaving the remaining 423 bp at the 3' end intact. In another particular aspect of the EHV of the invention, the insertion into ORF70 is characterized by a deletion of approximately 801 bp within ORF70 of wild-type EHV-1 strain ab4 (Genbank Accession No. AY665713.1), whereby the deleted portion of the wild-type ab4 genomic sequence is located between nucleotides 127681 and 128482 (SEQ ID NO:10) or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:10.

[0033] In another particular aspect of the EHV of the invention, the ORF70 insertion site encompasses a theoretical 801 bp deletion within ORF70 of wild-type EHV-1 strain ab4 (Genbank Accession No. AY665713.1), which is located between nucleotides 127681 and 128482 (SEQ ID NO: 10) of the genomic sequence of wild-type ab4 (Genbank Accession No. AY665713.1). In another particular aspect of the EHV of the invention, the insertion into ORF70(US4) is characterized by the deletion of an approximately 801 bp portion within ORF70(US4) of RacH (SEQ ID NO:11), or the deletion of a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:11. In another particular aspect of the EHV of the invention, the EHV comprises at least one flanking region selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to said flanking region.

[0034] In the present invention, the "flanking regions" direct the recombination of the expression cassette containing the FHV antigen coding sequence into the EHV-1 genome. These flanking regions are naturally present in EHV-1. The flanking regions have already been described in WO 2018 / 054837. In another particular aspect of the EHV of the invention, the EHV comprises (i) at least one US4 left flanking region selected from the group consisting of SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, and (ii) at least one US4 right flanking region selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17.

[0035] Promoter and Regulatory Sequences In another particular aspect of the EHV of the invention, the feline herpesvirus (FHV) antigen coding sequence is operably linked to a promoter sequence. In another particular aspect of the EHV of the invention, the gD coding sequence and / or the gB coding sequence is operably linked to a promoter sequence. As used herein, the term "promoter" or "promoter sequence" refers to a nucleotide sequence that allows RNA polymerase to bind and direct the transcription of a gene. Typically, promoters are located in the 5' untranslated region of a gene, close to the transcription start site of the gene. Sequence elements within a promoter that function in initiating transcription are often characterized by a consensus nucleotide sequence. Examples of promoters include, but are not limited to, promoters derived from bacteria, yeast, plants, viruses, and animals such as mammals (including horses, pigs, cattle, and humans), birds, or insects. Promoters may be inducible, repressible, and / or constitutive. An inducible promoter initiates increased transcription levels from DNA under its control in response to some change in culture conditions, such as a temperature shift (Ptashne, 2014. The Journal of Biological Chemistry Vol. 289, 9, 5417-5435). Examples of promoters well known to those skilled in the art include, for example, SV40 large T, HCMV and MCMV immediate early gene 1, human elongation factor alpha promoter, and baculovirus polyhedrin promoter.

[0036] As used herein, the term "operably linked" is used to describe the connection between a regulatory element and a nucleic acid sequence (e.g., a gene or antigen-encoding sequence). Typically, a nucleic acid sequence is placed under the control of one or more regulatory elements (e.g., but not limited to, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers). A nucleic acid sequence, gene, or coding region is said to be "operably linked" to, or "operably associated with," a regulatory element, meaning that the gene or coding region is controlled or influenced by the regulatory element. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Linking can be achieved by recombinant methods known in the art, such as ligation at appropriate restriction sites or blunt ends, or by using fusion PCR methodology. If suitable restriction sites are not present, synthetic oligonucleotide linkers or adapters can be used in accordance with conventional practice.

[0037] In another particular aspect of the EHV of the invention, the promoter sequence is selected from the group consisting of SV40 large T, HCMV and MCMV immediate early gene 1, human elongation factor alpha promoter, baculovirus polyhedrin promoter, p430 (SEQ ID NO: 18), or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to SEQ ID NO: 18. or a fragment of at least 350 contiguous nucleotides of SEQ ID NO: 18; p455 (SEQ ID NO: 19) or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, 99.95%, at least 99.98% or at least 99.99% sequence identity to SEQ ID NO: 19; or a promoter consisting of or comprising a fragment of at least 350 contiguous nucleotides of SEQ ID NO: 19; and The gB promoter (SEQ ID NO:24) or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to SEQ ID NO:24, or a promoter consisting of or comprising a fragment of at least 300 contiguous nucleotides of SEQ ID NO:24.

[0038] In another particular aspect of the EHV of the invention, the promoter sequence is a promoter consisting of or comprising p430 (SEQ ID NO:18), or a sequence having at least 95% sequence identity to SEQ ID NO:18, or a fragment of at least 400 contiguous nucleotides of SEQ ID NO:18. In another particular aspect of the EHV of the invention, the promoter sequence is a promoter consisting of or comprising p455 (SEQ ID NO:19), or a sequence having at least 95% sequence identity to SEQ ID NO:19, or a fragment of at least 400 contiguous nucleotides of SEQ ID NO:19. In any of the above-mentioned aspects of EHV, the promoter sequence is a promoter consisting of or comprising a truncated FHV-1 gB promoter (SEQ ID NO:24), or a sequence having at least 95% sequence identity to SEQ ID NO:24, or a fragment of at least 350 contiguous nucleotides of SEQ ID NO:24. In another particular aspect of the EHV of the invention, the EHV comprises one or more additional regulatory sequences, for example a termination signal, a polyadenylation signal, or a regulatory element such as an IRES.

[0039] A "termination signal" or "terminator," or "polyadenylation signal" or "polyA," or "transcription termination site" or "transcription termination element," is a signal sequence that causes cleavage of the 3' end of a eukaryotic mRNA at a specific site and post-transcriptional incorporation of a sequence of approximately 100-200 adenine nucleotides (a polyA tail) into the cleaved 3' end, thereby causing RNA polymerase to terminate transcription. The polyadenylation signal contains the sequence AATAAA approximately 10-30 nucleotides upstream of the cleavage site and sequences located downstream. Various polyadenylation elements are known, including 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 (WO2010010107). In another particular aspect of the EHV of the invention, the EHV comprises a polyadenylation signal. In another particular aspect of the EHV of the invention, the EHV comprises the polyadenylation signal BGHpA.

[0040] "Internal ribosome entry site" or "IRES" describes a sequence that functionally facilitates translation initiation independent of the gene 5' to the IRES, allowing two cistrons (open reading frames) to be translated from a single transcript in a cell. In eukaryotic cells, a polycistronic transcript with an IRES operably linked to the second or subsequent open reading frame of the transcript allows sequential translation of the downstream open reading frame, generating two or more polypeptides encoded by the same transcript. IRESs may vary in length and may be derived from various origins, such as encephalomyocarditis virus (EMCV), picornavirus (e.g., foot-and-mouth disease virus, FMDV, or poliovirus (PV)), or hepatitis C virus (HCV). Various IRES sequences and their use in vector construction have been described and are well known in the art.

[0041] In another particular aspect of the EHV of the invention, the EHV comprises an IRES element. In another particular aspect of the EHV of the invention, two FHV coding sequences flanked by an IRES element are inserted into a single insertion site. In another particular aspect of the EHV of the invention, FHV gD with an IRES and gB is inserted into the ORF1 / 3 site. In another particular aspect of the EHV of the invention, the EHV does not comprise a 2A peptide, such as an F2A peptide. The term "2a" or "2a peptide" refers to short oligopeptide sequences described as 2a and "2a-like" that function as linkers capable of mediating co-translational cleavage between proteins by a process defined as ribosomal skipping. Such 2a and "2a-like" sequences (derived from picornaviridae and other viruses or cellular sequences) can be used to concatenate multiple gene sequences into a single gene and ensure their co-expression within the same cell.

[0042] Specific constructs In another particular aspect of the EHV of the invention, the FHV gD coding sequence or a fragment thereof is inserted into ORF1 / 3 and the FHV gB coding sequence or a fragment thereof is inserted into ORF70. In another particular aspect of the EHV of the present invention, i) the FHV gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids, and is inserted into ORF1 / 3; and ii) the FHV gB coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity thereto, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids, and is inserted into ORF70.

[0043] Immunogenic composition or vaccine The present invention provides immunogenic compositions comprising the EHV described herein. The term "immunogenic composition" refers to a composition that comprises at least one antigen and elicits an immunological response in a host against the administered immunogenic composition. Such an immunological response can be a cellular immune response and / or an antibody-mediated immune response against the immunogenic composition of the present invention. Preferably, the immunogenic composition induces an immune response that, more preferably, confers protective immunity against one or more clinical signs of FHV infection. A host is also referred to as a "subject." Preferably, any host or subject described or referred to herein is a feline or cat. Typically, an "immunological response" includes, but is not limited to, one or more of the following: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells, and / or gamma delta T cells specific for one or more antigens contained in the immunogenic compositions of the invention. Preferably, the host will mount a protective or therapeutic immunological response. A "protective immunological response" or "protective immunity" will be indicated by either a reduction or absence of clinical signs normally exhibited by an infected host, a faster recovery time, and / or a reduction in pathogen titers or a shortened period of infectivity in the tissues, body fluids, or excretions of the infected host.

[0044] When the host mounts a protective immunological response such that resistance to new infection is enhanced and / or the clinical severity of disease is reduced, the immunogenic composition is described as a "vaccine." The present invention also provides vaccines comprising the EHV described herein. Additionally, the present invention provides a DIVA vaccine comprising an EHV as described herein. The term "DIVA (differentiation between infected and vaccinated animals)" refers to a vaccine that can be used to distinguish between naturally infected and vaccinated animals. In another particular aspect of the immunogenic composition, vaccine or DIVA vaccine of the invention, the immunogenic composition, vaccine or DIVA vaccine is bivalent. In another particular aspect of the immunogenic composition, vaccine or DIVA vaccine of the invention, the immunogenic composition, vaccine or DIVA vaccine is multivalent. In another particular aspect of the immunogenic composition, vaccine, or DIVA vaccine of the invention, the immunogenic composition, vaccine, or DIVA vaccine further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, adjuvants, immunostimulants, and combinations thereof.

[0045] "Diluents" can include water, saline, dextrose, ethanol, glycerol, etc. Isotonic agents can include sodium chloride, glucose, mannitol, sorbitol, and lactose, among others. Stabilizers can include albumin and alkali salts of ethylenediaminetetraacetic acid, among others. In another particular aspect of the immunogenic composition, vaccine or DIVA vaccine of the invention, the pharmaceutically acceptable carrier is water for injection, cell culture medium or a resuspension buffer. In another particular aspect of the immunogenic composition, vaccine or DIVA vaccine of the invention, the resuspension buffer is physiological solution or phosphate buffered saline. In another particular aspect of the immunogenic composition, vaccine or DIVA vaccine of the invention, the immunogenic composition, vaccine or DIVA vaccine is at least 1 x 10 of EHV. 4 ~1x10 9 TCID 50 , preferably 1x10 4 ~1x10 8 TCID 50 , and even more preferably 1x10 4 ~1x10 7 TCID 50 Includes.

[0046] kit The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, be constructed from metal or plastic foil (e.g., a blister pack). The pack or dispenser device may be accompanied by instructions for administration, preferably to animals, particularly felines (preferably cats). Such containers may be accompanied by a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the governmental agency for the manufacture, use, or sale for administration to animals. Additionally, the present invention provides kits comprising the immunogenic compositions, vaccines or DIVA vaccines described herein. In another particular aspect of the kits herein, the kit further comprises instructions for treating and / or preventing a disease in a feline animal. In another particular aspect of the kit herein, the kit further comprises instructions for the treatment and / or prevention of FHV. In another particular aspect of the kit herein, the kit further comprises a dispenser capable of administering the vaccine to an animal or feline.

[0047] Treatment method The present invention provides methods of immunization comprising administering to a feline an immunogenic composition, vaccine or DIVA vaccine described herein. The present invention provides a method of immunizing a feline, the method comprising administering to the feline an immunogenic composition, vaccine or DIVA vaccine described herein. Preferably, immunization results in a reduction in the incidence of the specific feline herpesvirus infection within the population or results in a reduction in the severity of clinical signs caused by or associated with the specific feline herpesvirus infection. Furthermore, immunization of felines with the immunogenic compositions provided herein results in the prevention of feline infection with feline herpesvirus. Even more preferably, immunization results in an effective, long-lasting immunological response against feline herpesvirus infection. It will be understood that the period lasts for more than two months, preferably more than three months, more preferably more than four months, more preferably more than five months, and more preferably more than six months. It will be understood that immunization will not be effective in all immunized animals. However, a period during which a majority of the animal population is effectively immunized is required.

[0048] Preferably, a group of felines would normally, i.e., without immunization, develop clinical signs normally attributed to or associated with feline herpesvirus infection. Whether a group of felines has been effectively immunized can be determined without difficulty by one of ordinary skill in the art. Preferably, immunization is effective if at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, even more preferably at least 95%, and most preferably 100% of a given group of animals exhibits at least a 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100% reduction in the incidence or severity of clinical signs compared to felines that were not immunized or that were immunized with an immunogenic composition available prior to the present invention but subsequently infected with a particular feline herpesvirus.

[0049] The present invention provides a method for treating or preventing clinical signs of FHV in a feline, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine described herein. The present invention provides a method for treating or preventing clinical signs of FHV in a feline, compared to an unimmunized control feline, comprising administering to said feline a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine as described herein. Advantageously, experimental data provided by the present invention disclose the safety and efficacy of the immunogenic compositions provided herein when administered to felines. Indeed, felines vaccinated with the immunogenic compositions provided herein exhibited reduced clinical signs associated with disease, such as reduced rhinitis, ocular discharge, or conjunctivitis, following challenge virus infection compared to unvaccinated felines. Preferably, clinical signs are reduced by at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%, compared to an untreated (non-immunized) feline that is subsequently infected with a particular feline herpesvirus.

[0050] The term "treatment and / or prevention" refers to a reduction in the incidence of a specific feline herpesvirus infection in a herd or a reduction in the severity of clinical signs caused by or associated with a specific feline herpesvirus infection. Thus, the term "treatment and / or prevention" also refers to a reduction in the number of animals infected with a specific feline herpesvirus in a herd (= a reduction in the incidence of a specific feline herpesvirus infection), or a reduction in the severity of clinical signs normally associated with or caused by FHV infection in a group of animals that have received an effective amount of the immunogenic composition provided herein compared to a group of animals that have not received the immunogenic composition provided herein. "Treatment and / or prevention" generally involves the administration of an effective amount of the immunogenic composition of the invention to an animal or group of animals in need thereof or that can benefit from such treatment / prevention. The term "treatment" refers to the administration of an effective amount of the immunogenic composition once an animal or at least some animals in the group have already been infected with such FHV (the animals have already shown some clinical signs caused by or associated with the FHV infection). The term "prevention" refers to administration to an animal before any infection with FHV or at least when this animal, or at least none of the animals in the group, have shown any clinical signs caused by or associated with the FHV. The terms "prevention" and "preventing" are used interchangeably in this application.

[0051] As used herein, the term "clinical signs" refers to signs of an animal infected with FHV. Examples of such clinical signs include, but are not limited to, shortness of breath, rhinitis, low-grade fever, depression, and loss of appetite. However, clinical signs also include, but are not limited to, clinical signs that are directly observable in live animals. Examples of clinical signs that are directly observable in live animals include nasal and ocular discharge, lethargy, coughing, wheezing, thumping, elevated body temperature, weight loss, dehydration, lameness, wasting, pale skin, and unthriftiness. As used herein, the term "effective amount," in the context of a composition, refers to a dose of an immunogenic composition capable of inducing an immune response that reduces the incidence or severity of an infection or associated disease in an animal. Such an effective amount can reduce the incidence of a specific feline herpesvirus infection in a population or reduce the severity of the clinical signs of a specific feline herpesvirus infection. In particular, an effective amount refers to colony-forming units (CFU) per dose. Alternatively, in the context of a therapy, the term "effective amount" refers to a dose of a therapy that is sufficient to reduce or ameliorate the severity or duration of a disease or disorder or one or more symptoms thereof, prevent the progression of a disease or disorder, cause regression of a disease or disorder, prevent the recurrence, development, onset, or progression of one or more symptoms associated with a disease or disorder, or enhance or improve prophylaxis or treatment by another therapy or therapeutic agent.

[0052] The present invention provides a method for treating or preventing FHV respiratory disease in a feline, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine described herein. The present invention provides a method for treating or preventing FHV respiratory disease in a feline, relative to an unimmunized control feline, comprising administering to said feline a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine as described herein. The term "respiratory disease" refers to an infection of the respiratory system and includes clinical signs such as ocular and nasal discharge, conjunctivitis, rhinitis, sneezing, coughing, and fever. Preferably, respiratory disease is reduced by at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%, compared to unimmunized control felines and felines of the same species subsequently infected with the particular feline herpesvirus.

[0053] The present invention provides a method for reducing FHV shedding in a feline, the method comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine described herein. The present invention provides a method for reducing FHV shedding in a feline relative to an unimmunized control feline, the method comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine as described herein. The term "shedding" refers to secretions such as nasal or ocular discharge, and also to aerosols generated by coughing or sneezing. Thus, shedding can be determined by testing the virus titer with a nasal or eye swab, or by testing the virus titer in the lungs. The term "shedding" also encompasses the transfer of virus to susceptible animals (i.e., sentinel animals). Methods for measuring virus shedding are within the knowledge of those skilled in the art. The term "reduced" means that shedding is reduced by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%, compared to an untreated (non-immunized) feline that is subsequently infected with a particular feline herpesvirus.

[0054] EP model The present invention provides an immunogenic composition, vaccine or DIVA vaccine as described herein for use in a method for immunizing a feline, said method comprising administering to said feline a therapeutically effective amount of said immunogenic composition, vaccine or DIVA vaccine. The present invention provides an immunogenic composition, vaccine or DIVA vaccine as described herein for use in a method for treating or preventing clinical signs of FHV in a feline, said method comprising administering to said feline a therapeutically effective amount of said immunogenic composition, vaccine or DIVA vaccine. The present invention provides an immunogenic composition, vaccine or DIVA vaccine as described herein for use in a method for treating or preventing FHV respiratory disease in a feline, said method comprising administering a therapeutically effective amount of the immunogenic composition, vaccine or DIVA vaccine to said feline. The present invention provides an immunogenic composition, vaccine or DIVA vaccine as described herein for use in a method for reducing FHV shedding in a feline, said method comprising administering a therapeutically effective amount of the immunogenic composition, vaccine or DIVA vaccine to said feline.

[0055] In another particular aspect of the method or use of the invention, the feline is selected from the group consisting of a cheetah, a puma, a jaguar, a leopard, a lion, a lynx, a tiger, a cat and a domestic cat. In another particular aspect of the method or use of the invention, the feline is a cat or a domestic cat. However, the immunogenic composition may be administered to the feline more than once, with the first dose being administered before the second (booster) dose. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is administered more than once. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is administered twice. As shown in the Examples, the immunogenic compositions provided herein have been shown to be effective after two doses. In addition to the first and second dose regimen, alternative embodiments further include subsequent doses. For example, in this aspect, a third, fourth, or fifth dose may be administered. Preferably, the subsequent third, fourth, or fifth dose regimen is administered in the same amount as the first dose, and the time frame between these doses corresponds to the time between the first and second doses, as described below. In one aspect of the invention, the feline is a cat. Preferably, the dose has a total volume of about 0.25 mL to 2.5 mL, more preferably about 0.25 mL to 1.5 mL, and even more preferably about 0.5 mL to 1 mL.

[0056] In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is administered to a feline that is 4, 5 or 6 weeks of age or older. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is administered to a feline 6 weeks of age or older. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is administered for the first time to a feline between 4 and 25 weeks of age. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is first administered to the feline between 6 and 20 weeks of age. In another particular aspect of the method or use of the invention, a first dose of the immunogenic composition, vaccine or DIVA vaccine is administered to a feline at least 4, 5 or 6 weeks of age, and a second dose is administered 2 to 8 weeks later. In another particular aspect of the method or use of the invention, a first dose of the immunogenic composition, vaccine or DIVA vaccine is administered to a feline 6 weeks of age or older, and a second dose is administered 2 to 8 weeks later. In another particular aspect of the method or use of the invention, after the second (booster) vaccination, further vaccinations are given annually (annual booster vaccinations).

[0057] The immunogenic composition, vaccine, or DIVA vaccine is preferably administered locally or systemically. Suitable administration routes conventionally used include oral or parenteral administration, such as intranasal, intravenous, intramuscular, intraperitoneal, subcutaneous, and inhalation. However, depending on the nature and mechanism of action of the compound, the immunogenic composition, vaccine, or DIVA vaccine may be administered by other administration routes. However, most preferably, the immunogenic composition, vaccine, or DIVA vaccine is administered intramuscularly, subcutaneously, or intranasally. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is administered intramuscularly, intradermally, subcutaneously, orally or intranasally. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is at least 1 x 10 per mL. 4 ~1x10 10 TCID 50 Includes EHVs. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine is at least 1 x 10 per mL. 6 ~1x10 9 TCID 50 Includes EHVs. In another particular aspect of the method or use of the invention, a dose of the immunogenic composition, vaccine or DIVA vaccine is 1 x 10 per mL 4 ~1x10 10 TCID 50 Includes EHVs. In another particular aspect of the method or use of the invention, a dose of the immunogenic composition, vaccine or DIVA vaccine is 1 x 10 per mL 6 ~1x10 9 TCID 50 Includes EHVs.

[0058] In another particular aspect of the method or use of the invention, the method results in an improvement in an efficacy parameter selected from the group consisting of reduced weight loss, reduced viral load in the lungs, reduced viremia, reduced lung lesions, reduced respiratory and alveolar epithelial lesions, reduced eye discharge, reduced conjunctivitis, reduced rhinitis, reduced and / or shortened viral shedding, reduced rectal temperature, reduced clinical symptoms (particularly respiratory symptoms), increased (neutralizing) induction of anti-FHV virus antibodies, increased stimulation of T cells against FHV, increased stimulation of B cells against FHV virus, and reduced inflammatory cytokines in the lungs (e.g. IL1β), or a combination thereof, compared to an unimmunized control group of the same species of feline.

[0059] The term "viral load" is well known to those skilled in the art. The term "viral load" is used interchangeably with the term "viral titer" herein. Viral load or viral titer is a measure of the severity of an active viral infection and can be determined by methods known to those skilled in the art. The determination can be based on the detection of viral proteins (e.g., by antibodies that bind to viral proteins) and further detection, or alternatively, the detection of viral nucleic acids by amplification methods such as RT-PCR. Monitoring virions associated with viral RNA in plasma by nucleic acid amplification methods is a widely used parameter for assessing the state and progression of retroviral diseases and the effectiveness of preventive or therapeutic interventions. Illustratively, viral load or viral titer can be calculated by estimating the viable amount of virus in the relevant body fluid (e.g., the number of RNA copies per milliliter of plasma). Preferably, the term "viral load" or "viral titer" is a measure of infectious units per volume of a viral preparation. Viral titer is the endpoint of biological procedures and is defined as the dilution at which a specific proportion of parallel tests show efficacy (Reed and Muench, 1938). Specifically, the 50% tissue culture infectious dose per milliliter (TCID50 / mL) indicates the dilution of a virus preparation that infects 50% of the number of cell cultures inoculated in parallel at that dilution. In another particular aspect of the method or use of the invention, the immunogenic composition, vaccine or DIVA vaccine protects against homologous and / or heterologous challenge with FHV. Experiments have shown that an EHV vaccine carrying FHV antigens (vaccine strain C-27) protects against a heterologous EHV vaccine (SGE strain, designated FVR 96-13).

[0060] Overall picture of EHV The present invention provides an EHV (Equine Herpesvirus) comprising an FHV gD coding sequence or a fragment thereof inserted into ORF1 / 3 and an FHV gB coding sequence or a fragment thereof inserted into ORF70. The present invention provides an EHV (Equine Herpesvirus), the EHV comprising: i) an FHV gD coding sequence inserted into ORF1 / 3, the FHV gD coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity thereto, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids; ii) an FHV gB coding sequence inserted into ORF70, the FHV gB coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:5, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids.

[0061] The present invention provides an EHV (Equine Herpesvirus) comprising an FHV gD coding sequence inserted into ORF1 / 3, the FHV gD coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:1, or a sequence having at least 95% sequence identity to said amino acid sequence; and an FHV gB coding sequence inserted into ORF70, the FHV gB coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:2, or a sequence having at least 95% sequence identity to said amino acid sequence. The present invention provides an immunogenic composition, vaccine or DIVA vaccine comprising EHV (Equine Herpesvirus), said EHV comprising: i) an FHV gD coding sequence inserted into ORF1 / 3, the FHV gD coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity thereto, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids; ii) an FHV gB coding sequence inserted into ORF70, the FHV gB coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids.

[0062] The present invention further provides an EHV, immunogenic composition, vaccine or DIVA vaccine described herein for use in therapy. The present invention further provides an EHV, immunogenic composition, vaccine or DIVA vaccine described herein for use as an immunogen or vaccine. The present invention further provides an EHV, immunogenic composition, vaccine or DIVA vaccine described herein for use as a medicament. The present invention further provides an EHV, immunogenic composition, vaccine or DIVA vaccine as described herein for the manufacture of a medicament. The present invention further provides the use of an EHV, immunogenic composition, vaccine or DIVA vaccine as described herein for the treatment and / or prevention of FHV infection in a feline.

[0063] Manufacturing method The present invention further provides a method for preparing a feline herpesvirus (EHV) (Equine Herpesvirus) comprising an FHV antigen coding sequence inserted into ORF70 (US4) and / or ORF1 / 3, said method comprising: i) providing EHVs; ii) providing an FHV antigen coding sequence; iii) inserting the FHV antigen coding sequence obtained from step ii) into the EHV ORF70 (US4) and / or ORF1 / 3 insertion site of step i); iv) obtaining said EHV comprising an FHV antigen coding sequence inserted into ORF70(US4) and / or ORF1 / 3. The present invention further provides a method for preparing a feline herpesvirus (EHV) (Equine Herpesvirus) comprising an FHV antigen coding sequence inserted into ORF70 (US4) and / or ORF1 / 3, said method comprising: i) providing EHVs; ii) providing an FHV gD, gB or gC coding sequence, the FHV gD, gB or gC coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity thereto, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300 or at least 350 consecutive amino acids; iii) inserting the FHV gD, gB or gC coding sequence obtained from step ii) into the EHV ORF70 (US4) and / or ORF1 / 3 insertion site of step i); iv) obtaining said EHV comprising an FHV gD, gB or gC coding sequence inserted into ORF70 (US4) and / or ORF1 / 3.

[0064] The term "obtaining" includes collecting, isolating, purifying and / or formulating (eg polishing, inactivating and / or blending) said EHV carrying an FHV antigen-encoding sequence. The term "harvesting" refers to the collection or recovery of the EHV carrying the FHV antigen-encoding sequence from transfected or infected cells, bacteria, or cell lines. Any conventional method known in the art can be used, for example, any separation method. Methods well known in the art include centrifugation or filtration (e.g., filtration using a semipermeable membrane with a certain pore size). The term "isolation" includes the process of isolating said EHV carrying an FHV antigen-encoding sequence. Methods for isolation from transfected or infected cells, bacteria or cell lines are known to those skilled in the art. These methods include physical and / or chemical methods, including, but not limited to, freeze-thaw cycles, sonication, etc.

[0065] Methods for "purifying" the EHV carrying the FHV antigen-encoding sequence from an isolate are known to those skilled in the art, for example, by methods described in Protein purification methods - a practical approach (E.L. V. Harris and S. Angel, eds., IRL Press at Oxford University Press). These methods include, but are not limited to, separation by centrifugation and / or filtration, precipitation, size exclusion (gel filtration) chromatography, affinity chromatography, metal chelate chromatography, ion exchange chromatography, covalent chromatography, hydrophobic interaction chromatography, etc. The vector can be obtained in a purified, pure form, or free or substantially free of other cellular material or culture medium, etc. After said isolation and / or purification, the antigen exhibits a purity of at least 80%, preferably 80% to 90%, more preferably 90% to 97%, and most preferably 97% or more, up to an absolutely pure form free from contaminants. In further respects, "obtaining" as used herein may also include further polishing steps as part of the final formulation process, such as buffer addition steps, inactivation steps, neutralization steps, etc. In another particular aspect of the method of the present invention for preparing an EHV comprising an FHV antigen coding sequence inserted into ORF70(US4) and / or ORF1 / 3, the EHV is attenuated. In another particular aspect of the method of the present invention for preparing an EHV comprising an FHV antigen coding sequence inserted into ORF70(US4) and / or ORF1 / 3, the EHV is RacH or RacH SE.

[0066] DIVA DIVA vaccines facilitate rapid and effective administration and allow differentiation between animals infected with field viruses (associated with the disease) and those that have been vaccinated. The immunogenic compositions, vaccines or DIVA vaccines of the present invention do not contain any FHV gG antigen coding sequence. In contrast, after animals are infected with wild-type feline herpesvirus, or receive a modified live vaccine, or a whole virus inactivated vaccine, the infected / vaccinated animals produce / have specific antibodies against FHV gG, but such specific antibodies against FHV gG cannot be detected in animals vaccinated with the immunogenic composition of the present invention. The immunogenic compositions, vaccines or DIVA vaccines of the invention comprise EHV and the EHV gD and EHV gC coding sequences, respectively, such that specific antibodies to EHV, such as antibodies to EHV gD and / or EHV gC, can be detected in animals vaccinated with the immunogenic compositions of the invention. In contrast, after an animal has been infected with a wild-type feline herpesvirus or received a modified live vaccine or a whole virus inactivated vaccine, the animal will be negative for EHV-specific antibodies, such as antibodies against EHV gD and / or EHV gC, whereas in animals vaccinated with the immunogenic composition of the present invention, such specific antibodies against EHV gD and EHV gC can be detected.

[0067] Exemplary immune and / or genomic analysis tests allow animals vaccinated with the immunogenic compositions of the present invention to be distinguished from animals infected with wild-type feline herpesvirus or animals that have received a modified live vaccine or a whole virus inactivated vaccine, because a) animals vaccinated with the immunogenic compositions of the present invention do not have any specific antibodies against FHV gD and any FHV gD coding sequence, respectively, or b) animals vaccinated with the immunogenic compositions of the present invention have specific antibodies against EHV gD and / or EHV gC, and the EHV gD coding sequence and / or EHV gC coding sequence, respectively. The terms "immunization test" and "genomic analysis test" are the basis for distinguishing between animals vaccinated with the immunogenic composition of the invention and animals infected with FHV.

[0068] Examples of immunoassays include any enzyme immunological or immunochemical detection method, such as ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), sandwich enzyme immunoassay, fluorescent antibody test (FAT), electrochemiluminescence sandwich immunoassay (ECLIA), dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) or solid-phase immunoassay, immunofluorescence test (IFT), immunohistochemistry, Western blot analysis, or any other suitable method available to those skilled in the art. Depending on the assay used, antigens or antibodies can be labeled with enzymes, fluorophores, or radioisotopes. See, for example, Coligan et al., Current Protocols in Immunology, John Wiley & Sons Inc., New York, NY (1994), and Frye et al., Oncogen 4: 1153-1157, 1987. The term "genomic analysis test" refers to a genomic analysis method based on polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time PCR (r-PCR) or real-time reverse transcription PCR (rRT-PCR), Templex-PCR, nucleic acid sequence-based amplification (NASBA), or isothermal amplification, which uses a polymerase and specific oligonucleotides as primers. The above amplification methods are well known in the art.

[0069] The present invention provides a method for distinguishing between felines vaccinated with an immunogenic composition, vaccine or DIVA vaccine described herein and felines infected with FHV, said method comprising: a) obtaining a sample from a feline; b) analyzing the sample with an immunological test, a cell culture-based assay, and / or a genomic analysis test. Advantageously, the experiments of the present application already explain how such an analysis of samples was performed: Examples 3 and 4 describe an IFA (immunofluorescence assay) for measuring the expression of gD and gB (immunoassay); in Examples 4 and 5, the assay readout for FHV SN and EHV SN is based on a cell culture-based assay, the CPE (cytopathic effect or cytopathogenic effect) assay.

[0070] Maes 2012 (ISRN Veterinary Science. (2-3):495830) also reviews various methods for diagnosing FHV infection. The most common laboratory diagnostic methods for demonstrating FHV or viral components in tissue homogenates or swabs include direct fluorescent antibody (FA) tests, virus isolation (VI), and PCR. Direct fluorescent antibody tests are performed on conjunctival or corneal tissue. Virus isolation (VI) or PCR uses extracts from oral, nasal, and conjunctival swabs as samples. VI detects infectious virus and is the gold standard for laboratory diagnosis. However, several PCR assays have been described that can also be used to detect FHV.

[0071] The term "sample" refers to a sample of a body fluid, a sample of separated cells, or a sample obtained from a tissue or an organ. Body fluid samples can be obtained by well-known techniques and preferably include blood, plasma, serum, or urine samples, more preferably blood, plasma, or serum samples. Tissue or organ samples can be obtained from any tissue or organ by, for example, biopsy. Separated cells can be obtained from body fluids or tissues or organs by separating techniques such as centrifugation or cell sorting. However, the term "sample" also encompasses oropharyngeal, conjunctival, or nasal swabs. The term "obtained" may include isolation and / or purification steps known to those skilled in the art, preferably using precipitation, columns, etc.

[0072] In one particular aspect of the differentiation method of the invention, the immunological test involves testing whether the sample contains antibodies that specifically recognize FHV gG. In another particular aspect of the differentiation method of the present invention, the feline is infected with FHV if antibodies that specifically recognize FHV gG are detected. In another particular aspect of the differentiation method of the invention, the genomic analysis test comprises testing whether the sample contains an FHV gG coding sequence. In another particular aspect of the differentiation method of the present invention, if an FHV gG coding sequence is detected, the feline is infected with FHV. In another particular aspect of the differentiation method of the present invention, the immunological test involves testing whether the sample contains antibodies that specifically recognize EHV gD and / or EHV gC. In another particular aspect of the differentiation method of the present invention, if antibodies that specifically recognize EHV gD and / or EHV gC are detected, the feline has been vaccinated with an immunogenic composition, vaccine or DIVA vaccine described herein. In another particular aspect of the differentiation method of the invention, the genomic analysis test comprises testing whether the sample contains EHV gD and / or EHV gC coding sequences. In another particular aspect of the differentiation method of the invention, if EHV gD and / or EHV gC coding sequences are detected, the feline has been vaccinated with an immunogenic composition, vaccine or DIVA vaccine described herein.

[0073] In another particular aspect of the differentiation method of the present invention, the immunological test is an EIA (enzyme immunoassay) or an ELISA (enzyme-linked immunosorbent assay), or the genomic analysis test is a PCR (polymerase chain reaction), an RT-PCR (reverse transcription polymerase chain reaction) or a real-time PCR (polymerase chain reaction). In another particular aspect of the differentiation method of the present invention, the feline is a cat. In another particular aspect of the differentiation method of the present invention, the sample is a serum sample. In another particular aspect of the differentiation method of the invention, the ELISA is an indirect ELISA, a sandwich ELISA, a competitive ELISA or a blocking ELISA. Thus, the test may involve, for example, cross-linking wells bearing FHV gG (or EHV gD and / or EHV gC) epitopes to a microwell assay plate. The cross-linking is preferably performed via an anchor protein (e.g., poly-L-lysine, etc.). Expression systems for obtaining FHV gG (or EHV gD and / or EHV gC) epitopes are well known to those skilled in the art. Alternatively, the FHV gG (or EHV gD and / or EHV gC) epitopes may be chemically synthesized.

[0074] Animals that were only vaccinated with the vaccine of the present invention did not produce antibodies against the FHV gG epitope. However, such animals produced antibodies against EHV gD and / or EHV gC. The result was that no antibodies bound to wells coated with the FHV gG epitope. However, such vaccinated animals produced antibodies against EHV gD and / or EHV gC. Therefore, if wells were coated with EHV gD and / or EHV gC epitopes, the antibodies would bind to such epitopes and give a positive detection signal. However, different ELISA techniques are well known to those skilled in the art. ELISA is exemplarily described by Wensvoort G. et al., 1988 (Vet. Microbiol. 17(2): 129-140), Robiolo B. et al., 2010 (J. Virol. Methods. 166(1-2): 21-27), and Colijn, EO et al., 1997 (Vet. Microbiology 59: 15-25).

[0075] Preferably, a test for distinguishing between animals infected with FHV, animals vaccinated with a modified live vaccine, or animals vaccinated with a whole virus inactivated vaccine and animals vaccinated only with the vaccine of the present invention is provided by isolating RNA from cells obtained from the sample defined above, and amplifying the cDNA using reverse transcriptase. PCR can be performed using specific primers for FHV gG. In such cases, if there is a positive PCR signal, the feline is infected with FHV. However, if the specific sequence of FHV gG cannot be amplified, the feline may not be infected with FHV but may have been vaccinated with the vaccine of the present invention. PCR can be performed using specific primers for EHV gD and / or EHV gC. In such cases, if there is a positive PCR signal, the feline has been vaccinated with the vaccine of the present invention. In another particular aspect of the differentiation method of the present invention, the genomic analysis test is PCR (polymerase chain reaction), RT-PCR (reverse transcription polymerase chain reaction) or real-time PCR (polymerase chain reaction). In another particular aspect of the differentiation method of the present invention, the cell culture-based assay is a CPE (cytopathic effect) assay. However, the technique of "cell culture based assays" or "CPE assays" is well known to those skilled in the art.

[0076] Disclosure The present disclosure further includes EHV (Equine Herpesvirus) comprising a feline herpesvirus (FHV) antigen coding sequence inserted into ORF70 (US4) and / or ORF1 / 3. The present disclosure further includes EHV (Equine Herpesvirus) comprising a single Feline Herpesvirus (FHV) antigen coding sequence inserted into ORF70 (US4) and / or a single Feline Herpesvirus (FHV) antigen coding sequence inserted into ORF1 / 3. The present disclosure further includes an Equine Herpesvirus (EHV) comprising a feline herpesvirus (FHV) antigen coding sequence encoding 1400 or fewer amino acids inserted into ORF70 (US4) and / or ORF1 / 3. In any of the above disclosures of EHV, the feline herpesvirus (FHV) antigen coding sequence is inserted into ORF70 (US4). In any of the above disclosures of EHV, the feline herpesvirus (FHV) antigen coding sequence is inserted into ORF1 / 3. In any of the above disclosures of EHV, the feline herpesvirus (FHV) antigen coding sequence is inserted into ORF70 (US4) and ORF1 / 3.

[0077] In any of the above disclosures of EHV, the FHV antigen coding sequence encodes 1400 or fewer amino acids. In any of the above disclosures of EHV, the FHV antigen coding sequence encodes 1200 or fewer amino acids. In any of the above disclosures of EHV, the FHV antigen coding sequence encodes 1000 or fewer amino acids. In any of the above disclosures of EHV, the FHV antigen coding sequence encodes between 200 and 1200 amino acids. In any of the above disclosures of EHV, the FHV antigen coding sequence encodes between 200 and 1000 amino acids. In any of the above disclosures of EHV, the FHV antigen coding sequence comprises no more than 4200 nucleic acids. In any of the above disclosures of EHV, the FHV antigen coding sequence comprises no more than 3600 nucleic acids. In any of the above disclosures of EHV, the FHV antigen coding sequence comprises no more than 3000 nucleic acids. In any of the above disclosures of EHV, the FHV antigen coding sequence encodes between 600 and 3600 nucleic acids. In any of the above disclosures of EHV, the FHV antigen coding sequence encodes between 600 and 3000 nucleic acids.

[0078] In any of the above disclosures of EHV, the FHV antigen coding sequence is one FHV antigen coding sequence. In any of the above disclosures of EHV, the FHV antigen coding sequence is a single FHV antigen coding sequence. In any of the above disclosures of EHV, the FHV antigen coding sequence is not two or more FHV antigen coding sequences. In any of the above disclosures of EHV, the FHV antigen coding sequence is a glycoprotein coding sequence or a fragment thereof. In any of the above disclosures of EHV, the FHV antigen coding sequence is an FHV gD (glycoprotein D) coding sequence or a fragment thereof, and / or an FHV gB (glycoprotein B) coding sequence or a fragment thereof, and / or an FHV gC (glycoprotein C) coding sequence or a fragment thereof. In any of the above disclosures of EHV, the FHV antigen coding sequence is an FHV gD coding sequence or a fragment thereof. In any of the above disclosures of EHV, the FHV antigen coding sequence is an FHV gB coding sequence or a fragment thereof. In any of the above disclosures of EHV, the FHV antigen coding sequence is an FHV gC coding sequence or a fragment thereof.

[0079] In any of the above disclosures of EHV, the fragment of the glycoprotein coding sequence encodes at least 100, at least 150, at least 200, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids. In any of the above disclosures of EHV, the fragment of the glycoprotein coding sequence comprises at least 300, at least 450, at least 600, at least 900, at least 1050, at least 1200, at least 1500, at least 1800, at least 2100, or at least 2400 nucleic acids. In any of the above disclosures of EHV, the fragment of the glycoprotein coding sequence encodes at least 100, at least 200, at least 300, or at least 350 amino acids. In any of the above disclosures of EHV, the fragment of the glycoprotein coding sequence comprises at least 300, at least 600, at least 900, or at least 1050 nucleic acids. In any of the above disclosures of EHV, the fragment of the FHV gD coding sequence encodes at least 100, at least 200, at least 250, at least 300, or at least 350 amino acids. In any of the above disclosures of EHV, the fragment of the FHV gD coding sequence comprises at least 300, 600, 750, 900, or 1050 nucleic acids.

[0080] In any of the above disclosures of EHV, the fragment of the FHV gB coding sequence encodes at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids. In any of the above disclosures of EHV, the fragment of the FHV gB coding sequence comprises at least 300, at least 450, at least 600, at least 900, at least 1200, at least 1500, at least 1800, at least 2100, or at least 2400 nucleic acids. In any of the above disclosures of EHV, the fragment of the FHV gC coding sequence encodes at least 100, at least 150, at least 200, at least 300, at least 400, or at least 500 amino acids. In any of the above disclosures of EHV, the fragment of the FHV gC coding sequence comprises at least 300, at least 450, at least 600, at least 900, at least 1200, or at least 1500 nucleic acids. In any of the above disclosures of EHV, the FHV antigen coding sequence is an FHV gD coding sequence, and / or an FHV gB coding sequence, and / or an FHV gC coding sequence based on the wild-type feline herpesvirus strain C-27 (ACCN: FJ478159).

[0081] In any of the above disclosures of EHV, the FHV antigen coding sequence is an FHV gD coding sequence, and the FHV gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in AAB30980.1, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids. In any of the above disclosures of EHV, the FHV antigen coding sequence is an FHV gB coding sequence, The gB coding sequence consists of, or comprises, a nucleic acid sequence encoding the amino acid sequence set forth in AAB28559.3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids.

[0082] In any of the above disclosures of EHV, the FHV antigen coding sequence is a glycoprotein coding sequence, and the glycoprotein coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids. In any of the above disclosures of EHV, the FHV antigen coding sequence is a gD, gB or gC coding sequence, and the gD, gB or gC coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300 or at least 350 consecutive amino acids.

[0083] In any of the above disclosures of EHV, the FHV antigen-encoding sequence is a glycoprotein consisting of or comprising the nucleic acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 600, at least 900, or at least 1050 consecutive nucleic acids. In any of the above disclosures of EHV, the FHV antigen-encoding sequence is gD, gB or gC consisting of or comprising the nucleic acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 600, at least 900 or at least 1050 consecutive nucleic acids.

[0084] In any of the above disclosures of EHV, the gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids. In any of the above disclosures of EHV, the gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said amino acid sequence.

[0085] In any of the above disclosures of EHV, the gB coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids. In any of the above disclosures of EHV, the gB coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence.

[0086] In any of the above disclosures of EHV, the gC coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, or at least 500 consecutive amino acids. In any of the above disclosures of EHV, the gC coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:3, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence.

[0087] In any of the above disclosures of EHV, the gD coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 600, at least 900 or at least 1050 consecutive nucleic acids. In any of the above disclosures of EHV, the gD coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence.

[0088] In any of the above disclosures of EHV, the gB coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 450, at least 600, at least 900, at least 1200, at least 1500, at least 1800, at least 2100, or at least 2400 consecutive nucleic acids. In any of the above disclosures of EHV, the gB coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence.

[0089] In any of the above disclosures of EHV, the gC coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence, or a fragment of said nucleic acid sequence having at least 300, at least 450, at least 600, at least 900, at least 1200 or at least 1500 consecutive nucleic acids. In any of the above disclosures of EHV, the gC coding sequence consists of or comprises the nucleic acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9, or a sequence having at least at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to said nucleic acid sequence.

[0090] In any of the above disclosures of EHV, said EHV is a vector. In any of the above disclosures of EHV, said EHV is an EHV vector. In any of the above disclosures of EHV, said EHV is attenuated. In any of the above disclosures of EHV, the EHV is genetically engineered. In any of the above disclosures of EHV, the EHV is recombinant. In any of the above disclosures of EHV, said EHV is selected from the group consisting of EHV-1, EHV-3, EHV-4, EHV-8 and EHV-9. In any of the above disclosures of EHV, the EHV is EHV-1. In any of the above disclosures of EHV, said EHV is RacH or RacH SE. In any of the above disclosures of EHV, the insertion into ORF70 (US4) is characterized by a partial deletion, truncation, substitution or modification in ORF70 (US4), while US5 (ORF71) remains functional.

[0091] In any of the above disclosures of EHV, the insertion into ORF70 is characterized by a deletion of approximately 801 bp in ORF70 of wild-type EHV-1 strain ab4 (Genbank Accession No. AY665713.1), whereby the deleted portion of the wild-type ab4 genomic sequence is located between nucleotides 127681 and 128482 (SEQ ID NO: 10) or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In any of the above disclosures of EHV, the insertion into ORF70(US4) is characterized by a deletion of approximately 801 bp in ORF70(US4) of RacH (SEQ ID NO:11), or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:11.

[0092] In any of the above disclosures of EHVs, the EHV comprises at least one flanking region selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to said flanking region. In any of the above disclosures of EHVs, the EHV comprises (i) at least one US4 left flanking region selected from the group consisting of SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, and (ii) at least one US4 right flanking region selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17. In any of the above disclosures of EHV, the feline herpesvirus (FHV) antigen coding sequence is operably linked to a promoter sequence. In any of the above disclosures of EHV, the gD coding sequence and / or the gB coding sequence is operably linked to a promoter sequence.

[0093] In any of the above disclosures of EHV, the promoter sequence is selected from the group consisting of SV40 large T, HCMV and MCMV immediate early gene 1, human elongation factor alpha promoter, baculovirus polyhedrin promoter, p430 (SEQ ID NO: 18), or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to SEQ ID NO: 18. or a fragment of at least 350 contiguous nucleotides of SEQ ID NO: 18; p455 (SEQ ID NO: 19) or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, 99.95%, at least 99.98% or at least 99.99% sequence identity to SEQ ID NO: 19; or a promoter consisting of or comprising a fragment of at least 350 contiguous nucleotides of SEQ ID NO: 19; and The gB promoter (SEQ ID NO:24) or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity to SEQ ID NO:24, or a promoter consisting of or comprising at least 300 contiguous nucleotides of SEQ ID NO:24. In any of the above disclosures of EHV, the promoter sequence is p430 (SEQ ID NO: 18), or a sequence having at least 95% sequence identity to SEQ ID NO: 18, or a promoter consisting of or comprising at least 400 contiguous nucleotides of SEQ ID NO: 18.

[0094] In any of the above disclosures of EHV, the promoter sequence is p455 (SEQ ID NO:19), or a sequence having at least 95% sequence identity to SEQ ID NO:19, or a promoter consisting of or comprising at least 400 contiguous nucleotides of SEQ ID NO:19. In any of the above disclosures of EHV, the EHV is a promoter consisting of or comprising a truncated FHV-1 gB promoter (SEQ ID NO:24), or a sequence having at least 95% sequence identity to SEQ ID NO:24, or at least 350 contiguous nucleotides of SEQ ID NO:24. In any of the above disclosures of EHV, the EHV comprises one or more additional regulatory sequences, for example, a termination signal, a polyadenylation signal, or a regulatory element such as an IRES. In any of the above disclosures of EHV, said EHV comprises a polyadenylation signal. In any of the above disclosures of EHV, said EHV comprises the polyadenylation signal BGHpA. In any of the above disclosures of EHV, said EHV comprises an IRES element. In any of the above disclosures of EHV, two FHV coding sequences flanked by an IRES element are inserted into a single insertion site. In any of the above disclosures of EHV, FHV gD with an IRES and gB is inserted into the ORF1 / 3 site. In any of the above disclosures of EHV, said EHV does not comprise a 2A peptide, such as an F2A peptide. In any of the above disclosures of EHV, the FHV gD coding sequence or a fragment thereof is inserted into ORF1 / 3 and the FHV gB coding sequence or a fragment thereof is inserted into ORF70.

[0095] In any of the above disclosures of EHV: i) the FHV gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids, and is inserted into ORF1 / 3; and ii) the FHV gB coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity thereto, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids, and is inserted into ORF70.

[0096] The present disclosure further includes immunogenic compositions comprising the EHV of the present disclosure. The present disclosure further includes vaccines comprising the EHV of the present disclosure. The present disclosure further includes a DIVA vaccine comprising an EHV of the present disclosure. In any of the above disclosures of immunogenic compositions, vaccines or DIVA vaccines, said immunogenic compositions, vaccines or DIVA vaccines are bivalent. In any of the above disclosures of immunogenic compositions, vaccines or DIVA vaccines, said immunogenic compositions, vaccines or DIVA vaccines are multivalent. In any of the above disclosures of immunogenic compositions, vaccines or DIVA vaccines, said immunogenic compositions, vaccines or DIVA vaccines further comprise a pharmaceutically acceptable carrier. In any of the above disclosures of immunogenic compositions, vaccines or DIVA vaccines, the pharmaceutically acceptable carrier is water for injection, cell culture medium or resuspension buffer. In any of the above disclosures of immunogenic compositions, vaccines or DIVA vaccines, the resuspension buffer is physiological solution or phosphate buffered saline. In any of the above disclosures of immunogenic compositions, vaccines or DIVA vaccines, the immunogenic compositions, vaccines or DIVA vaccines contain 1x10 of EHV. 4 ~1x10 9 TCID 50 , preferably 1x10 4 ~1x10 8 TCID 50 , and even more preferably 1x10 4 ~1x10 7 TCID 50 Includes.

[0097] The present disclosure further includes kits comprising the immunogenic compositions, vaccines, or DIVA vaccines of the present disclosure. In any of the above disclosures of kits, the kit further comprises instructions for treating and / or preventing a disease in a feline animal. In any of the above disclosures of kits, the kit further comprises instructions for the treatment and / or prevention of FHV. In any of the above disclosures of kits, the kit further comprises a dispenser capable of administering the vaccine to an animal or feline. The present disclosure further includes methods of immunization comprising administering to a feline an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure. The present disclosure further includes a method of immunizing a feline, the method comprising administering to said feline an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure.

[0098] The present disclosure further includes a method for treating or preventing clinical signs of FHV in a feline, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure. The present disclosure further includes a method for treating or preventing clinical signs of FHV in a feline relative to an unimmunized control feline, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure. The present disclosure further includes a method for treating or preventing FHV respiratory disease in a feline, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure. The present disclosure further includes a method for treating or preventing FHV respiratory disease in a feline, compared to an unimmunized control feline, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure. The present disclosure further includes a method for reducing FHV shedding in a feline, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure. The present disclosure further includes a method for reducing FHV shedding in a feline relative to an unimmunized control feline, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine, or DIVA vaccine described herein.

[0099] The present disclosure further includes an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure for use in a method for immunizing a feline, the method comprising administering a therapeutically effective amount of the immunogenic composition, vaccine, or DIVA vaccine to the feline. The present disclosure further includes an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure for use in a method for treating or preventing clinical signs of FHV in a feline, the method comprising administering a therapeutically effective amount of the immunogenic composition, vaccine, or DIVA vaccine to a feline. The present disclosure further includes an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure for use in a method for treating or preventing respiratory disease in a feline caused by FHV, the method comprising administering a therapeutically effective amount of the immunogenic composition, vaccine, or DIVA vaccine to a feline. The present disclosure further includes an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure for use in a method for reducing FHV shedding in a feline, the method comprising administering a therapeutically effective amount of the immunogenic composition, vaccine, or DIVA vaccine to the feline.

[0100] In any of the above disclosures of methods or uses, said feline is selected from the group consisting of cheetah, puma, jaguar, leopard, lion, lynx, tiger, cat and domestic cat. In any of the above disclosures of methods or uses, said feline is a cat or a domestic cat. In any of the above disclosed methods or uses, the immunogenic composition, vaccine or DIVA vaccine is administered more than once. In any of the above disclosed methods or uses, the immunogenic composition, vaccine or DIVA vaccine is administered twice. In any of the above disclosed methods or uses, the immunogenic composition, vaccine or DIVA vaccine is administered to a feline that is 4, 5 or 6 weeks of age or older. In any of the above disclosed methods or uses, the immunogenic composition, vaccine or DIVA vaccine is administered to a feline 6 weeks of age or older. In any of the above disclosed methods or uses, the immunogenic composition, vaccine or DIVA vaccine is first administered to a feline between 4 and 25 weeks of age. In any of the above disclosed methods or uses, the immunogenic composition, vaccine or DIVA vaccine is first administered to a feline between 6 and 20 weeks of age.

[0101] In any of the above disclosed methods or uses, a first dose of the immunogenic composition, vaccine or DIVA vaccine is administered to a feline at least 4, 5 or 6 weeks of age, and a second dose is administered 2 to 8 weeks later. In any of the above disclosed methods or uses, a first dose of the immunogenic composition, vaccine or DIVA vaccine is administered to a feline 6 weeks of age or older, and a second dose is administered 2 to 8 weeks later. In any of the above disclosed methods or uses, after the second (booster) vaccination, further vaccinations are given annually (annual booster vaccinations). In any of the above disclosed methods or uses, said immunogenic composition, vaccine or DIVA vaccine is administered intramuscularly, intradermally, subcutaneously, orally or intranasally. In any of the above disclosures of methods or uses, the immunogenic composition, vaccine or DIVA vaccine is administered at a concentration of 1 x 10 per mL. 4 ~1x10 10 TCID 50 Including EHVs In any of the above disclosures of methods or uses, the immunogenic composition, vaccine or DIVA vaccine is administered at a concentration of 1 x 10 per mL. 6 ~1x10 9 TCID 50 Includes EHVs. In any of the above disclosures of methods or uses, a dose of the immunogenic composition, vaccine or DIVA vaccine is 1 x 10 per mL. 4 ~1x10 10 TCID 50 Includes EHVs. In any of the above disclosures of methods or uses, a dose of the immunogenic composition, vaccine or DIVA vaccine is 1 x 10 per mL. 6 ~1x10 9 TCID 50 Includes EHVs.

[0102] In any of the above disclosed methods or uses, the method results in an improvement in an efficacy metric selected from the group consisting of reduced weight loss, reduced viral load in the lungs, reduced viremia, reduced lung, respiratory and alveolar epithelial lesions, reduced eye discharge, reduced conjunctivitis, reduced rhinitis, reduced and / or shortened viral shedding, reduced rectal temperature, reduced clinical symptoms (particularly respiratory symptoms), increased (neutralizing) induction of anti-FHV virus antibodies, increased stimulation of T cells against FHV, increased stimulation of B cells against FHV virus, and reduced inflammatory cytokines in the lungs (e.g., IL1β), or a combination thereof, compared to an unimmunized control group of the same species as the feline. In any of the above disclosed methods or uses, said immunogenic composition, vaccine or DIVA vaccine protects against homologous and / or heterologous challenge with FHV. The present disclosure further includes an EHV (Equine Herpesvirus) comprising an FHV gD coding sequence or a fragment thereof inserted into ORF1 / 3 and an FHV gB coding sequence or a fragment thereof inserted into ORF70. The present disclosure further includes an EHV (Equine Herpesvirus) comprising an FHV gD coding sequence or a fragment thereof inserted into ORF1 / 3 and an FHV gB coding sequence or a fragment thereof inserted into ORF70.

[0103] The present disclosure further includes EHV (Equine Herpesvirus), wherein said EHV is i) an FHV gD coding sequence inserted into ORF1 / 3, the FHV gD coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity thereto, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids; ii) an FHV gB coding sequence inserted into ORF70, the FHV gB coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids.

[0104] The present disclosure further includes an EHV (Equine Herpesvirus), the EHV comprising an FHV gD coding sequence inserted into ORF1 / 3, the FHV gD coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 95% sequence identity to said amino acid sequence, and an FHV gB coding sequence inserted into ORF70, the FHV gB coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 95% sequence identity to said amino acid sequence. The present disclosure further includes an immunogenic composition, vaccine or DIVA vaccine comprising EHV (Equine Herpesvirus), wherein said EHV is i) an FHV gD coding sequence inserted into ORF1 / 3, the FHV gD coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity thereto, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300, or at least 350 consecutive amino acids; ii) an FHV gB coding sequence inserted into ORF70, the FHV gB coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:2, or a sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98%, or at least 99.99% sequence identity to said amino acid sequence, or a fragment of said amino acid sequence having at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 consecutive amino acids.

[0105] The present disclosure further includes an EHV, immunogenic composition, vaccine, or DIVA vaccine of the present disclosure for use in therapy. The present disclosure further includes an EHV, immunogenic composition, vaccine, or DIVA vaccine of the present disclosure for use as an immunogen or vaccine. The present disclosure further includes an EHV, immunogenic composition, vaccine, or DIVA vaccine of the present disclosure for use as a medicament. The present disclosure further includes an EHV, immunogenic composition, vaccine, or DIVA vaccine of the present disclosure for the manufacture of a medicament. The present disclosure further includes the use of an EHV, immunogenic composition, vaccine or DIVA vaccine of the present disclosure for the treatment and / or prevention of FHV infection in a feline. The present disclosure further includes a method for preparing a feline herpesvirus (EHV) (Equine Herpesvirus) comprising an FHV antigen coding sequence inserted into ORF70 (US4) and / or ORF1 / 3, said method comprising: i) providing EHVs; ii) providing an FHV antigen coding sequence; iii) inserting the FHV antigen coding sequence obtained from step ii) into the EHV ORF70 (US4) and / or ORF1 / 3 insertion site of step i); iv) obtaining said EHV comprising an FHV antigen coding sequence inserted into ORF70(US4) and / or ORF1 / 3.

[0106] The present disclosure further includes a method for preparing a feline herpesvirus (EHV) (Equine Herpesvirus) comprising an FHV antigen coding sequence inserted into ORF70 (US4) and / or ORF1 / 3, said method comprising: i) providing EHVs; ii) providing an FHV gD, gB or gC coding sequence, the FHV gD, gB or gC coding sequence consisting of or comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.98% or at least 99.99% sequence identity thereto, or a fragment of said amino acid sequence having at least 100, at least 200, at least 300 or at least 350 consecutive amino acids; iii) inserting the FHV gD, gB or gC coding sequence obtained from step ii) into the EHV ORF70 (US4) and / or ORF1 / 3 insertion site of step i); iv) obtaining said EHV comprising an FHV gD, gB or gC coding sequence inserted into ORF70 (US4) and / or ORF1 / 3.

[0107] In any of the above disclosures of methods for preparing an EHV, the EHV is attenuated. In any of the above disclosures of methods for preparing an EHV, the EHV is RacH or RacH SE. The present disclosure further includes a method of distinguishing between a feline vaccinated with an immunogenic composition, vaccine, or DIVA vaccine of the present disclosure and a feline infected with FHV, the method comprising: a) obtaining a sample from a feline; b) analyzing the sample with an immunological test, a cell culture-based assay, and / or a genomic analysis test. In any of the above disclosed differentiation methods, the immunological test comprises testing whether the sample contains antibodies that specifically recognize FHV gG. In any of the above disclosed differentiation methods, if antibodies that specifically recognize FHV gG are detected, the feline is infected with FHV. In any of the above disclosed differentiation methods, the genomic analysis test comprises testing whether the sample contains an FHV gG coding sequence. In any of the above disclosed differentiation methods, if an FHV gG coding sequence is detected, the feline is infected with FHV.

[0108] In any of the above disclosed differentiation methods, the immunological test comprises testing whether the sample contains antibodies that specifically recognize EHV gD and / or EHV gC. If, in any of the above disclosed differentiation methods, antibodies that specifically recognize EHV gD and / or EHV gC are detected, the feline has been vaccinated with the immunogenic composition, vaccine or DIVA vaccine of the present disclosure. In any of the above disclosures of differentiation methods, the genomic analysis test comprises testing whether the sample contains EHV gD and / or EHV gC coding sequences. If EHV gD and / or EHV gC coding sequences are detected in any of the above disclosed differentiation methods, the feline has been vaccinated with an immunogenic composition, vaccine or DIVA vaccine of the present disclosure. In any of the above disclosures of the differentiation method, the immunological test is an EIA (enzyme immunoassay) or an ELISA (enzyme-linked immunosorbent assay), or the genomic analysis test is a PCR (polymerase chain reaction), an RT-PCR (reverse transcription polymerase chain reaction) or a real-time PCR (polymerase chain reaction). In any of the above disclosures of the differentiation method, said feline is a cat. In any of the above disclosed methods of differentiation, the sample is a serum sample. In any of the above disclosures of differentiation methods, the ELISA is an indirect ELISA, a sandwich ELISA, a competitive ELISA, or a blocking ELISA. In any of the above disclosures of the differentiation method, the genomic analysis test is PCR (polymerase chain reaction), RT-PCR (reverse transcription polymerase chain reaction) or real-time PCR (polymerase chain reaction). In any of the above disclosures of differentiation methods, said cell culture based assay is a CPE (cytopathic effect) assay. The following drawings form part of the present specification and are included to further support certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0109] [Figure 1] Schematic diagram of p1_3-p430-FHVgD(co) [Figure 2] Schematic diagram of p455-FHVgB(co) [Figure 3] Schematic diagram of rEHV-1-p430-FHVgD(co)-p455-FHVgB(co). [Figure 4] Schematic diagram of p1_3-p430-FHVgD(co)F2AgB(co) [Figure 5] Schematic diagram of rEHV-1-p430-FHVgD(co)F2AgB(co). [Figure 6] Schematic diagram of p455-FHVgB(n) [Figure 7] Schematic diagram of rEHV-1-p430-FHVgD [Figure 8] Schematic diagram of rEHV-1-p455-FHVgB(n) [Figure 9] Schematic diagram of pFHgB-FHVgB(n) [Figure 10] Schematic diagram of rEHV-1-p430-FHVgD(co)-pFHgB-FHVgB(n). [Figure 11] Schematic diagram of p1_3-p430-FHVgD(co)IRESgB(n) [Figure 12] Schematic diagram of rEHV-1-p430-FHVgD(co)IRESgB(n). [Figure 13] FHV-1 SN results from swine serology testing using treatment group geometric mean antibody titers [Figure 14] EHV-1 SN results from swine serology testing using treatment group geometric mean antibody titers [Figure 15] Clinical sign results in feline challenge tests for cats with moderate to severe signs lasting for 2 days or more (score > 1) [Figure 16] Average weight (kg) by group in feline challenge study [Figure 17] Geometric mean EHV-1 SN titers and individual animal EHV-1 SN titers for vaccinated groups only in the feline challenge study (× = 50% endpoint titers for individual animals within a group). [Figure 18] Geometric group means of FVR SN antibody titers and individual animal FVR SN antibody titers for groups 1 and 2 in the feline challenge study (× = individual values ​​for group 1 animals; filled circles = individual values ​​for group 2 animals).

[0110] Array Overview The following sequences of the present invention are now described and disclosed in detail: Glycoproteins: SEQ ID NO: 1 FHVgD amino acid sequence obtained from strain C-27 SEQ ID NO: 2 FHVgB amino acid sequence obtained from strain C-27 SEQ ID NO: 3 FHVgC amino acid sequence obtained from strain C-27 SEQ ID NO: 4 Codon-optimized FHVgD nucleotide sequence obtained from FHV-1 strain C-27 SEQ ID NO:5 Native FHVgD nucleotide sequence obtained from FHV-1 strain C-27 SEQ ID NO:6 Codon-optimized FHVgB nucleotide sequence obtained from FHV-1 strain C-27 SEQ ID NO:7 Native FHVgB nucleotide sequence obtained from strain C-27 SEQ ID NO:8 Codon-optimized FHVgC nucleotide sequence obtained from FHV-1 strain C-27 SEQ ID NO:9 Native FHVgC nucleotide sequence obtained from strain C-27

[0111] ORF70 insertion site: SEQ ID NO: 10 801 bp deletion within ORF70 of wild-type EHV-1 strain ab4 (Genbank accession number AY665713.1) SEQ ID NO: 11 801 bp portion within ORF70 (US4) of RacH SEQ ID NO: 12 US4 left flanking region (417 bp) SEQ ID NO: 13 US4 right flanking region (431 bp) SEQ ID NO: 14 US4 left flanking region (417 bp) SEQ ID NO: 15 US4 right flanking region (431 bp) SEQ ID NO: 16 US4 left flanking region (283 bp) SEQ ID NO: 17 US4 right flanking region (144 bp) promoter: SEQ ID NO: 18 p430 promoter SEQ ID NO: 19 p455 promoter SEQ ID NO: 24 Truncated FHV-1 gB promoter Primer SEQ ID NO: 20 to SEQ ID NO: 23 Primers [Example]

[0112] The following examples are included to support preferred embodiments of the invention. It will be understood by those of skill in the art that the techniques disclosed in the examples which follow are representative of techniques discovered by the inventors to function well in the practice of the invention, and as such can be considered to constitute preferred modes for the practice of the invention. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0113] Example 1: Generation of recombinant EHV-1 (rEHV-1-p430-FHVgD(co)-p455-FHVgB(co)) carrying FHV-1 glycoprotein D at the ORF1 / 3 insertion site and FHV-1 glycoprotein B at the ORF70 insertion site The synthetic, codon-optimized FHVgD coding sequence (SEQ ID NO: 4) was digested with restriction endonucleases and ligated into the p1_3-mCMV (mouse CMV promoter described in Dorsch-Hasler et al. 1985) vector, which contains the mCMV promoter and flanking regions for EHV-1 ORF1 / 3 homologous recombination, digested with the same restriction endonucleases, to yield p1_3-mCMV-FHVgD(co). The EHV-4 gG430 promoter p430 (SEQ ID NO: 18) was excised from the pgG430-MCS1 vector using MluI and EcoRI restriction endonucleases and ligated into p1_3-mCMV-FHVgD(co) to yield p1_3-p430-FHVgD(co) (Figure 1). The synthetic codon-optimized FHVgB coding sequence (SEQ ID NO: 6) was digested with FseI and KpnI restriction endonucleases and cloned into the p455 shuttle vector containing the EHV-4 major capsid protein (MCP) promoter p455 (SEQ ID NO: 19) and flanking regions for EHV-1 ORF70 homologous recombination digested with the same restriction endonucleases, resulting in p455-FHVgB(co) (Figure 2). To generate recombinant EHV-1 viruses carrying two FHV-1 antigens at two different insertion sites, we used the RED recombination system to insert expression cassettes for codon-optimized FHV-1 gD and codon-optimized FHV-1 gB into the ORF1 / 3 and ORF70 insertion sites, respectively, to generate rEHV-1-p430-FHVgD(co)-p455-FHVgB(co) (Figure 3). After kanamycin selection, the rEHV-1-p430-FHVgD(co)-p455-FHVgB(co) BAC DNA was transfected into A1-ST cells to rescue the recombinant virus, rEHV-1-p430-FHVgD(co)-p455-FHVgB(co).

[0114] Example 2: Generation of recombinant EHV-1 carrying FHV-1 glycoproteins D and B at the ORF1 / 3 insertion site (rEHV-1-p430-FHVgD(co)F2AgB(co)) Polymerase chain reaction (PCR) was performed on p1_3-mCMV-FHVgD(co) using primers F2A oligo (Table 1; SEQ ID NO: 20) and FHVgD-F (Table 1; SEQ ID NO: 21) to amplify a 347-bp fragment containing 270 bp of the 3' end of the FHV gD(co) coding sequence, a void for the stop codon, the codon-optimized foot-and-mouth disease virus (FMDV) 2A peptide sequence (F2A), and 23 bp of the 5' end of the FHV gB(co) coding sequence. Table 1: Primers for construction of p1_3-mCMV-FHVgD(co)F2AgB(co) TIFF0007756860000001.tif43170

[0115] A second PCR was performed with primers FHVgB-F (Table 1; SEQ ID NO: 22) and 3'-FHVgB-R (Table 1; SEQ ID NO: 23) to amplify the complete FHV gB(co) coding sequence (2,487 bp in length) from p455-FHVgB(co). Both PCR fragments were then ligated by overlap extension PCR (OE-PCR) to generate a fragment containing an XbaI site at the 5' end and a SalI site at the 3' end. The fused coding sequence was digested with XbaI and SalI restriction endonucleases and cloned into the multiple cloning site 1 (MCS1) of p1_3-mCMV-FHVgD(co) via the XbaI and SalI restriction sites, yielding p1_3-mCMV-FHVgD(co)F2AgB(co). The gG430 promoter of EHV-4 was excised from the pgG430-MCS1 vector using MluI and EcoRI restriction endonucleases and ligated into p1_3-mCMV-FHVgD(co)F2AgB(co) digested with the same restriction endonucleases, resulting in p1_3-p430-FHVgD(co)F2AgB(co) (Figure 4). By en passant mutagenesis using the RED recombination system (Tischer et al. 2006. Biotechnol. Tech. 40, 191-197), expression cassettes for codon-optimized FHV-1 gD and codon-optimized FHV-1 gB were inserted into the ORF1 / 3 insertion site and ligated via the F2A cleavage site to generate rEHV-1-p430-FHVgD(co)F2AgB(co) (Figure 5). After kanamycin selection, the rEHV-1-p430-FHVgD(co)F2AgB(co) BAC DNA was transfected into A1-ST cells to rescue the recombinant virus, rEHV-1-p430-FHVgD(co)F2AgB(co).

[0116] Example 3: Generation of recombinant EHV-1 with codon-optimized FHV-1 glycoprotein D at the ORF1 / 3 insertion site (rEHV-1-p430-FHVgD(co)) or native FHV-1 glycoprotein B at the ORF70 insertion site (rEHV-1-p455-FHVgB(n)) The synthetic native FHVgB coding sequence (SEQ ID NO: 7) was digested with NcoI and KpnI restriction endonucleases and cloned into the p455 plasmid digested with the same restriction endonucleases, resulting in p455-FHVgB(n) (Figure 6). The codon-optimized FHV-1 gD obtained from p1_3-FHVgD(co) was inserted into the ORF1 / 3 insertion site by en passant mutagenesis using the RED recombination system (Tischer et al. 2006. Biotechnol. Tech. 40, 191-197) (exemplary as described in WO 2018 / 054837) to generate the BAC DNA of rEHV-1-p430-FHVgD(co) (Figure 7), which was subsequently transfected into A1-ST cells to rescue the rEHV-1-p430-FHVgD(co) virus. Similarly, en passant mutagenesis was used to insert native FHV-1 gB obtained from p455-FHVgB(n) into the ORF70 insertion site (as exemplarily described in WO 2018 / 054837) to generate the BAC DNA of rEHV-1-p455-FHVgB(n) (Figure 8), which was subsequently transfected into A1-ST cells to rescue the rEHV-1-p455-FHVgB(n) virus.

[0117] Example 4: Generation of recombinant EHV-1 with codon-optimized FHV-1 glycoprotein D in the ORF1 / 3 insertion site with the p430 promoter and native FHV-1 glycoprotein B in the ORF70 insertion site with the native FHV-1 gB promoter (rEHV-1-p430-FHVgD(co)-pFHgB-FHVgB(n)), and generation of recombinant EHV-1 with FHV-1 glycoprotein D and glycoprotein B in the ORF1 / 3 insertion site (rEHV-1-p430-FHVgD(co)IRESgB(n)). A synthetic 671 bp fragment containing the native promoter for FHV gB (SEQ ID NO: 24), generated by digestion with Bsu36I and SalI restriction endonucleases, was ligated to p455-FHVgB(n) digested with the same restriction endonucleases, yielding pFHgB-FHVgB(n) (Figure 9). The codon-optimized FHV-1 gD obtained from p1_3-p430-FHVgD(co) was inserted into the ORF1 / 3 insertion site by en passant mutagenesis using the RED recombination system (Tischer et al. 2006. Biotechnol. Tech. 40, 191-197) (exemplary description is given in WO 2018 / 054837). Subsequently, RED recombination was performed to insert native FHV-1 gB driven by the native FHV-1 gB promoter into the ORF70 insertion site (exemplary description is given in WO 2018 / 054837), generating the BAC DNA rEHV-1-p430-FHVgD(co)-pFHgB-FHVgB(n) (Figure 10). This BAC DNA was transfected into A1-ST cells to rescue the rEHV-1-p430-FHVgD(co)-pFHgB-FHVgB(n) virus. A synthetic 3,687-bp fragment containing the 3' end of the codon-optimized FHV-1 gD, the EMCV IRES, and the native FHV-1 gB coding sequence was digested with PacI and XbaI restriction endonucleases and ligated to p1_3-p430-FHVgD(co) digested with the same restriction endonucleases, yielding p1_3-p430-FHVgD(co)IRESgB(n) (Figure 11). By en passant mutagenesis using the RED recombination system (Tischer et al. 2006. Biotechnol. Tech. 40, 191-197), a codon-optimized FHV-1 gD expression cassette and a native FHV-1 gB expression cassette were inserted into the ORF1 / 3 insertion site and linked via the EMCV IRES to generate the BAC DNA of rEHV-1-p430-FHVgD(co)IRESgB(n) (Figure 12). This BAC DNA was transfected into A1-ST cells, and the rEHV-1-p430-FHVgD(co)IRESgB(n) virus was rescued.

[0118] Example 5: Recombinant EHV-1 viruses expressing FHV-1 glycoprotein D and / or glycoprotein B Limiting dilutions were performed on A1-ST cells containing rEHV-1-p430-FHVgD(co)-p455-FHVgB(co) and rEHV-1-p430-FHVgD(co)F2AgB(co) vectors, and clonal populations of viruses were obtained by removing the viral GFP marker. Next-generation (NextGen) sequencing verified correct insertion of the expression cassette into the EHV-1 RacH bacmid backbone. Transgene expression in infected cells was analyzed by immunofluorescence assay (IFA). TCID in A1-ST cells was 1.25 mg / mL. 50 Peak titers, determined as / mL, were within the same range as those of the parental virus rEHV-1 RacH, indicating that transgene expression did not adversely affect virus replication (not shown).

[0119] In vitro characterization of rEHV-1 FHVgD / FHVgB viruses - IFA The recombinant viruses showed no significant differences in plaque size, growth kinetics, or titer compared with the parental EHV-1 strain RacH. Expression of FHVgD and FHVgB by rEHV-p430-FHVgD(co)-p455-FHVgB(co) and rEHV-p430-FHVgD(co)F2AgB(co) viruses was assessed by IFA in A1-ST cells infected with EHV-1 RacH, FHV-1 strain F2, rEHV-p430-FHVgD(co)-p455-FHVgB(co), and rEHV-p430-FHVgD(co)F2AgB(co). Cells were stained with mouse anti-FHVgD monoclonal antibody clone 215C1M, mouse anti-FHVgB monoclonal antibody clone 218E4S, feline anti-FVR serum, and mouse anti-EHV-1 monoclonal antibody clone 16H9 (all owned by Boehringer Ingelheim). Alexa Fluor 594 goat anti-mouse IgG (Life Technologies, Carlsbad, CA) was used as the secondary antibody for the primary monoclonal antibodies, and FITC-conjugated goat anti-cat IgG (Jackson ImmunoResearch, West Grove, PA) was used as the secondary antibody for the feline anti-FVR serum. As expected, expression of FHVgD and FHVgB was only detected in A1-ST cells infected with the recombinant viruses (Table 2). The staining for FHV gD expression in cells infected with rEHV-p430-FHVgD(co)F2AgB(co) was weaker than that in cells infected with rEHV-p430-FHVgD(co)-p455-FHVgB(co). Expression of FHVgD and FHVgB by rEHV-p430-FHVgD(co) and rEHV-p455-FHVgB(n) viruses was assessed by IFA in A1-ST cells infected with rEHV-p430-FHVgD(co) and rEHV-p455-FHVgB(n).Cells were stained with mouse anti-FHVgD monoclonal antibody clone 215C1M (proprietary of Boehringer Ingelheim), mouse anti-FHVgB monoclonal antibody clone 218E4S (proprietary of Boehringer Ingelheim), and FITC-conjugated anti-EHV-1 ready-to-use caprine polyclonal antiserum (VMRD, Pullman, WA). Alexa Fluor 594 goat anti-mouse IgG (Life Technologies, Carlsbad, CA) was used as a secondary antibody for the primary monoclonal antibodies. As expected, expression of FHV gD was detected only in A1-ST cells infected with rEHV-1-p430-FHVgD(co), and expression of FHV gB was detected only in A1-ST cells infected with rEHV-1-p455-FHVgB(n) (Table 2 ).

[0120] Table 2: Expression of FHV gD and FHV gB in A1-ST cells - IFA results TIFF0007756860000002.tif62170N=negative; W+=weak staining; +=average staining Conclusions of Example 4: Constructs containing FHV gD and FHV gB antigens showed expression of both proteins. The FHV gD monovalent construct showed expression of FHV gD, and the FHV gB monovalent construct showed expression of FHV gB.

[0121] Example 6: Testing of a recombinant EHV-1 vector vaccine expressing FHV-1 gD and gB in vivo in pigs To evaluate the serological response of recombinant EHV-1 vaccines expressing FHV-1 gD and FHV-1 gB, serological studies were conducted in pigs. A total of eight pigs, approximately 6-7 weeks of age, were randomly assigned to three treatment groups (Table 3). Table 3: Swine serology test design TIFF0007756860000003.tif46170

[0122] Treatment consisted of two experimental vaccines: rEHV-p430-FHVgD(co)-p455-FHVgB(co) (Group 1; 3 pigs, titer: 8.02 TCID 50 / mL) and rEHV-p430-FHVgD(co)F2AgB(co) (Group 2; 3 pigs, titer: 8.30 TCID 50 / mL), as well as a recombinant EHV-1 virus with a non-FHV antigen as a negative control (Group 3; 2 pigs, titer: 7.80 TCID 50 The vaccine consisted of 2 mL of immunization time (min / mL). On Day 0, pigs were administered a 2 mL intramuscular (IM) and 2 mL intranasal (IN) dose of the appropriate vaccine. On Day 14, all animals received a booster dose of 2 mL IM and 2 mL IN of the appropriate vaccine. See Table 4 for the study schedule. Table 4: Swine Serology Testing Event Schedule TIFF0007756860000004.tif66102

[0123] TIFF0007756860000005.tif48170Table 5: SN results for FHV-1 from swine serology testing TIFF0007756860000006.tif57170

[0124] TIFF0007756860000007.tif27170Table 6: SN results for EHV-1 from swine serology testing TIFF0007756860000008.tif56170

[0125] Furthermore, to demonstrate the presence of FHVgD-specific antibodies and FHVgB-specific antibodies, HI was performed to confirm FHVgD antibodies, and IFA was performed to confirm FHVgD-specific antibodies and FHVgB-specific antibodies. HI (hemagglutination inhibition) assay Because FHV gD is known to induce hemagglutination of feline RBCs (Maeda et al. 1998. J Vet Med Sci;60:881-888), HI assays were performed on test serum samples to test for the presence of FHVgD-specific antibodies. The antigen for hemagglutination was FHVgD obtained from A1-ST cells infected with rEHV-p430-FHVgD(co)-p455-FHVgB(co) and lysed with 0.1% CHAPS. After a booster vaccination on D14, pigs vaccinated with rEHV-p430-FHVgD(co)-p455-FHVgB(co) exhibited 2- to 4-fold higher HI antibody titers and higher concentrations of specific antibodies to FHVgD than pigs vaccinated with rEHV-p430-FHVgD(co)F2AgB(co) (Table 7). Sera obtained from animals in group 3 did not exhibit HI activity throughout the study.

[0126] Table 7: FHV-1 HI results from swine serology testing TIFF0007756860000009.tif52170 IFA (Immunofluorescence Assay) To test the test sera for FHVgD-specific and FHVgB-specific antibodies, A1-ST cells were transfected with the shuttle vectors p1_3-p430-FHVgD(co) and p455-FHVgB(co), respectively. A third set of cells was not transfected to serve as a negative cell control. Table 8 shows the results of this assay. Table 8: Serum IFA results of porcine serology testing TIFF0007756860000010.tif56170N=negative; W+=weak staining; +=average staining; S+=strong staining

[0127] Complementing the HI antibody titers observed from pigs in Group 1, these same serum samples also showed strong IFA staining of cells transfected with the FHV gD shuttle vector, and were found to show even stronger IFA staining after the second vaccination at D14. In contrast, the same serum from Group 1 only weakly stained cells transfected with the FHV gB shuttle vector. Serum from pigs in Group 2 had low HI antibody titers to gD. The same serum sample from Group 2 was found to moderately stain cells transfected with the FHV gD shuttle vector. Similar to the anti-gB IFA reaction observed with serum from pigs in Group 1, serum samples from pigs in Group 2 showed punctate staining but only weakly stained cells transfected with the FHV gB shuttle vector. Serum samples obtained from pigs in Group 3, inoculated with an unrelated rEHV-1 virus, showed negative or only weak, non-punctate staining (intensity due to background staining) in cells transfected with either the FHV gD shuttle vector or the FHV gB shuttle vector. The background staining observed from pigs in Group 3 was considered an overall baseline for the negative IFA reactivity of pigs in Groups 1 and 2. Conclusions of Example 5: In summary, both constructs were functional and demonstrated serum neutralizing antibodies, HI antibody titers and IFA staining for FHV-1.

[0128] Example 7: Testing of a recombinant EHV-1 vector vaccine expressing FHV-1 gD and gB in vivo in cats To evaluate the serological response to recombinant EHV-1 vaccines expressing FHV-1 gD and FHV-1 gB, a challenge study was conducted in the target species, cats. On day 0, a total of 10 cats, approximately 7-9 weeks of age, were randomly assigned to two treatment groups (Table 9). Table 9: Cat challenge test design TIFF0007756860000011.tif46170

[0129] Treatment began on day 44 with feline herpesvirus type 1 (CVB BUA No. 2019020, titer: 6.50 TCID), also known as feline rhinotracheitis virus (FVR) 96-13. 50 Five control cats in Group 1 were challenged with rEHV-p430-FHVgD(co)-p455-FHVgB(co) (titer: 7.86 TCID 50 / mL) and then on day 44, FVR 96-13 (CVB BUA No. 2019020, titer: 6.50 TCID 50 The study consisted of five cats in Group 2 that were challenged with a 0.5 mL dose of vaccine (1 mL / mL). On Day 0, cats in Group 2 were administered a 0.5 mL dose of vaccine subcutaneously, followed by a booster vaccination by the same volume and route of administration on Day 21 of the study. On Day 44, all cats in the study were challenged with equal doses of 1 mL of challenge virus administered via both the intranasal and oropharyngeal routes. See Table 10 for the study schedule.

[0130] Table 10: Event schedule for cat challenge testing TIFF0007756860000012.tif141137

[0131] The primary variable was clinical signs of disease, and the secondary variables were fever, weight loss, and serology. Acceptance criteria for the primary variable included clinical signs for ≥2 days postchallenge in unvaccinated controls to meet the case definition, and >80% of controls meeting the case definition for a successful challenge. Acceptance criteria for the secondary variable included a reduction in fever in vaccinated animals compared to unvaccinated controls, cats remaining seronegative before vaccination, demonstrating seroconversion after vaccination, and control cats remaining seronegative until challenge. Clinical observations were conducted daily after challenge. Cats were weighed pre-study, pre-challenge, and at the end of the study to determine weight loss. Rectal temperatures were measured twice pre-challenge as a baseline and daily for the first 7 days postchallenge in all cats. Rectal temperatures were then measured in cats exceeding 39.5°C until recovery. All prechallenge swabs were submitted to the University of Georgia Athens Veterinary Diagnostic Laboratory (UGA VDL) for screening using feline respiratory panel PCR. Serum samples obtained from the study were tested for serum neutralizing FHV-1 and EHV-1 antibody titers using a CPE-based readout. All cats in the control group showed moderate to severe clinical signs lasting for 2 days or more (Figure 11). Rhinitis was the most common clinical sign observed across all groups. Ocular signs and conjunctivitis were not observed in the vaccinated groups. Four of the five animals in Group 1 showed other clinical signs, such as audible rales, mouth breathing, and blood on nostrils due to sneezing. None of the vaccinated animals showed these findings. No local or systemic adverse events were observed in the vaccinated cats. Control cats lost weight after challenge, while the vaccinated group maintained the same weight or gained slightly more (Figure 12).Three cats in the control group developed fever on day 4 post-challenge, while none of the vaccinated animals developed fever (data not shown). All vaccinated animals developed EHV-1 serum neutralizing antibody titers after the first vaccination and a booster response was seen after the second vaccination (Figure 13). The challenge control group was seronegative for EHV-1 neutralizing antibodies throughout the study period (data not shown). All vaccinated animals developed EHV-1 serum neutralizing antibody titers after the second vaccination (Figure 14). Vaccination induced a successful priming of the neutralizing antibody response, as indicated by the spike in serum neutralizing antibody titers in vaccinated animals post-challenge (day 55).

[0132] Conclusions of Example 6: Overall, vaccination with rEHV-p430-FHVgD(co)-p455-FHVgB(co) resulted in a reduction in the severity and duration of clinical signs of disease compared with unvaccinated control animals. Furthermore, after challenge, animals vaccinated with rEHV-p430-FHVgD(co)-p455-FHVgB(co) had higher serum neutralizing antibody titers to FHV-1, indicating a positive vaccine effect.

Claims

1. 1. An EHV (Equine Herpesvirus) vector comprising a feline herpesvirus (FHV) antigen coding sequence inserted into ORF70 (US4) and a feline herpesvirus (FHV) antigen coding sequence inserted into ORF1 / 3, wherein the FHV antigen coding sequence inserted into ORF70 (US4) and the FHV antigen coding sequence inserted into ORF1 / 3 are different from each other and are selected from an FHV gD (glycoprotein D) coding sequence or a fragment thereof, and an FHV gB (glycoprotein B) coding sequence or a fragment thereof; the FHV gD coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:1, or a sequence having at least 90% sequence identity to said amino acid sequence; An EHV vector, wherein the FHV gB coding sequence consists of or comprises a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, or a sequence having at least 90% sequence identity to said amino acid sequence.

2. The EHV vector according to claim 1, wherein the FHV antigen coding sequence inserted into the ORF70 (US4) and the FHV antigen coding sequence inserted into the ORF1 / 3 are each one FHV antigen coding sequence.

3. 3. The EHV vector according to claim 1 or 2, wherein the EHV is attenuated and / or recombinant.

4. The EHV vector according to any one of claims 1 to 3, wherein the EHV is EHV-1 and / or RacH or RacH SE.

5. An immunogenic composition, vaccine or DIVA vaccine comprising an EHV vector according to any one of claims 1 to 4.

6. The immunogenic composition, vaccine or DIVA vaccine of claim 5, further comprising a pharmaceutically acceptable carrier.

7. 7. A method for immunizing a feline, comprising administering to said feline an immunogenic composition, vaccine or DIVA vaccine according to claim 5 or 6.

8. A method for treating or preventing clinical signs of FHV in a feline animal compared to an unimmunized control group of felines, comprising administering to the feline animal a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine described in claim 5 or 6.

9. A method for treating or preventing respiratory disease in felines caused by FHV compared to an unimmunized control group of felines, comprising administering to the feline a therapeutically effective amount of an immunogenic composition, vaccine or DIVA vaccine described in claim 5 or 6.

10. The method of any one of claims 7 to 9, wherein the immunogenic composition, vaccine or DIVA vaccine is administered more than once.

11. The method of any one of claims 7 to 10, wherein the immunogenic composition, vaccine or DIVA vaccine is administered intramuscularly, intradermally, subcutaneously, orally, or intranasally.

Images